Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.     

Ionic liquids used in and analyzed by capillary and microchip electrophoresis

This review, covering reports published from 2001 to December 2008, shows how ionic liquids (ILs) have made significant contributions in the improvement of capillary and microchip electrophoresis (CE and μCE) for the separation and detection of analytes such as phenols and aromatic acids, metal ions, medicines, enantiomers, biological materials, etc. Furthermore, CE methods applied in the sensitive and accurate determination of physico-chemical properties of ILs have been summarized. Accordingly, research vacancies and future development trends in these areas are discussed.     

Investigation of selected parameters of capillary zone electrophoresis method for analysis of isolates of outer membrane vesicles

The capillary zone electrophoresis (CZE) has recently been proposed by our group as a novel technique for outer membrane vesicles (OMVs) characterization (J. Chromatography 1621 (2020) 461047). In present work the impact of selected parameters of CZE method on OMVs isolates analysis was assessed. It was shown that the extension of sample injection plug length significantly improves the detectability of macromolecular aggregates in CZE. Moreover, a negligible adsorption of OMVs to both uncoated and polymer-modified (poly(DMA-GMA-MAPS)) capillary walls was proven. Finally, the relaxation effect as well as deformation/polarization of vesicles were demonstrated to affect OMVs electrophoretic mobility. The significance of these findings was discussed.     

Simultaneous electrochemical and electrochemiluminescence detection for microchip and conventional capillary electrophoresis

A simultaneous electrochemical (EC) and electrochemiluminescence (ECL) detection scheme was introduced to both microchip and conventional capillary electrophoresis (CE). In this dual detection scheme, tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) was used as an ECL reagent as well as a catalyst (in the formation of Ru(bpy)3(3+)) for the EC detection. In the Ru(bpy)3(2+)-ECL process, Ru(bpy)3(3+) was generated and then reacted with analytes resulting in an ECL emission and a great current enhancement in EC detection due to the catalysis of Ru(bpy)3(3+). The current response and ECL signals were monitored simultaneously. In the experiments, dopamine and three kinds of pharmaceuticals, anisodamine, ofloxacin, and lidocaine, were selected to validate this dual detection strategy. Typically, for the EC detection of dopamine with the presence of Ru(bpy)3(2+), a approximately 5 times higher signal-to-noise ratio (S/N) can be achieved than that without Ru(bpy)3(2+), during the simultaneous EC and ECL detection of a mixture of dopamine and lidocaine using CE separation. The results indicated that this dual EC and ECL detection strategy could provide a simple and convenient detection method for analysis of more kinds of analytes in CE separation than the single EC or ECL detection alone, and more information of analytes could be achieved in analytical applications simultaneously.     

Vapor and liquid phase detection of cyanide on a microchip

A capillary electrophoresis microchip is used to selectively and sensitively monitor cyanide levels in both vapor (HCN((g))) and aqueous (NaCN in drinking water) phases. Laser-induced fluorescence detection is applied using a violet diode laser to monitor the fluorescent isoindole derivative formed by the reaction of cyanide with 2,3-naphthalenedicarboxaldehyde (NDA) and taurine. Air sampling of hydrogen cyanide is achieved using a miniature impinger (2 mL), giving collection efficiencies as high as 79% for a sampling rate of 1.0 L/min and a 10 s sampling time (relative standard deviation RSD: 2.7% for n = 5). Following the addition of NDA and taurine to either the vapor phase impinger sample or an aqueous drinking water sample, the NDA/cyanide derivative can be detected in just over 40 s on the microchip, giving a detection limit of 0.56 microg/L and a linear dynamic range from 0.56 microg/L-2.4 mg/L. The detection limit for hydrogen cyanide in air was determined to be 2.3 ppb (mole%). On-chip derivatization of cyanide by NDA was successful, although a 50% decrease in signal intensity was observed due to insufficient time for completion of the reaction on the microchip. A number of different interferents were examined, and only iron(II) and chlorine showed any interference due to their capability for masking the presence of cyanide by reacting with free cyanide in solution.     

Carbon nanotube and diamond as electrochemical detectors in microchip and conventional capillary electrophoresis

As two important polymorphs of carbon, carbon nanotube (CNT) and diamond have been widely employed as electrode materials for electrochemical sensing. This review focuses on recent advances and the key strategies in the fabrication and application of electrochemical detectors in microchip and conventional capillary electrophoresis (CE) using CNT and boron-doped diamond. The subjects covered include CNT-based electrochemical detectors in microchip CE, CNT-based electrochemical detectors in conventional CE, boron-doped diamond electrochemical detectors in microchip CE, and boron-doped diamond electrochemical detectors in conventional CE. The attractive properties of CNT and boron-doped diamond make them very promising materials for the electrochemical detection in microchip and conventional CE systems and other microfluidic analysis systems.     

Separation of DNA fragments for fast diagnosis by microchip electrophoresis using programmed field strength gradient

We evaluated a novel strategy for fast diagnosis by microchip electrophoresis (ME), using programmed field strength gradients (PFSG) in a conventional glass double-T microfluidic chip. The ME-PFSG allows for the ultrafast separation and enhanced resolving power for target DNA fragments. These results are based on electric field strength gradients (FSG) that use an ME separation step in a sieving gel matrix poly-(ethylene oxide). The gradient can develop staircase or programmed shapes FSG over the time. The PFSG method could be easily used to increase separation efficiency and resolution in ME separation of specific size DNA fragments. Compared to ME that uses a conventional and constantly applied electric field (isoelectrostatic) method, the ME-PFSG achieved about 15-fold faster analysis time during the separation of 100 bp DNA ladder. The ME-PFSG was also applied to the fast analysis of the PCR products, 591 and 1191 bp DNA fragments from the 18S rRNA of Babesia gibsoni and Babesia caballi.     

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.     

Ultraviolet absorbance detection of colchicine and related alkaloids on a capillary electrophoresis microchip

The separation and UV absorbance detection of four toxic alkaloids, colchicine, thiocolchicine, colchicoside, and thiocolchicoside, on a microchip-based capillary electrophoresis device are reported. To increase the sensitivity of UV absorbance detection, optical cells with extended path lengths were integrated into the separation channel during the microfabrication process. The absorbance values realized on the microchip using these optical cells were proportional to the increase in average depths according to the Beer-Lambert Law, resulting in sensitivity enhancements by as much as five times. Linearity of response was observed from 5.0 to 500 mg L⁻¹ of colchicine, with detection limits ranging from 2 to 6 mg L⁻¹ depending upon the specific alkaloid and the dimension of the optical cell. The extraction of colchicine from spiked milk samples was performed and an average recovery rate of 83% with a relative standard deviation of 3.8% was determined using the optimized conditions on the microchip.     

Investigation of iron oxide nanoparticles by capillary zone electrophoresis

The electrophoretic behavior of γ-Fe₂O₃ nanoparticles was studied in aqueous solutions of Na₂SO₄-NaOH (pH 10.8) and of Na₂SO₄-Na₃cit (pH 7.1) as running electrolytes. Two electrophoretic zones (smooth and with spikes) due to colloidal and suspended particles of approximately the same size range were formed during the runs. The suspension stability and size distribution were shown to depend on the composition of electrolyte used for dispersing the solids. The effects of electric field strength, injection time, injection pressure as well as sodium citrate concentration were studied and particle electrophoretic mobilities were calculated. Electron micrographs of particles studied were obtained. Preparation of reference samples based on the colloidal γ-Fe₂O₃ has been discussed.     

Speciation analysis of inorganic arsenic by microchip capillary electrophoresis coupled with hydride generation atomic fluorescence spectrometry

A novel method for speciation analysis of inorganic arsenic was developed by on-line hyphenating microchip capillary electrophoresis (chip-CE) with hydride generation atomic fluorescence spectrometry (HG-AFS). Baseline separation of As(III) and As(V) was achieved within 54 s by the chip-CE in a 90 mm long channel at 2500 V using a mixture of 25 mmol l(-1) H3BO3 and 0.4 mmol l(-1) CTAB (pH 8.9) as electrolyte buffer. The precisions (RSD, n=5) ranged from 1.9 to 1.4% for migration time, 2.1 to 2.7% for peak area, and 1.8 to 2.3% for peak height for the two arsenic species at 3.0 mg l(-1) (as As) level. The detection limits (3sigma) for As(III) and As(V) based on peak height measurement were 76 and 112 microg l(-1) (as As), respectively. The recoveries of the spikes (1 mg l(-1) (as As) of As(III) and As(V)) in four locally collected water samples ranged from 93.7 to 106%.     

Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry

The mitochondrial membrane potential (DeltaPsi(m)) is an important indicator of the energetic state of both the mitochondria and the cells. To develop a sensitive, convenient, and rapid method for the measurement of DeltaPsi(m), we carried out cell fluorescence assays using the Agilent 2100 bioanalyzer system which, unlike the conventional flow cytometry, is based on microfluidic technology employing fluorescence detection with a 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)) fluorescent probe. The use of DiOC(6)(3) in the fluorometer was shown to be feasible for monitoring variations in DeltaPsi(m) in the mitochondria isolated from rat liver and treated with rotenone, succinate, ADP, and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Flow cytometry analysis showed severe reduction of fluorescence intensity in Jurkat cells after treatment with 1.0 and 10 microM FCCP. However, fluorescence microscopy demonstrated obvious accumulation of fluorescence in the mitochondria and induction of diffuse cytoplasmic fluorescence not localized to the mitochondria in these cells. The dose response range of DiOC(6)(3) in the Agilent 2100 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm-2.0 microM. Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 microM DiOC(6)(3) was observed after treatment with 10 microM FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring DeltaPsi(m) in cell assays.     

Genotyping the -6A/G functional polymorphism in the core promoter region of angiotensinogen gene by microchip electrophoresis

Angiotensinogen (AGT) gene has been regarded as one of the candidate genes for essential hypertension. In our study, the role of AGT gene as a putatively predisposing gene for hypertension was evaluated by genotyping a A (-6) G polymorphism in the core promoter region in 123 patients with essential hypertension and 103 healthy controls. A microchip electrophoresis method coupled with polymorphism chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay was used for genotyping the A (-6) G single-nucleotide polymorphism. The separation and detection of the digested PCR amplicons were completed just in 280 s or less. The genotype frequency fulfilled the criteria of the Hardy-Weinbery equilibrium (X2 = 3.067, P > 0.05). The results showed a higher frequency of the -6 A allele (0.70) in the normotensive subjects, which is higher than those reported in Germany (0.47) and Czech (0.40) populations, but similar to that found in Japanese populations (0.73). The frequencies of genotype AA, AG, and GG were 0.46, 0.49, and 0.05 in hypertensive subjects, and 0.44, 0.53, and 0.03 in control subjects. There is no significant difference in the distributions of the genotype and allele between the two groups (X2 = 0.88, P > 0.05; X2 = 0.024, P > 0.05). These findings differ from some of the results obtained in other ethnic groups, indicating the potential importance of ethnic origin in the assessment of genetic risk identifiers for a complex disease.     

Fabrication and performance of a three-dimensionally adjustable device for the amperometric detection of microchip capillary electrophoresis

A microchip CE-amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an AD cell containing a one-dimensionally adjustable disk detection electrode in a Plexiglas holder. It facilitates the precise 3-D alignment between the channel outlet and the detection electrode without a complicated 3-D manipulator. The performance of this unique system was demonstrated by separating five aromatic amines (1,4-phenyldiamine, aniline, 2-methylaniline, 4-chloroaniline, and 1-naphthylamine) of environmental concern. Factors influencing their separation and detection processes were examined and optimized. The five analytes have been well separated within 140 s in a 74 cm long separation channel at a separation voltage of +2500 V using a 10 mM phosphate buffer (pH 3.5). Highly linear response is obtained for the five analytes over the range 20-200 microM with the detection limits ranging from 0.46 to 1.44 microM, respectively. The present system demonstrated long-term stability and reproducibility with RSDs of less than 5% for the peak current (n = 9). The new approach for the microchannel-electrode alignment should find a wide range of applications in CE, flowing injection analysis, and other microfluidic analysis systems.     

Determination of vitamin C and preservatives in beverages by conventional capillary electrophoresis and microchip electrophoresis with capacitively coupled contactless conductivity detection

The separation and detection of commonly used preservatives (benzoate, sorbate) and vitamin C by both conventional CE and microchip electrophoresis with capacitively coupled contactless conductivity detection is presented. The separation was optimized by adjusting the pH-value of the buffer and the use of hydroxypropyl-beta-CD (HP-beta-CD) and CTAB as additives. For conventional CE, optimal separation conditions were achieved in a histidine/tartrate buffer at pH 6.5, containing 0.025% HP-beta-CD and 0.1 mM CTAB. LOD ranged from 0.5 to 3 mg/L (S/N = 3) and the RSDs for migration time and peak area were less than 0.1 and 2%, respectively. A considerable reduction of analysis time can be accomplished by using microchip electrophoresis without significant loss in sensitivity under optimal separation conditions. A histidine/tartrate buffer at pH 6.5, incorporating 0.06% HP-beta-CD and 0.25 mM CTAB, gave detection limits ranging between 3 and 10 mg/L and satisfactory reproducibilities of < or =0.4% for the migration time and < or =3.5% for the peak area. The methods developed are useful for the quantitative determination of food additives in real samples such as soft drinks and vitamin C tablets.     

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR-123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh-123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mM borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic NB4 cells induced by arsenic trioxide (As(2)O(3)) at low concentration (1-2 microM). Buthionine sulfoximine (BSO), in combination with As(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of As(2)O(3) and hydrogen peroxide (H(2)O(2)) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.     

Electrochemical detection of phenolic compounds using cylindrical carbon-ink electrodes and microchip capillary electrophoresis

A simple method to fabricate cylindrical carbon electrodes for use in capillary electrophoresis (CE) microchips is described. The electrodes were fabricated using a metallic wire coated with carbon ink. Several experimental variables were studied in order to establish the best conditions to fabricate the electrode. Finally, the electrodes were integrated in a poly(dimethylsiloxane) microchip and used for the analysis of phenolic compounds. Using the optimum conditions, the analysis of a mixture of dopamine, epinephrine, catechol, and 4-aminophenol was achieved in less than 240 s, showing good linear responses (R² = 0.999) in the 0.1-190 μM range, and limits of detection (without the use of stacking or a decoupler) of 140 and 105 nM for dopamine and epinephrine, respectively.     

Selective detection of cytochrome C by microchip electrophoresis based on an aptamer strategy

The release of cytochrome C (Cyt C) plays an important role in apoptosis. In this study, selective and sensitive detection of Cyt C based on an aptamer strategy coupled with MCE was developed. Following the binding of a specific aptamer to Cyt C, the aptamer exhibited an irregular state, reducing the binding affinity of a fluorescent probe, and thus preventing the aptamer‐Cyt C complexes from detection within the MCE. The height of the detection peak of the residual aptamer linearly decreased, and therefore, the difference in peak height of residual aptamer compared to that of the initial aptamer was used to quantify the captured protein concentration. Experimental conditions such as incubation time, pH, temperature, and ionic strength were optimized. A measurement of Cyt C concentration by MCE was achieved within 135 s, with a limit of detection as low as 0.4 nM. The proposed method has high selectivity and good stability for the detection of Cyt C. The experimental results demonstrate that this method is quick, consumes only a small quantity of sample, is highly selectivity and exhibits high sensitivity.     

Rapid and efficient isotachophoretic preconcentration in free solution coupled with gel electrophoresis separation on a microchip using a negative pressure sampling technique

We present a novel isotachophoresis–gel electrophoresis (ITP–GE) microchip system designed for rapid and efficient isotachophoretic preconcentration coupled with gel electrophoresis separation by using a negative pressure sampling technique. The overall ITP–GE procedure involves only three steps: sample loading, ITP preconcentration and GE separation and was controlled by a simple and compact negative pressure sampling device, which is composed of a vacuum vessel, a three-way electromagnetic valve and a single high voltage power supply. During the sample loading stage, a negative pressure was applied via a three-way electromagnetic valve in headspace of the two sealed sample waste reservoirs (SWs). A sandwiched sample zone between a leading and a terminating electrolyte zone was formed in the channel intersection in less than 1 s. Once the three-way electromagnetic valve was switched to connect SWs to ambient atmosphere to release vacuum in SWs, ITP preconcentration in free solution and GE separation in the 4% hydroxyethylcellulose (HEC) sieving material were consequently activated under the electric potentials applied. The performance of present approach was evaluated by using DNA fragments as model analytes. Compared to conventional cross microchip GE using electrokinetic pinched injection, an average signal enhancement of 185-fold was obtained with satisfactory resolution. The results demonstrated the ITP–GE approach possessing an exciting potential of high sensitivity and short sampling time with significant simplification in operation and instrumentation.