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We give an overview on recent developments in high-throughput microfluidic sample-introduction techniques based on a capillary sampling probe and a slotted-vial array (SVA). We discuss the advantages and the potential of SVA-based sample-introduction systems as well as their applications in miniaturized flow-injection analysis, sequential-injection analysis, capillary electrophoresis and liquid-liquid extraction. We illustrate the advantages and the potential of SVA-based sample-introduction systems using results obtained recently.

In the aqueous phase, ethidium bromide (EB) intercalates into the double helix structure of dsDNA (ds= double-stranded) with a notable enhancement in fluorescence and resonance light scattering (RLS). However, when dsDNA was extracted into an ionic liquid (IL), 1 -butyl-3-methylimidazolium hexafluorophosphate (BmimPF 6 ), an abnormal RLS arising from the interactions of IL-DNA-EB was observed, with a substantial decrease of the recorded RLS. The cationic Bmim + groups of BmimPF 6 intercalate into the DNA helix structure, in which they interact with the P-O bonds of phosphate groups in DNA strands and result in a reduction of the base-pair interstice along with transformation of DNA conformations that consequently prohibits the intercalation of EB with DNA. Thus, in the IL phase, the interactions between ethidium and DNA were dominated by electrostatic interactions and hydrogen bonding, leading to a congregation of EB entities around the DNA strands that results in an increase of absorption by ethidium, and consequently the inner filter effect leads to a reduction of the RLS. The present observation has been applied to the direct quantification of DNA in an ionic-liquid phase after DNA from human whole blood was extracted into BmimPF 6 .

On the basis of oxidative decoloration of bromopyrogallol red(BPR) with H 2 O 2 , catalyzed by horseradish peroxidase(HRP), and the sequential injection renewable surface technique(SI-RST), a highly sensitive optical-fiber sensor spectrophotometric method for the enzymatic determination of hydrogen peroxide was proposed. By coupling with a glucose oxidase(GOD) -catalyzed reaction, the method was used to determine glucose in human serum. The considerations in system and flow cell design, and factors that influence the determination performance are discussed. With 100 μL of sample loaded and 0.6 mg of bead trapped, the linear response range from 5.0 × 10 −8 to 5.2 × 10 −6 mol/L BPR with a detection limit(3σ) of 2.5 × 10 −8 mol/L BPR, and a precision of 1.1% RSD( n = 11) and a throughput of a 80 samples per hour can be achieved. Under the conditions of a 8.7 × 10 −6 mol/L BPR substrate, 0.04 unit/mL HRP, 600 s reaction time and a reaction temperature of 37°C, the linear response range for H 2 O 2 was from 5.0 × 10 −8 to 7.0 × 10 −6 mol/L with a detection limit(3σ) of 1.0 × 10 −8 mol/L and a precision of 3.7% RSD ( n = 11). The linear response range by coupling with a GOD-catalyzed reaction was from 1.0 × 10 −7 to 1.0 × 10 −5 mol/L. The method was directly applied to determine glucose in human serum. Glucose contents obtained by the proposed procedure were compared with those obtained by using the phenol-4-AAP method, the error was found to be less than 3%.

A novel high-throughput method without cytolysis for determination of enzyme activity inside single cells was developed by a combination of chemical cell perforation and an intracellular enzyme-catalyzed reaction. Peroxidase (PO) inside human neutrophils was chosen as the model system. Cells were perforated with digitonin to form micropores on the cell membrane. The perforated cells, with physiological buffer saline of pH 7.4 containing hydroquinone (H2Q) and H2O2, were continuously propelled by pressure through a capillary as the microsampler and microreactor. Small molecules H2Q and H2O2 could diffuse into the cell interior through the micropores on the cell membrane, and the large molecule PO remained in the cell interior. Intracellular PO converted H2Q into benzoquinone (BQ). BQ diffused out from the cell interior to the cell surface through the micropores and formed a BQ zone around the cell. The process proceeded in the capillary during cell movement. The BQ zones around every moving perforated cell were continuously delivered to the capillary outlet by hydraulic flow and detected. An average detection rate of1 cell/min was obtained.

The performance of a micropump operating on evaporation and capillary effects, developed for microfluidic (lab-on-a-chip) systems, was studied employing it as the fluid drive in a microfluidic flow injection (FI) system, with chemiluminescence (CL) detection. The micropump featured simple structure, small dimensions, low fabrication cost and stable and adjustable flow-rates during long working periods. Using a micropump with 6.6 cm 2 evaporation area, with the ambient temperature and relative humidity fluctuating within 2 h in the ranges 20–21 °C and 30–32%, respectively, an average flow-rate of 3.02 μL/min was obtained, with a precision better than 1.2% R.S.D. ( n = 61). When applied to the microchip FI-CL system using the luminol/hexacyanoferrate/H 2 O 2 reaction, a precision of 1.4% R.S.D. ( n = 11) was obtained for luminol at a sampling frequency of 30 h −1 .

A robust and simple approach for microfabricated chip based liquid-liquid extraction was developed for on-chip sample pretreatment. The chip based extraction system was composed of two microfabricated glass plates with a microporous membrane sandwiched in between. A simple bonding approach using epoxy was used to achieve bonding and sealing of the L-L extraction chip. Gravity was employed to drive the aqueous and organic flows through separate channels in the extraction system, separated by the membrane. During extraction, the analyte in an aqueous sample stream was transferred through the membrane into the organic stream. The fluorescence intensity of the analyte extracted into the organic stream was monitored in situ by a laser induced fluorescence detection system. The performance of the system was demonstrated using an aqueous solution of butyl rhodamine B (BRB) and isobutanol as sample and extractant, respectively. The system proved to be an efficient means for achieving chip based microporous membrane liquid-liquid extraction. The precision of fluorescence measurements was 1.5% R.S.D. (n=4). A linear response range of 1x10(-7) to 1 x 10(-4) M BRB was obtained with a regression equation: I=8.00 x 10(6) C + 4.91. An enrichment factor of ca. 3 was obtained with an extraction efficiency of 69%.

A simple and robust static adsorptive (dynamic) coating process using 2% hydroxyethylcellulose was developed for surface modification of poly(methyl methacrylate) (PMMA) microfluidic chips for DNA separations, suitable for usage over extended periods, involving hundreds of runs. The coating medium was also used as a sieving matrix for the DNA separations following the coating process. Four consecutive static treatments, by simply filling the PMMA chip channels with sieving matrix once every day, were required for obtaining a stable coating and optimum performance. The performance of the coated chips at different phases of the coating process was studied by consecutive gel electrophoretic separations with LIF detection using a PhiX-174/HaeIII DNA digest sample. The coated chip, with daily renewal of the sieving matrix, showed high stability in performance during a 25-day period of systematic study, involving more than 100 individual runs. The performance of the coated chip also remained almost the same after 3 months of continuous usage, during which over 200 separations were performed. The average precision of migration time for the 603-bp fragment was 1.31% RSD (n = 6) during the 25-day study, with a separation efficiency of 6.5 x 10(4) plates (effective separation length 5.4 cm).

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 (

The signal-to-noise level of light emitting diode (LED) fluorimetry using a liquid-core-waveguide (LCW)-based microfluidic capillary electrophoresis system was significantly enhanced using a synchronized dual wavelength modulation (SDWM) approach. A blue LED was used as excitation source and a red LED as reference source for background-noise compensation in a microfluidic capillary electrophoresis (CE) system. A Teflon AF-coated silica capillary served as both the separation channel and LCW for light transfer, and blue and red LEDs were used as excitation and reference sources, respectively, both radially illuminating the detection point of the separation channel. The two LEDs were synchronously modulated at the same frequency, but with 180 degrees -phase shift, alternatingly driven by a same constant current source. The LCW transferred the fluorescence emission, as well as the excitation and reference lights that strayed through the optical system to a photomultiplier tube; a lock-in amplifier demodulated the combined signal, significantly reducing its noise level. To test the system, fluorescein isothiocyanate (FITC)-labeled amino acids were separated by capillary electrophoresis and detected by SDWM and single wavelength modulation, respectively. Five-fold improvement in S/N ratio was achieved by dual wavelength modulation, compared with single wavelength modulation; and over 100-fold improvement in S/N ratio was achieved compared with a similar LCW-CE system reported previously using non-modulated LED excitation. A detection limit (S/N=3) of 10nM FITC-labeled arginine was obtained in this work. The effects of modulation frequency on S/N level and on the rejection of noise caused by LED-driver current and detector were also studied.

A micro-sequential injection spectrophotometric procedure for DNA assay was developed based on the employment of a lab-on-valve (LOV) meso-fluidic analytical system. A small amount of crystal violet solution (10mul) was de-colored inside the flow cell of the LOV at the presence of 5mul lambda-DNA/HindIII within a certain pH range, and the absorbance decrease of crystal violet solution at 591nm was measured via optical fibers and was employed as the basis of quantification. A uni-variant approach was adopted for the optimization of experimental parameters, including buffer pH, concentration and volume of crystal violet solution, reaction time and sample/reagent loading flow rates. A linear calibration graph was obtained within 0.2-6.0mugml(-1), along with a detection limit of 0.07mugml(-1). The procedure was applied for the determination of lambda-DNA/HindIII in synthetic samples in comparison with a documented procedure.

Reactive oxygen species (ROS) are known to not only mediate the damage of cellular constituents but also to regulate cellular signaling. Analysis of ROS is essential if we wish to understand the mechanisms of cellular alterations. In this paper, a microfluidic chip-based approach to the determination of ROS in single erythrocyte was developed by using a simple crossed-channel glass chip with integrated operational functions, including cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser-induced fluorescence (LIF) detection. Non-fluorescent dihydrorhodamine 123 (DHR 123), which can be oxidized intracellularly by ROS to the fluorescent rhodamine 123 (Rh 123), was used as the fluorogenic reagent. The effect of pH on the migration time of Rh 123 and detection sensitivity was discussed. The present method minimized dilution of intracellular ROS during reaction with DHR 123 and determination. As a result, an extremely low detection limit of 0.8 amol has been achieved. The time required for complete analysis of one human erythrocyte was less than 3 min. A migration time precision of 4.1% RSD was obtained for six consecutively-injected cells. Upon stimulation with 4 mmol/l H2O2 for 10 min, the intracellular ROS concentration was found to increase on average by about a factor of 8.4.

A novel electrochemical method with a microfluidic device was developed for analysis of single cells. In this method, cell injection, loading and cell lysis, and electrokinetic transportation and detection of intracellular species were integrated in a microfluidic chip with a double-T injector coupled with an end-channel amperometric detector. A single cell was loaded at the double-T injector on the microfluidic chip by using electric field. Then, the docked cell was lysed by a direct current electric field strength of 220 V/cm. The analyte of interest inside the cell was electrokinetically transported to the detection end of separation channel and was electrochemically detected. External standardization was used to quantify the analyte of interest in individual cells. Ascorbic acid (AA) in single wheat callus cells was chosen as the model compound. AA could be directly detected at a carbon fiber disk bundle electrode. The selectivity of electrochemical detection made the electropherogram simple. The technique described here could, in principle, be applied to a variety of electroactive species within single cells.

A miniaturized capillary electrophoresis (CE) system with UV-Vis detection was coupled to a flow injection (FI) system for achieving high throughput continuous sample introduction. The cassette of a commercial CE instrument was modified to hold a 6.5 cm long silica capillary and a flow-through waste reservoir. The cassette was inserted into the flow-cell chamber of a commercial UV detector, with the light beam focused on the capillary and collected by two ball lenses on the cassette. The capillary inlet, left outside the cassette and detector, was positioned on the top of a vertical 3.5 mm diameter glass rod, in close contact with an electrode. Samples injected through the FI system dropped freely on top of the pillar, covering the capillary inlet and electrode. Continuous sample introduction was achieved for CE separations under non-interrupted separation voltage, which was isolated from the FI system through the discontinuity of droplets. The newly developed interface and UV detection system was used for fast separation of sulphamethoxazole (SMZ) and trimethoprim (TMP) in sulphatrim tablets, achieving a high throughput of over 48 h−1, and a low carryover of 2%. Separation efficiencies of 8 μm plate height and detection limits of 1.0 mg l−1 for SMZ and 0.5 mg l−1 (3σ) for TMP were obtained.

A three-layer poly(dimethylsiloxane) (PDMS)/glass microfluidic system for performing on-chip solid-phase enzymatic reaction and chemiluminescence (CL) reaction was used for the determination of glucose as a model analyte. A novel method for the immobilization of controlled-pore-glass based reactive particles on PDMS microreactor beds was developed, producing an on-chip solid-phase reactor that featured large reactive surface and low flow impedance. Efficient mixing of reagent/sample/carrier streams was achieved by incorporating chaotic mixer structures in the microfluidic channels. A conventional sequential injection (SI) system was adapted for direct coupling with the microfluidic system, and combined with hydrostatic delivery of reagents to achieve efficient and reproducible sample introduction at 10 μl levels. A detection limit of 10 μM glucose (3σ), and a precision of 3.1% RSD (n=7, 0.2 mM glucose) were obtained using the SI-microfluidic-CL system integrated with a glucose oxidase (GOD) reactor. Carryover was

The combined flow injection (FI)–capillary electrophoresis (CE) system was further exploited by coupling to an electrogenerated chemiluminescence (ECL) detection system. A low-cost miniaturized CE system was developed on a chip platform to provide easy interface both with FI sample introduction and with ECL detection. A falling-drop interface was employed to perform FI split-flow sample introduction while achieving electrical isolation from the CE high voltage. A plexiglas reservoir at the capillary outlet served as both the reaction and detection cell for the ECL reaction, with Ru(bpy) 3 2+ reagent continuously flowing through the cell. An optical fiber was positioned within the reservoir close to the capillary outlet for transferring the ECL emission to the PMT. The relative positions of the capillary outlet, working electrode and optical fiber as well as reagent renewal flow-rate were optimized to achieve both good sensitivity and separation efficiency under non-interrupted sampling conditions, involving large numbers of samples. An on-column joint often used in other works for isolating the ECL detection system from the CE separation voltage was not found necessary. The performance of the system was illustrated by the baseline separation of proline, valine and phenylalanine with a high throughput of 50 h −1 and plate height of 14 μm for proline under 147 V cm −1 field strength. Detection limits (3 σ ) were 1.2, 50 and 25 μM and peak height precisions were 1.4, 5.4 and 4.3% R.S.D. ( n =9) for proline, valine and phenylalanine, respectively.

Microfabrication techniques used for the production of MEMS (micro electro-mechanical systems) have been successfully used to produce highly efficient microfluidic capillary electrophoresis chip systems. A limitation of this approach are the difficulties associated with the creation of the micrometer-sized structures in glass or other substrates, which currently involve specialized and expensive lithographic and etching processes. A further limitation is that hitherto most microfluidic chips are not designed for continuous introduction of a series of different samples, which limits the overall throughput of such systems. This article reviews the development of a microfluidic system for rapid CE separations, produced at a low cost of less than a dollar each, using equipment and materials readily available in the ordinary laboratory. Applications of the system, after coupling to flow-injection and/or sequential-injection sample introduction, for the determination of FITC- labeled amino acids by laser-induced fluorescence, trace metals by chemiluminescence, carbohydrates by amperometry, and inorganic and organic anions by indirect UV absorbance are exemplified to illustrate the performance and versatility of the microfluidic system.

A microchip-based capillary electrophoresis (CE) system with amperometric detection, combined with flow injection (FI) sample introduction, was constructed from components readily available in the analytical laboratory. An H-channel configuration was used with separation capillary positioned between two tubular side-arms, and a falling-drop interface connected to one side-arm was developed to achieve electrical isolation between the FI and CE systems. End-column amperometric detection was accomplished with separation voltage decoupled from the detection system, employing a microdisk working electrode positioned immediately outside the capillary outlet in the other side-arm, which functioned as a large-volume reservoir. Sample dispersion in the FI system and the FI–CE interface was minimized by intercalating the sample zone between two air segments. Performance of the FI–CE amperometric system was demonstrated by separation of sugars. Separation of sucrose and glucose was performed with a 5 cm, 25 μm i.d. capillary with 1.7 kV separation voltage in about 60 s (40 μm plate height for glucose), achieving a sampling frequency of over 65 h−1. Responses for sucrose and glucose were linear in the range of 10–1000 μM with sensitivities of 0.011 and 0.025 nA μM−1. Detection limits (S/N=3) were 2 μM for sucrose and 1 μM for glucose. Peak height precisions were 2.1 and 2.4% R.S.D. (n=9) for sucrose and glucose, respectively.

An overview on developments of chemiluminescence (CL) detection in capillary electrophoresis (CE) systems is presented covering the related publications till early 2000. Contributions are reviewed both in relation to developments in instrumental design, CL chemistry and application field. Future prospects for development are discussed.

A sequential injection system for on-line sorbent extraction preconcentration in electrothermal atomic absorption spectrometry was developed for the determination of trace cadmium in seawater and other water samples. The system is characterized by simple manifold design and improved robustness. The Cd-APDC complex was sorbed on a 15-μl micro-column (located at the tip of the furnace sampling probe) packed with C 18 . After sequentially metering segments of ethanol, washing acid, sample and APDC complexing reagent into a 2.5-m-long, 1-mm-i.d. holding coil, the flow was reversed and directed to the column. The complexing reaction, sample loading and column washing were then achieved within the single reversed syringe stroke. The sorbed analyte was then eluted into the furnace with 50 μl ethanol. Mutual mixing between sample, washing acid, and eluent were prevented by separating the zones with small air segments during metering. Progressive tightening of the column was avoided by a short back-suction through the column after each operational cycle. With 1 ml sample loaded, and 50 μl eluate introduced into the furnace, an enrichment factor of 19 was obtained with a detection limit of 0.5 ng l −1 Cd (3σ), a precision of 2.1% R.S.D. ( n =11), and a throughput of 12 h −1 . Good recoveries were obtained for spiked water samples and good agreement of results with standard reference seawater and river water samples were achieved.

The important role of flow injection (FI) techniques for the automation, acceleration and miniaturization of solution handling in sample pretreatment as well as some recent trends in the development of the field are discussed, illustrated mainly by recent achievements in the author’s laboratory, including: (a) sample pretreatment for vapor generation and electrothermal AAS based on sequential injection (SI) techniques, with low reagent consumption and enhanced ruggedness; (b) combination of FI and SI sample pretreatment (filtration, dialysis, gas diffusion, column sorption) with capillary electrophoresis (CE) giving enhanced reproducibility and efficiency; (c) application of on-line microdialysis in in vivo monitoring of blood glucose in test animals and (d) application of on-line microdialysis and solvent extraction in continuous monitoring of drug dissolution processes with high resolution of process events. Future perspectives of FI sample pretreatment are discussed, emphasizing the improvement in ruggedness of the equipment and methods, the combination and synchronization of different means for liquid propulsion, and the development of miniaturized systems.

A bias-free sample injection method based on electroosmotic-flow (EOF) traction was developed for the combined flow injection-capillary electrophoresis system. A Nafion joint implemented at a capillary fracture close to the capillary inlet was used for isolating the electric field during the split-flow sample injection, the sample solution being drawn into the capillary by EOF without bias effects. An electronic time relay system was adopted to control the switching of high voltage between the capillary inlet end and the joint. The peak area ratios of two model compounds, dimethyl sulfoxide and benzoic acid obtained using the proposed system were identical to those with gravity injection. Relative standard deviations of 0.5–2.5% (n = 32) were achieved for peak height and peak area evaluation in the consecutive injections of a series of replicate sample solutions at a sampling frequency of 15 h−1 . Similar performance of the system was obtained with a reversed EOF background electrolyte after reversing the electrode polarity for a mixture of the anions, Cl−, SO42−, NO3− and F− using indirect UV detection. The performance of the system for the analysis of real samples was demonstrated by bias-free sample injection in the separation of caffeine, theobromine and theophylline in beverage samples with satisfactory recoveries in the 92–107% range.

A flow injection (FI) system was developed incorporating on-line solvent extraction separation and stopped-flow dialysis systems for automated continuous monitoring of multi-vessel drug dissolution processes. The system employed PTFE pump tubes for solvent delivery, and combined a coaxial phase segmentor, conical cavity gravitational phase separator with a microdialysis sampling system to produce a reliable extraction system capable of stable non-stop operation over extended period of time. The system was evaluated for continuous monitoring in multi-vessel drug dissolution testing of ethambutol hydrochloride tablets using bromocresol green as reagent and chloroform as extractant. With the six-vessel dissolution system used, a sampling frequency of 15/h for each dissolution vessel was achieved with a total of 90 determinations per hour, giving an average precision of 3.6% RSD (n = 11). Results showed good agreement with those obtained using the Chinese Pharmacopoeia standard method.

An automatic system for drug-dissolution studies based on the sequential-injection (SI) technique is described and used for simultaneous monitoring of dissolution profiles of aspirin, phenacetin and caffeine in compound aspirin tablets. The partial least squares calibration technique was used for simultaneous determination of the three components. 300 μl test solution in dissolution vessel was aspirated into a PTFE holding coil via on-line filters by a syringe pump. After delivering an aliquot (10 μl) of each aspirated solution into the transferring line connected to the detector, the residual dissolution solution in the holding coil was fed back to the dissolution vessel to clear the filter and decrease sample consumption. The sampled aliquot was carried and stopped in the spectrophotometric detector and scanned within 220 and 310 nm. Fifteen samples run in triple-replicates were processed per hour with a total of 45 measurements.

A sequential injection micro-sample introduction system was coupled to a microfluidic-chip based capillary electrophoresis system through a split–flow sampling interface integrated on the micro-chip. The microfluidic system measured 20×70×3 mm in dimension, and was produced using a non-lithographic approach with components readily available in the analytical laboratory. In the H-configuration channel design the horizontal separation channel was a 75 μm I.D.×60 mm quartz capillary, with two vertical side arms produced from plastic tubing. The conduits were embedded in silicon elastomer with a planar glass base. Sequential introduction of a series of samples with about 2.5% carryover was achieved at 48 h−1 throughput with samples containing a mixture of fluorescein isothiocyanate (FITC)-labeled amino acids using SI sample volumes of 3.3 μl and carrier flow-rate of 2.0 ml min−1. Baseline separation was achieved for FITC-labeled arginine, phenylalanine, glycine and FITC (laser induced fluorescence detection) in sodium tetraborate buffer (pH 9.2) within 8–80 s, at separation lengths of 25–35 mm and electrical field strengths of 250–1500 V cm−1, with plate heights in the 0.7–3 μm range.

An overview is given of the recent development of automated flow injection (FI) systems for continuous monitoring in drug dissolution testing. The advantages and potentials of FI systems involving sequential sampling from multi-dissolution vessels, with the application of chemometric methods for multi-component determination, on-line separation by filtration, microdialysis, solvent extraction, and capillary electrophoresis, and indirect determinations based on atomic absorption spectrometry are discussed and illustrated by recent results obtained in the authors' group.

The combined flow injection–capillary electrophoresis (FI–CE) system, described previously in this series, was used in connection with a drug dissolution testing system to automatically monitor the dissolution process of multicomponents. Samples from the dissolution medium were withdrawn at fixed intervals through an on-line membrane filter to load the sample loop of an injection valve, from which 25 μl samples were injected into a carrier buffer and transported into a split–flow interface coupling the FI and CE systems. Trimethoprim (TMP) and sulphamethoxazole (SMZ), the two active components in a sulphatrim tablet formulation dissolved in 0.1 mol l−1 HCl, were introduced into a short silica separation capillary of 14.5 cm effective length by electrokinetic means, separated at a constant voltage of 1.0 kV by capillary zone electrophoresis, using a phosphate running buffer (pH 6.5), and the separated constituents recorded continuously, using an UV detector at 224 nm. The signals were recorded within a testing period of 65 min. By partially overlapping the separation zones of neighboring samples, a high sample throughput of 60 h−1 was achieved with single-vessel dissolution and 48 (24 duplicates) h−1 with dual-vessel dissolution. The reproducibility of the FI–CE system, obtained using TMP and SMZ standards within the testing period was 1.6 and 0.8% relative standard deviation (RSD) with peak height evaluation, and 2.0 and 1.1% RSD (n=72), respectively, for peak area. Good agreement of results was obtained between those using the reported method and a standard liquid chromatography method.

Recent progress in flow injection on-line separation and preconcentration techniques for electrothermal atomic absorption spectrometry (ETAAS) are reviewed, stressing the advancements made within the past 2 or 3 years. Important trends and potentials for future development are discussed, including the use of air-transport and air-segmentation in on-line separation systems, the use of knotted reactors as a sorption medium, and other designs for on-line coprecipitation and solvent extraction systems to improve the robustness and efficiency of on-line separation systems for ETAAS.

A dual stage preconcentration system based on flow injection on-line ion-exchange and solvent-extraction has been developed for flame atomic absorption spectrometry. Lead is taken as a model trace element. A column packed with Amberlite IRC-718 cation exchanger is incorporated into the FI manifold. The analyte is retained on the column by time-based sample loading and eluted by 1 mol/L HNO3. The eluate is subsequently merged with potassium iodide and tetrabutylammonium bromide (TBABr), and isobutyl methyl ketone (IBMK). Lead is extracted on-line into IBMK as the ion-pair formed between the iodoplumbate anion and tetrabutylammonium cation. The organic phase is separated from the aqueous phase by a gravity phase separator. 50 μL of concentrate is introduced into the nebulizer-burner system of the spectrometer. An enhancement factor of 550 is achieved with a 30 mL sample consumption at a sampling frequency of 30/h. The precision (relative standard deviation) is 2.4% at 10 μg/L level and the detection limit is 0.3 μg/L (3 σ). The method was successfully applied to the determination of lead in water samples.

An automated system for drug-dissolution studies based on the sequential-injection (SI) technique is described and evaluated for monitoring of dissolution profiles of ibuprofen tablets, sustained-release capsules and controlled-release matrix tablets. The system was composed of a computer-controlled syringe pump and a multi-position selection valve and coupled to a spectrophotometric detector. An 800 μl test solution in dissolution vessel was aspirated sequentially into a PTFE holding coil via on-line filters. After delivering an aliquot (40 μl) of each aspirated solution into the transferring line connected to the detector, the residual dissolution solution in the holding coil was fed back to the dissolution vessel to clear the filter and decrease sample consumption. The sampled aliquot was carried to the detector and monitored at 222 nm. Every hour, seven samples run in hexa-replicates were processed with a total of 42 measurements. The system was continuously run for 12 h with an overall relative standard deviation of 2.18% (n=72) at 50% final concentration level of the dissolution solution, which demonstrates the stability and robustness of the automated SI system.

A combined flow injection (FI)–capillary electrophoresis (CE) system with a split-flow interface reported previously was used for on-line preconcentration of pseudoephedrine by solid phase extraction on a micro-column packed with C18. Aqueous samples were loaded on the column at 1.0 ml min−1 for 90 s and eluted by a mixed eluent of 40% 62.5 mM acetate buffer (pH 4.0) and 60% acetonitrile at 0.2 ml min−1. A zone-sampling approach was employed to sample 45 μl of the most concentrated zone of the eluate, which was injected into the FI–CE split-flow interface using a phosphate buffer carrier, with a 25 μl water zone preceding the sampled eluate zone to achieve enhanced field amplification effects. Sensitivity enhancements of 60 fold (180 fold with 4 min sample loading) were thus achieved with a sample throughput of 9 h−1 and 4.7% relative standard deviations (n = 10, 2 μg ml−1 pseudoephedrine) for peak area evaluation, without significant degradation of column efficiency.

The combined flow injection (FI)-capillary electrophoresis (FI-CE) system described in Part I of this series was developed for achieving chiral separation of intermediate racemates in the synthesis of chloramphenicol. Hydroxypropyl-β-cyclodextrin, β-cyclodextrin and heptakis-(2,2-di-o-methyl)-β-cyclodextrin were employed as chiral selectors in the separation buffers. Baseline resolution of enantiomers of the rac-threo bases 2-amino-1-(p-nitrophenyl-1,3-propanediol) was achieved using the combined system demonstrating significantly improved precision and sample throughput compared to conventional CE with manual electrokinetic sample introduction, while achieving similar resolution and column efficiencies. A series of samples (the rac-threo bases) was injected continuously without current interruption, achieving sample throughputs at least a factor of five higher than conventional CE sample introduction.

A combined flow injection (FI)-capillary electrophoresis (CE) system was developed using conventional FI and CE equipment. A flow-through reservoir equipped with a conical inlet was used as an interface to connect the FI system to the capillary inlet. A grounded electrode was inserted in the reservoir. Split-sampling was achieved in the electrokinetic mode when an injected sample zone in the microliter range was transported into the conical inlet by a carrier solution which also served as the separation buffer. Using benzoic acid as a model the combined system demonstrated significantly improved precision in peak height, peak area, and migration time as compared to conventional manual electrokinetic sample introduction while achieving similar sensitivity and column efficiency. Using a test sample containing magnolol and benzoic acid, a series of samples was injected continuously without current interruption, achieving sample throughputs at least three times higher than conventional CE sample introduction.

An automated nanoliter sample introduction system was combined to a liquid-core waveguide (LCW)-based microfluidic CE system for high-throughput analysis of DNA fragments. The main component of the sample introduction system was a motor-driven plate, on which a circular array of bottom-slotted vials containing sample/buffer solutions was placed. A 7 cm-long LCW capillary served as both the sample probe and separation channel. The inlet terminal of the capillary could pass through the slots of the vials for electrokinetic sample introduction, and the capillary outlet was immersed in the solution of a reservoir, behind which a PMT facing directly to the outlet was positioned. A diode laser was used as excitation source for LCW LIF detection. Performance of the system was demonstrated through the separation of DNA fragments. Baseline separation was achieved for all 11 fragments of PhiX174-HaeIII digest DNA with a throughput of 33/h. Theoretical plate number for 603 bp fragment was 7.3x10(6)/m, corresponding to a plate height 0.14 microm. The detection limitation for 603 bp fragment was 0.4 ng/microL with a precision of 2.2% RSD for the peak height. Automated sample changing and introduction were achieved with only 0.3 nL gross sample consumption for each cycle.

Ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF6), as a green solvent, was successfully used for the direct extraction of dsDNA. The extraction efficiency and the distribution coefficient values indicated that trace amounts of DNA at the levels of5 ng microL-1 facilitate quantitative fast extraction, while proteins and metal species do not interfere. A total of 30% of the DNA in ionic liquid at approximately 20 ng microL-1 was back extracted into aqueous phase in phosphate-citrate buffer with a single-stage extraction. The extraction is demonstrated to be endothermic with an enthalpy of 34.3 kJ moL-1. The extraction mechanisms were proposed and verified by 31P NMR and FT-IR spectra. Interactions between cationic 1-butyl-3-methylimidazolium (Bmim+) and P-O bonds of phosphate groups in the DNA strands take place both in the dissolved BmimPF6 in aqueous phase and at the interface of the two phases. This interaction consequently led to the transformation of DNA conformations, along with a reduction of ethidium resonance light scattering at 510 nm, and a procedure for DNA quantification in ionic liquid was developed based on this observation.

A microfluidic chip-based sequential injection system with trapped droplet liquid-liquid extraction preconcentration and chemiluminescence detection was developed for achieving high sensitivity with low reagent and sample consumption. The microfabricated glass lab-chip had a 35 mm long extraction channel, with 134 shrunken opening rectangular recesses (L 100 microm x W 50 microm x D 25 microm) arrayed within a 1 mm length on both sides of the middle section of the channel. Ketonic peroxyoxalate ester solution was filled in the recesses forming organic droplets, and keeping the aqueous sample solution flowing continuously in the extraction channel; analytes were transferred from the aqueous phase into the droplets through molecular diffusion. After liquid-liquid extraction preconcentration, catalyst and hydrogen peroxide solutions were introduced into the channel, and mixed with analytes and peroxyoxalate ester to emit chemiluminescence light. The performance of the system was tested using butyl rhodamine B, yielding a precision of 4% RSD (n = 5) and a detection limit of 10(-9) M. Within a 17 min analytical cycle, the consumptions of sample and peroxyoxalate solutions were 2.7 microL and 160 nL, respectively.

An automated microfluidic sequential injection analysis system that efficiently manipulates sample and reagent solutions in the nanoliter range in approximately 10 s per analytical cycle is described. The system consisted of a 6-cm-long, typically 75-microm i.d., fused-silica capillary (which functioned as a sampling probe and reactor as well as a flow-through detection cell), a horizontally oriented waste reservoir that provided liquid level differences for inducing gravity-driven flows, an autosampling device holding samples and reagents with horizontally fixed slotted microvials, and a laser-induced fluorescence detection system. Sample and reagent zones were sequentially introduced via gravity-driven flow by scanning the capillary tip (functioning as the sampling probe) through the vial slots, while vials containing sample, reagent, and carrier were sequentially rotated to the probe by programmed movement of the vial holders. Sequentially injected nanoliter zones were rapidly mixed by convection and diffusion within the carrier flow, demonstrating a behavior that conformed well to the Taylor dispersion model, and zone penetration effects were characterized and optimized under Taylor's dispersion theory guidelines. For the determination of fluorescein, a high throughput of 400 h(-1) was achieved, rapidly producing calibration curves (five points) within 45 s. Owing to its adaptability to the Taylor's dispersion model, the system was used also for measuring diffusion coefficients of fluorescent species. Potentials for using the system in enzyme inhibition assays were demonstrated by a reaction involving the conversion of fluorescein digalactoside to fluorescent hydrolysates via beta-galactosidase and the inhibition of beta-galactosidase by diethylenetriaminepentaacetic acid.

In this work, a simple LIF detection system based on an orthogonal optical arrangement for microfluidic chips was developed. Highly sensitive detection was achieved by detecting the fluorescence light emitted in the microchannel through the sidewall of the chip to reduce scattered light interference from the laser source. A special crossed-channel configuration, with a 1.5-mm distance from the separation channel to the sidewall of the glass chip, was designed in order to facilitate collection of emitted fluorescence light through the sidewall. The significant difference in intensity distribution of scattered laser light on the chip plane observed in this study was fully exploited to optimize S/N ratio of detected signals by rejection of scattered light, both through systematic measurements and employing ray-tracing simulation. A fluorescence collection angle of 45 degrees in the chip plane gave the best result, with a scattered light intensity 1/38 of that obtained at an angle of 90 degrees. Sodium fluorescein and fluorescein isothiocyanate-labeled amino acids were used as model samples to demonstrate the performance of the LIF system. A detection limit (S/N = 3) of 1.1 pM fluorescein was obtained, which is comparable to that of optimized confocal LIF systems for chip-based capillary electrophoresis. Apart from the high detection power, the system also has the advantages of simple optical structure, compactness, and ease in building.

A simple method for injecting well-defined non-biased sample plugs into the separation channel of a microfluidic chip-based capillary electrophoresis system was developed by a combination of flows generated by negative pressure, electrokinetic and hydrostatic forces. This was achieved by using only a single syringe pump and a single voltage supply at constant voltage. In the loading step, a partial vacuum in the headspace of a sealed sample waste reservoir was produced using a syringe pump equipped with a 3-way valve. Almost instantaneously, sample was drawn from the sample reservoir across the injection intersection to the sample waste reservoir by negative pressure. Simultaneously, buffer flow from the remaining two buffer reservoirs pinched the sample flow to form a well-defined sample plug at the channel intersection. In the subsequent separation stage, the vacuum in headspace of the sample waste reservoir was released to terminate all flows generated by negative pressure, and the sample plug at the channel intersection was electrokinetically injected into the separation channel under the potential applied along the separation channel. The liquid levels of the four reservoirs were optimized to prevent sample leakage during the separation stage. The approach considerably simplified the operations and equipment for pinched injection in chip-based CE, and improved the throughput. Migration time precisions of 3.3 and 1.5% RSD for rhodamine123 (Rh123) and fluorescein sodium (Flu) in the separation of a mixture of Flu and Rh123 were obtained for 56 consecutive determinations with peak height precisions of 6.2% and 4.4% RSD for Rh123 and Flu, respectively.

A miniaturized CE system has been developed for fast DNA separations with sensitive fluorimetric detection using a rectangle type light-emitting diode (LED). High sensitivity was achieved by combining liquid-core waveguide (LCW) and lock-in amplification techniques. A Teflon AF-coated silica capillary on a compact 6x3 cm baseplate served as both the separation channel for CE separation and as an LCW for light transmission of fluorescence emission to the detector. An electronically modulated LED illuminated transversely through a 0.2 mm aperture, the detection point on the LCW capillary without focusing, and fluorescence light was transmitted to the capillary outlet. To simplify the optics and enhance collection of light from the capillary outlet, an outlet reservoir was designed, with a light transmission window, positioned directly in front of a photomultiplier tube (PMT), separated only by a high pass filter. Automated sample introduction was achieved using a sequential injection system through a split-flow interface that allowed effective release of gas bubbles. In the separation of a phiX174 HaeIII DNA digest sample, using ethidium bromide as labeling dye, all 11 fragments of the sample were effectively resolved in 400 s, with an S/N ratio comparable to that of a CE system with more sophisticated LIF.

Octadecyl immobilized surface was, for the first time, proved to be a superb precipitate-collecting medium. Surface charge effect was assumed to dominate the adsorption of cadmium hydroxide precipitate, facilitated by electrostatic interaction between the negatively charged C18 bead surface and positively charged cadmium hydroxide clusters. Residual silanol groups on the C18-immobilized silica surface did not contribute to precipitate adsorption. A novel procedure for ultratrace cadmium preconcentration was proposed by incorporating a renewable microcolumn in a lab-on-valve system. Cd(OH)(2) precipitate was adsorbed onto the C18 surface, which was afterward eluted with 20 microL of nitric acid (1%) and quantified with detection by electrothermal atomic absorption spectrometry. An enrichment factor of 28 and a limit of detection of 1.7 ng L(-1), along with a sampling frequency of 13 h(-1) were obtained with a sample consumption of 600 microL within the concentration range of 0.01-0.2 microg L(-1), achieving a precision of 2.1% RSD at the 0.05 microg L(-1) level. The enrichment factor was further enhanced to 44 by increasing the sample volume to 1200 microL. The procedure was validated by analyzing cadmium in three certified reference materials, that is, river sediment (CRM 320), sea lettuce (CRM 279), and frozen cattle blood (GBW 09140). Good agreement between the obtained results and the certified values was achieved.

In this work, a simple, robust, and automated microfluidic chip-based FIA system with gravity-driven flows and liquid-core waveguide (LCW) spectrometric detection was developed. The high-throughput sample introduction system was composed of a capillary sampling probe and an array of horizontally positioned microsample vials with a slot fabricated on the bottom of each vial. FI sample loading and injection were performed by linearly moving the array of vials filled alternately with 50-microL samples and carrier, allowing the probe inlet to enter the solutions in the vials through the slots sequentially and the sample and carrier solution to be introduced into the chip driven by gravity. The performance of the system was demonstrated using the complexation of o-phenanthroline with Fe(II) as a model reaction. A 20-mm-long Teflon AF 2400 capillary (50-microm i.d., 375-microm o.d.) was connected to the chip to function as a LCW detection flow cell with a cell volume of 40 nL and effective path length of 1.7 cm. Linear absorbance response was obtained in the range of 1.0-100 microM Fe(II) (r2=0.9967), and a good reproducibility of 0.6% RSD (n=18) was achieved. The sensitivity was comparable with that obtained using conventional FIA systems, which typically consume 10,000-fold more sample. The highest sampling throughput of 1000 h-1 was obtained by using injection times of 0.08 and 3.4 s for sample and carrier solution, respectively, with a sample consumption of only 0.6 nL for each cycle.

Microfluidic devices have been fabricated on polycarbonate (PC) substrates by use of a hot embossing method using a silicon master template. By adding auxiliary lines around the functional channel on the silicon master, its lifetime was significantly prolonged and the bonding strength of the PC cover plate to the microfluidic chip was greatly improved. More than 300 polycarbonate microfluidic chips have been replicated with the same silicon mold. CE separation of Phi X-174/HaeIII DNA restriction fragments, with high resolution efficiency and good reproducibility, was achieved on these devices using the low-viscosity sieving matrix HPMC-50. Temperature was found to have a significant effect on separation efficiency.

A simple, room-temperature bonding process was developed for the fabrication of glass microfluidic chips. High-quality bonding with high yields (95%) was achieved without the requirement of clean room facilities, programmed high-temperature furnaces, pressurized water sources, adhesives, or pressurizing weights. The plates to be bonded were sequentially prewashed with acetone, detergent, high-flow-rate (10-20 m/s) tap water, and absolute ethyl alcohol and were soaked in concentrated sulfuric acid for 8-12 h. The plates were again washed in high-flow-rate tap water for 5 min and, finally, with demineralized water. The plates were bonded by bringing the cleaned surfaces into close contact under a continuous flow of demineralized water and air-dried at room temperature for more than 3 h. This bonding process features simple operation, good smoothness of the plate surface, and high bonding yield. The procedures can be readily applied in any routine laboratory. The bonding strength of glass chips thus produced, measured using a shear force testing procedure, was higher than 6 kg/cm(2). The mechanism for the strong bonding strength is presumably related to the formation of a hydrolyzed layer on the plate surfaces after soaking the substrates in acid or water for extended periods. Microfluidic chips bonded by the above procedure were tested in the CE separation of fluorescein isothiocyanate-labeled amino acids.

The development of efficient sample introduction and pretreatment systems for microfluidic chip-based analytical systems is important for their application to real-life samples. In this work, world-to-chip interfacing was achieved by a novel flow-through sampling reservoir featuring a guided overflow design. The flow-through reservoir was fabricated on a 30 x 60 x 3 mm planar glass chip of crossed-channel design used for capillary electrophoresis separations. The 20-microL sample reservoir was produced from a section of plastic pipet tip and fixed at one end of the sampling channel. Sample change was performed by pumping 80-microL samples sandwiched between air segments at approximately 0.48 mL/min flow rate through the flow-through reservoir, introduced from an access hole on the bottom side of the chip. A filter paper collar wrapped tightly around the reservoir guided the overflowing sample solution into a plastic trough surrounding the reservoir and then to waste. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine, glycine, phenylalanine, and glutamic acid with LIF detection, by continuously introducing a train of different samples through the system without electrical interruption. Employing a separation channel of 4 cm (2-cm effective separation length) and 1.4-kV separation voltage, maximum throughputs of 80/h were achieved with4.1% carryover and precisions ranging from 1.5% for arginine to 2.6% RSD (n = 11) for glycine. The sampling system was tested in the continuous monitoring of the derivatizing process of amino acids by FITC over a period of 4 h, involving 166 analytical cycles. An outstanding overall precision of 4.8% RSD (n = 166) was achieved for the fluorescein internal standard.

A microfluidic chip for the determination of potassium in blood was developed, employing a multiphase laminar flow approach for separation of potassium ions from the blood matrix by diffusion into a buffer acceptor stream. Both sample and acceptor streams were driven by gravity, and the potassium was determined using an ion-selective membrane electrode integrated on the chip.

A novel miniaturized capillary electrophoresis (CE) system is described where a Teflon AF-coated silica capillary serves both as the separation channel and as a transversely illuminated liquid core waveguide. This device uniquely uses flow injection (FI)-based split-flow sample introduction through a falling-drop interface. An H-channel structure fixed on a microscope glass slide utilizes a horizontal separation capillary with tubular sidearms on each end that serve as inlet and outlet flow-through electrode reservoirs. The inlet reservoir also functions as a falling-drop interface for coupling to the FI system. A blue LED is used as excitation source. A large-core optical fiber takes the emitted fluorescence to an inexpensive PMT with two layers of green plastic used for optical filtering. No focusing arrangement is needed. Continuous FI introduction of a series of 30-microL samples containing a mixture of of fluorescein isothiocyanate (FITC)-labeled amino acids allowed a throughput rate up to 144 samples/ h, with approximately 2% carryover and good precision (3.2% RSD). Baseline separation was achieved for FITC-labeled arginine, phenylalanine, glycine, and FITC in sodium tetraborate buffer (pH 9.5) with plate heights of 5.4-5.5 microm and plate numbers of 2.34 x 10(4)-2.37 x 10(4) under electrical field strengths of 214 V/cm for injection and 500 V/cm for separation (14-cm capillary, 48-microm i.d.). Detection limits (S/N = 3) were 1.3 microM for arginine and 1.9 microM for phenylalanine and glycine.

A capillary electrophoresis (CE) system with chemiluminescence (CL) detection was combined with flow injection (FI) sample introduction on a chip platform. A falling-drop interface was applied to perform FI split-flow sample introduction while achieving electrical isolation from the CE high voltage. A tubular reservoir at the capillary outlet served as both the CL reaction and detection cell for the luminol–peroxide–metallic ion chemiluminescent reaction, with the luminol included in the separation buffer and CL reagent H2O2 continuously introduced into the outlet reservoir. An optical fiber was positioned within the outlet reservoir directly opposite, and 300 µm away from, the capillary outlet for collecting and transferring the generated CL to the PMT. The peak height signals and the separation efficiency were almost independent of the reagent flow-rate, making the system a robust one. The performance of the system was illustrated by the separation of Co(II) and Cu(II), achieving baseline separation in 60 s. Detection limits (3σ) were 1.25 × 10−8 and 2.3 × 10−6 mol dm−3 for Co(II) and Cu(II), respectively. Peak height precision was 1.9% RSD (n=9) at the 10−7 mol dm−3 Co level.

A sequential injection system for the determination of mercury by vapor generation atomic absorption spectrometry (VGAAS) using tetrahydoborate reductant was developed, characterized by prevention of sample and reagent mixing in the holding coil using small air segments and initiation of the vapor generation in a flow-through gas-liquid separator. Extremely small volumes of reductant of 15-30 mul (0.2-1.0% NaBH(4)) and sample acidity as low as 0.05 mol l(-1) HCl were sufficient for achieving performance similar to flow injection (FI) VGAAS systems. A sample throughput of 90 h(-1) was achieved with 400 mul samples with a precision of 2.0% RSD at 10 mug l(-1)Hg, and a detection limit of 0.1 mug l(-1) (3sigma). Reagent consumption was reduced by a factor of 25 in comparison to the FI-VGAAS system. Good agreement with the certified value was obtained for the determination of mercury in seawater in a standard reference sample.

A simple and robust flow injection on-line microdialysis system for multivessel drug dissolution testing is described. Microdialysis probes were used for sampling from the dissolution media. A stopped-flow dialysis mode with a 50-s stopped-flow period followed by 10-s injection at a perfusion rate of 2.8 ml min −1 for each probe was used to achieve high resolution of dissolution events using relatively simple equipment and operation. The precisions obtained for simultaneous monitoring of dissolution profiles for six tablets were all better than 0.9% (RSD n =80) and the overall sampling frequency of the system was 360 and 60 h −1 for each test vessel. The dissolution profiles of isoniazid fast-release tablet from three sources were determined to demonstrate the performance of the system.

A novel flow injection on-line microdialysis system for in vivo monitoring of glucose in subcutaneous tissue fluid and blood is described. An implantable loop-type microdialysis probe was used for subcutaneous sampling, and a flow-through microdialyzer was used for intravenous sampling by pumping of the blood from the tested rabbit through the microdialyzer located outside the living system at a flow rate of 10 microL/min. The perfusion rate of the dialysate was 20 microL/min. The glucose in the dialysate was detected on-line with a flow injection chemiluminescence system after passing through an immobilized glucose oxidase reactor. The calibration of the detector system (including reactor) and monitoring of baseline drifts were performed simultaneously to improve the reliability of the monitoring process. The dialysate sample volume was 20 microL, and the sample throughout was 28 h-1. The variation of glucose level in subcutaneous tissue fluid and blood of the rabbits was monitored after the administration of glucose or insulin to demonstrate the favorable resolution and reliability of the system for in vivo on-line monitoring.