University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Analytical Chemistry

  • Publisher: American Chemical Society
  • FAD Code: ANCH
  • CODEN: ANCHAM
  • ISSN: 0003-2700
  • Abbreviation: Anal. Chem.
  • DOI Prefix: 10.1021/ac
  • Language: English
  • Comments: Fulltext from 1929 V1

Citations 973

"Application Of Raman Spectroscopy And Sequential Injection Analysis For PH Measurements With Water Dispersion Of Polyaniline Nanoparticles"
Anal. Chem. 2007 Volume 79, Issue 2 Pages 608-611
Tom Lindfors and Ari Ivaska

Abstract: A novel method for pH measurements between pH 7.5 and 10.4 is reported in this paper. The method combines Raman spectroscopy and the automated sequential injection analysis system (SIA) and makes use of the acid-base properties of a commercially available water dispersion of polyaniline (PANI) nanoparticles with a mean particle size of 46 nm. The useful pH range of the PANI nanoparticles is broader than for conventional acid-base indicators, due to the 1:2 reaction stoichiometry of the proton-ligand complex of the nanoparticles. The pH measurements were conducted with the 633-nm laser excitation wavelength by calculating the difference between the Raman intensities of the primary and reference wavenumbers. In this study, the pH-sensitive CH=CH stretching band at 1422 cm-1 and C-H in-plane bending band of the quinoid form at 1163 cm-1 were chosen as the primary wavenumbers. The presented method is fast and allows pH to be measured with a precision of 0.2 pH unit. The advantage of the proposed method is that the hysteresis effect, which is usually observed for PANI films, can be overcome with the PANI nanoparticles, because a fresh nanoparticle solution is used in each measurement It should be pointed out, that this work is a fundamental study showing the applicability of Raman spectroscopy in combination with the SIA technique for pH measurements in specific applications, where the conventional glass pH electrode cannot be used. © 2007 American Chemical Society.

"Piezoelectric Quartz Crystal Microbalance Sensor For Trace Aqueous Cyanide Ion Determination"
Anal. Chem. 2007 Volume 79, Issue 1 Pages 251-255
Yegor G. Timofeyenko, Jeffrey J. Rosentreter, and Susan Mayo

Abstract: Using selective reaction chemistry, our present research has developed an online, real-time sensor capable of monitoring toxic cyanide at both drinking water standard and environmental regulatory concentrations. Through the use of a flow cell, aqueous samples containing cyanide are reacted with a gold electrode of a piezoelectric crystal to indirectly sense cyanide concentration by the dissolution of metallic gold. The quartz crystal is an AT-cut wafer sandwiched between two neoprene O-rings within the liquid flow cell. The presence of cyanide in solution results in the selective formation of a soluble dicyano-gold complex according to the Elsner reaction: 4Au + 8CN- + 2H 2O + O2 ? 4Au(CN)2- + 4OH-. The resulting loss of gold from the electrode is detected by the piezoelectric crystal as a resonant frequency change. Since free cyanide is a weak acid (pKa = 9.3), available protons compete for cyanide ligands. Therefore, increased sample pH provides higher sensitivity. The detection limits at pH 12 are 16.1 and 2.7 ppb for analysis times of 10 min and 1 h, respectively. The incorporation of the flow cell improves both analyte sensitivity and instrument precision, with an average signal intensity drift of only 5% over a 2-h analysis. The calibrations show excellent linearity over a variety of cyanide concentrations ranging from low ppb to hundreds of ppm. This detection method offers the advantage of selectively detecting cyanides posing a biohazard while avoiding detection of stable metal cyanides. This aspect of the system is based on competitive exchange of available metals and gold with cyanide ligands. Stable metal cyanide complexes possess a higher formation constant than cyanoaurate. This detection system has been configured into a flow injection analysis array for simple adaptation to automation. Anions commonly found in natural waters have been examined for interference effects. Additionally, the sensor is free from interference by aqueous cyanide analogues including thiocyanate. The developed detection system provides rapid cyanide determinations with little sample preparation or instrument supervision. © 2007 American Chemical Society.

"Application Of Ganglioside-sensitized Liposomes In A Flow Injection Immunoanalytical System For The Determination Of Cholera Toxin"
Anal. Chem. 2007 Volume 79, Issue 1 Pages 246-250
Ja-an Annie Ho, Li-Chen Wu, Ming-Ray Huang, Yong-Jen Lin, Antje J. Baeumner, and Richard A. Durst

Abstract: Cholera, an acute infectious disease associated with water and seafood contamination, is caused by the bacterium Vibrio cholerae, which lives and colonizes in the small intestine and secretes cholera toxin (CT), a causative agent for diarrhea in humans. Based on earlier lateral flow assays, a flow injection liposome immunoanalysis (FILIA) system with excellent sensitivity was developed in this study for the determination of CT at zeptomole levels. Ganglioside (GM1), found to have specific affinity toward CT, was inserted into the phospholipid bilayer during the liposome synthesis. These GM1-sensitized, sulforhodamine B (SRB) dye-entrapping liposomes were used as probes in the FILIA system. Anti-CT antibodies were immobilized in its microcapillary. CT was detected by the formation of a sandwich complex between the immobilized antibody and GM1 liposomes. During the assay, the sample was introduced first into the column, and then liposomes were injected to bind to all CT captured by the antibody in the microcapillary. Subsequently, the SRB dye molecules were released from the bound liposomes via the addition of the detergent octyl glucopyranoside. The released dye molecules were transported to a flow-through fluorescence detector for quantification. The FILIA system was optimized with respect to flow rate, antibody concentration, liposome concentration, and injected sample volume. The calibration curve for CT had a linear range of 10^-16 to 10^-14 g mL-1. The detection limit of this immunosensor was 6.6 x 10^-17 g mL-1 in 200-?L samples (equivalent to 13 ag or 1.1 zmol).

"Hyphenating Multisyringe Flow Injection Lab-on-valve Analysis With Atomic Fluorescence Spectrometry For On-line Bead Injection Preconcentration And Determination Of Trace Levels Of Hydride-forming Elements In Environmental Samples"
Anal. Chem. 2006 Volume 78, Issue 24 Pages 8290-8298
Xiangbao Long, Manuel Miró, Elo Harald Hansen, José Manuel Estela, and Víctor Cerdà

Abstract: In this work the third generation of flow injection analysis, that is, the so-called micro-lab-on-valve (?LOV) approach, is proposed for the first time for the separation, pre-concentration, and monitoring of metalloids as hyphenated with atomic fluorescence spectrometry (AFS). This was made feasible by interfacing the micromachined LOV-module with AFS by a multisyringe flowing stream network for on-line post-column derivatization of the eluate aimed at generation of hydride species. The potential of this new hyphenated technique for environmental assays was ascertained via determination of ultratrace level concentrations of total inorganic arsenic in freshwater. Employing quantitative preoxidation of As(III) to As(V) in the samples by means of permanganate, the method involves pre-concentration of arsenate at pH 10 on a renewable anion exchanger, namely, Q-Sepharose, packed in a LOV microcolumn. The analyte species is afterward stripped out and concurrently prereduced by a 300 µL eluent plug containing 6 mol L-1 HCl and 10% KI. The eluate is downstream merged with a metered volume of sodium tetrahydroborate (0.3% w/v) for generation of arsine, which is subsequently quantified by AFS. The flow system facilitates on-column reduction of the retained arsenic with no need for application of programmable stopped flow. Yet, the high concentration of reductant and extreme pH conditions for elution hinder the sorbent to be reused due to gradual deactivation of the functional moieties, so that maximum benefit can be taken from the application of the bead renewable strategy. The proposed procedure is characterized by a high tolerance to metal species and interfering hydride-forming elements. In fact, ratios of Se(IV) to As ? 5000 and Sb(V) to As ? 500 are tolerated at the 10% interference level. Under the optimized experimental conditions, a detection limit (3?) of 0.02 ng mL-1 As, a dynamic linear range of 0.05-2.0 ng mL-1 As (by tailoring the AFS gain), an enrichment factor of 8.8 for arsenate, and a precision better than 6.0% at the 0.1 ng mL-1 level were obtained for the bead-injection mode whenever the loading sample volume was affixed at 3.0 mL. The reliability and accuracy of the automated procedure was ascertained by determining total inorganic arsenic in both spiked environmental waters and certified reference materials of variable matrix complexity (TMDA-54.3 and ERM-CAO10) at the low ng mL-1 level. No significant differences were found between the experimental results and the certified values at a significance level of 0.05. © 2006 American Chemical Society.

"Chemiluminescent Method For Detection Of Eutrophication Sources By Estimation Of Organic Amino Nitrogen And Ammonium In Water"
Anal. Chem. 2006 Volume 78, Issue 21 Pages 7504-7510
S. Meseguer-Lloret, C. Molins-Legua, J. Verdú-Andrés, and P. Campíns-Falcó

Abstract: An automatic method has been developed for the estimation of organic amino nitrogen (CH2-NH) and ammonium in water samples. We propose a continuous flow system in which nitrogen compounds react with hypochlorite reagent to produce chloramines. Subsequently, the mixture is mixed with luminol, generating a chemiluminescence signal. The signal emission at 425 nm, registered as a function of time, decreases as nitrogen concentration increases, due to the decrease on hypochlorite concentration. A large number of nitrogen compounds have been assayed and their sensitivities compared, in milligrams per liter nitrogen. The ammonium calibration graph, expressed as N, can be used for most of the assayed compounds. The linear interval was 0.24-4 mg L-1 N, with the detection limit 0.07 mg L-1 N. The chemiluminescence method was applied to the analysis of several kinds of real water samples, natural, lake, irrigation ditch, fountain, residual, and seawater in order to detect possible sources of eutrophication. The accuracy (% relative error) and precision were satisfactory, with mean values of 5 ± 4 and 3 ± 2, respectively. This procedure has been used to estimate nitrogen content in samples before and after Kjeldahl treatment In the same samples, the N found for the untreated samples provided a good estimation of the N Kjeldahl. Sixty samples per hour can be analyzed, and the procedure can also be used for in situ monitoring. © 2006 American Chemical Society.

"1D And 2D Temperature Imaging With A Fluorescent Ruthenium Complex"
Anal. Chem. 2006 Volume 78, Issue 21 Pages 7499-7503
Oscar Filevich and Roberto Etchenique

Abstract: Temperature imaging based on the fluorescence of the complex [Ru(bpy) 3]2+ is described. The method allows precise temperature measurement on unidimensional flow injection reactors and bidimensional measurement on dishes for biological and biochemical assays. The fluorescence dependence on temperature is linear, achieving a resolution of 0.05 K with a simple two-point calibration. The large Stokes shift of [Ru(bpy) 3]2+ makes it easy to use a simple CCD camera without special filters. Large or small area fields can be achieved by changing the optics of the camera. High spatial resolution is possible by using any fluorescence microscope. © 2006 American Chemical Society.

"A Gas-phase Chemiluminescence-based Analyzer For Waterborne Arsenic"
Anal. Chem. 2006 Volume 78, Issue 20 Pages 7088-7097
Ademola D. Idowu, Purnendu K. Dasgupta, Zhang Genfa, Kei Toda, and John R. Garbarino

Abstract: We show a practical sequential injection/zone fluidics-based analyzer that measures waterborne arsenic. The approach is capable of differentiating between inorganic As(III) and As(V). The principle is based on generating AsH 3 from the sample in a confined chamber by borohydride reduction at controlled pH, sparging the chamber to drive the AsH3 to a small reflective cell located atop a photomultiplier tube, allowing it to react with ozone generated from ambient air, and measuring the intense chemiluminescence that results. Arsine generation and removal from solution results in isolation from the sample matrix, avoiding the pitfalls encountered in some solution-based analysis techniques. The differential determination of As(III) and As(V) is based on the different pH dependence of the reducibility of these species to AsH3. At pH ?1, both As(III) and As(V) are quantitatively converted to arsine in the presence of NaBH4. At a pH of 4-5, only As(III) is converted to arsine. In the present form, the limit of detection (S/N = 3) is 0.05 ?g/L As at pH ?1 and 0.09 ?g/L As(III) at pH ?4-5 for a 3 mL sample. The analyzer is intrinsically automated and requires 4 min per determination. It is also possible to determine As(III) first at pH 4.5 and then determine the remaining As in a sequential manner; this requires 6 min. There are no significant practical interferences. A new borohydride solution formulation permits month-long reagent stability. © 2006 American Chemical Society.

"Microfluidic Sequential Injection Analysis In A Short Capillary"
Anal. Chem. 2006 Volume 78, Issue 18 Pages 6404-6410
Wen-Bin Du, Qun Fang, and Zhao-Lun Fang

Abstract: An automated microfluidic sequential injection analysis system that efficiently manipulates sample and reagent solutions in the nanoliter range in ~10 s per analytical cycle is described. The system consisted of a 6 cm long, typically 75-µm 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 hr-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 β-galactosidase and the inhibition of β-galactosidase by diethylenetriaminepentaacetic acid.

"Electrochemical Detection Of Arsenic(III) Using Indium-implanted Boron-doped Diamond Electrodes"
Anal. Chem. 2006 Volume 78, Issue 18 Pages 6291-6298
Tribidasari A. Ivandini, Rika Sato, Yoshihiro Makide, Akira Fujishima, and Yasuaki Einaga

Abstract: Indium-modified, boron-doped diamond electrodes fabricated by an ion implantation method have been developed for electrochemical detection of arsenite (As(III)). Ir+ ions were implanted with an energy of 800 keV and a dose of 1015 ion cm-2. An annealing treatment at 850°C for 45 min in H2 plasma (80 Torr) was required to rearrange metastable diamond produced by an implantation process. Characterization was investigated by SEM, AFM, Raman, and X-ray photoelectron spectroscopy. Cyclic voltammetry and flow injection analysis with amperometric detection were used to study the electrochemical reaction. The electrodes exhibited high catalytic activity toward As(III) oxidation with the detection limit (S/N = 3), sensitivity, and linearity of 20 nM (1.5 ppb), 93 nA ?M -1 cm-2, and 0.999, respectively. The precision for 10 replicate determinations of 50 ?M As(III) was 4.56% relative standard deviation. The advantageous properties of the electrodes were its inherent stability with a very low background current. The electrode was applicable for analysis of spiked arsenic in tap water containing a significant amount of various ion elements. The results indicate that the metal-implanted method could be promising for controlling the electrochemical properties of diamond electrodes. © 2006 American Chemical Society.

"Lab-on-valve System Integrating A Chemiluminescent Entity And In Situ Generation Of Nascent Bromine As Oxidant For Chemiluminescent Determination Of Tetracycline"
Anal. Chem. 2006 Volume 78, Issue 16 Pages 5900-5905
Mei Yang, Ying Xu, and Jian-Hua Wang

Abstract: A novel configuration of a lab-on-valve (LOV) system was fabricated and applied for chemiluminescence (CL) detection by integrating a demountable Z-type flow cell onto the LOV unit. A bismuthate immobilized microcolumn was incorporated in one port of the LOV for in situ oxidation of KBr and generation of bromine as oxidant for the bromine-hydrogen peroxide-tetracycline (TC) chemiluminescent reaction. The nascent bromine reacts with hydrogen peroxide and produces a weak CL signal, the intensity of which was significantly enhanced in the presence of TC following an energy-transfer mechanism. A novel procedure for tetracycline quantification was therefore developed based on the present system. When compared with the reported flow injection-CL methods for TC, this procedure not only provided an improved detection limit of 2.0 ?g L -1 but also minimized sample and reagent consumption. A linear range of 6.0-10 000 ?g L-1 was derived along with RSD values of 5.9 (at the concentration level of quantification limit) and 2.2% (at 50 ?g L -1), and a sampling frequency of 120 h-1 was achieved. The system was validated with a National Standard Procedure (GB/T 18932.4-2002, HPLC with UV detection) by measuring TC contents in commercial milk samples. © 2006 American Chemical Society.

"Capillary Electrophoresis Absorption Detection Using Fiber-loop Ring-down Spectroscopy"
Anal. Chem. 2006 Volume 78, Issue 16 Pages 5685-5692
Runkai Li, Hans-Peter Loock, and Richard D. Oleschuk

Abstract: The application of phase-shift, fiber-loop, ring-down spectroscopy (PS-FLRDS) as an on-line detector for capillary electrophoresis (CE) of biomolecules is demonstrated. CE was conducted using a custom-designed capillary/fiber interface coupled to an absorption detector, which is based on the ring-down of an optical signal in a closed fiber waveguide loop. The ring-down times were obtained by measuring the phase difference between intensity modulated light entering and exiting the fiber loop. The incorporation of a microlens to enhance transmission through the sample gap led to an improvement of the sensitivity by up to 80% compared to the square-cut fiber and a reduction in the detection limit. The performance of the PS-FLRDS absorption technique as an online detector was characterized by flow injection through a capillary. Good repeatability and linear response were obtained, and the detection limit using the lensed fiber/capillary interface system was determined to be ?min = 1.6 cm-1 for an absorption path of ?30 ?m. PS-FLRDS coupled to CE was also applied to the analysis of human serum albumin (HSA) by using a NIR dye as a noncovalent label. The excess free dye and the dye/protein complex were resolved. The labeling coefficient was determined to be ?6, and good repeatability of peak areas (RSD = 8.7%) was obtained for the analysis of HSA. Furthermore, an excellent linear response (R2 > 0.99) was obtained between the peak areas and concentrations of HSA. The detection limit of labeled HSA was determined to be 1.67 ?M. © 2006 American Chemical Society.

"Continuous-flow PI-based Sorting Of Proteins And Peptides In A Microfluidic Chip Using Diffusion Potential"
Anal. Chem. 2006 Volume 78, Issue 11 Pages 3528-3536
Yong-Ak Song, Stephanie Hsu, Anna L. Stevens, and Jongyoon Han

Abstract: Efficient sample preparation tools are the key to measuring molecular signals in a complex biological system. A novel continuous-flow isoelectric point (pI)-based sorting technique has been developed for proteins and peptides in a microfluidic chip format. It can sort biomolecules at a relatively high flow rate of up to 10 µL/min and does not require carrier ampholytes, which can create molecular backgrounds for subsequent analysis. Furthermore, the electrophoretic field required to run the pI-based sorting is generated by the diffusion of buffer ions in situ, at the liquid junction between two laminar flows within the microfluidic channel. Utilizing the diffusion potential in combination with a pH difference between the buffers, we demonstrated a separation of binary mixtures of pI markers and proteins without applying any external field. The sorting resolution and its efficiency are sufficiently high for sample preparation and could be further improved by optimizing buffers or with an additional transverse electric field. Once fully developed, it can potentially be a pI-based sample fractionation tool for proteomic analysis of complex biomolecule samples. © 2006 American Chemical Society.

"Electrochemical Oxidation Of Oxalic Acid At Highly Boron-doped Diamond Electrodes"
Anal. Chem. 2006 Volume 78, Issue 10 Pages 3467-3471
Tribidasari A. Ivandini, Tata N. Rao, Akira Fujishima, and Yasuaki Einaga

Abstract: Electrochemical oxidation of oxalic acid has been investigated at bare, highly boron-doped diamond electrodes. Cyclic voltammetry and flow injection analysis with amperometric detection were used to study the electrochemical reaction. Hydrogen-terminated diamonds exhibited well-defined peaks of oxalic acid oxidation in a wide pH range. A good linear response was observed for a concentration range from 50 nM to 10 ?M, with an estimated detection limit of ?0.5 nM (S/N = 3). In contrast, oxygen-terminated diamonds showed no response for oxalic acid oxidation inside the potential window, indicating that surface termination contributed highly to the control of the oxidation reaction. An investigation with glassy carbon electrodes was conducted to confirm the surface termination effect on oxalic acid oxidation. Although a hydrogen-terminated glassy carbon electrode showed an enhancement of signal-to-background ratio in comparison with untreated glassy carbon, less stability of the current responses was observed than that at hydrogen-terminated diamond. © 2006 American Chemical Society.

"Universal Response In Liquid Chromatography Using Charged Aerosol Detection"
Anal. Chem. 2006 Volume 78, Issue 9 Pages 3186-3192
Tadeusz Górecki, Frederic Lynen, Roman Szucs, and Pat Sandra

Abstract: A new, empirical approach is introduced to correct for the varying response of aerosol-based detectors with the varying composition of the mobile phase during gradient elution in HPLC. A Corona charged aerosol detector was used in the experiments. The detector is characterized by a nearly universal response at a given, constant mobile-phase composition for sufficiently nonvolatile analytes. A second pump was used to deliver an exactly inverse gradient compared to the analytical HPLC system, and both flows were mixed in a tee piece before introduction to the Corona detector. The approach proposed made it possible to extend the universal response from isocratic to gradient elution conditions in HPLC, vastly improving the usefulness of this detection technique. The constant response of the detector obtained in this way was first demonstrated in flow injection analysis. Very similar calibration curves were obtained for six sulfonamide drugs after mobile-phase compensation. The approach was also applied to gradient elution with excellent results. The data were characterized by good precision ranging from 4% RSD at 10 mg/L to 1.6% RSD at 780 mg/L. The average limit of detection with a 2-?L injection was 0.5 mg/L, corresponding to 1 ng injected on the column. The approach proposed allows quantification of unknown compounds, e.g., in pharmaceutical mixtures. Measurement of analytes at a relative concentration of 0.05% versus the main component is demonstrated. © 2006 American Chemical Society.

"Optical PH Measurements With Water Dispersion Of Polyaniline Nanoparticles And Their Redox Sensitivity"
Anal. Chem. 2006 Volume 78, Issue 9 Pages 3019-3026
Tom Lindfors, Leo Harju, and Ari Ivaska

Abstract: A new method for optical pH and redox measurements with a commercially available water dispersion of polyaniline (PANI) nanoparticles (mean particle size, 46 nm) is presented. The pH measurements are based on the acid-base equilibrium of PANI and were carried out either by combining both the automated sequential injection analysis (SIA) and UV-visible spectrophotometric techniques or with a fiber-optic light guide. In the former case, the detection was done in continuous mode at ? = 800 nm by using the SIA technique for transporting the sample to a flow-through cell, which was placed in the light path of the photometer. With the fiber-optic light guide, the detection was done in batch mode ? = 400 and 580 nm. In both methods, fresh pH reagent (PANI) solution was used in each measurement, thus overcoming the problem with hysteresis (memory effect), which is usually observed with PANI films. The PANI nanoparticles were characterized with UV-visible spectroscopy in pH buffer solutions between pH 2-12 and a protonation constant of logK H0.5L0.5H,L = 4.4 was calculated from these data. Fast pH measurements can be done between pH 6 and 10.5 depending on the measuring technique. It is possible to determine pH with an accuracy of 0.1 pH unit between pH 8 and 10.5 (RSD, 0.5-2%). Redox transitions typical for PANI films were also observed for water solutions of PANI nanoparticles in the presence of the hexacyanoferrate(II/III) and the iron(II/III) oxalate redox couples. The absorbance at ? = 875 nm is linearly dependent on the logarithm of the concentration ratio (0.1-10) of the iron oxalate redox couple. © 2006 American Chemical Society.

"Continuous Flow Analytical Microsystems Based On Low-temperature Co-fired Ceramic Technology. Integrated Potentiometric Detection Based On Solvent Polymeric Ion-selective Electrodes"
Anal. Chem. 2006 Volume 78, Issue 9 Pages 2985-2992
Nuria Ibanez-Garcia, Manel Bautista Mercader, Zaira Mendes da Rocha, Carlos Antonio Seabra, Mário Ricardo Góngora-Rubio, and Julián Alonso Chamarro

Abstract: In this paper, the low-temperature co-fired ceramics (LTCC) technology, which has been commonly used for electronic applications, is presented as a useful alternative to construct continuous flow analytical microsystems. This technology enables not only the fabrication of complex three-dimensional structures rapidly and at a realistic cost but also the integration of the elements needed to carry out a whole analytical process, such as pretreatment steps, mixers, and detection systems. In this work, a simple and general procedure for the integration of ion-selective electrodes based on liquid ion exchanger is proposed and illustrated by using ammonium- and nitrate-selective membranes. Additionally, a screen-printed reference electrode was easily incorporated into the microfluidic LTCC structure allowing a complete on-chip integration of the potentiometric detection. Analytical features of the proposed systems are presented. © 2006 American Chemical Society.

"Automated On-line Renewable Solid-phase Extraction-liquid Chromatography Exploiting Multisyringe Flow Injection-bead Injection Lab-on-valve Analysis"
Anal. Chem. 2006 Volume 78, Issue 8 Pages 2832-2840
José Benito Quintana, Manuel Miró, José Manuel Estela, and Víctor Cerdà

Abstract: In this paper, the third generation of flow injection analysis, also named the lab-on-valve (LOV) approach, is proposed for the first time as a front end to high-performance liquid chromatography (HPLC) for on-line solid-phase extraction (SPE) sample processing by exploiting the bead injection (BI) concept. The proposed microanalytical system based on discontinuous programmable flow features automated packing (and withdrawal after single use) of a small amount of sorbent (<5 mg) into the microconduits of the flow network and quantitative elution of sorbed species into a narrow band (150 µL of 95% MeOH). The hyphenation of multisyringe flow injection analysis (MSFIA) with BI-LOV prior to HPLC analysis is utilized for on-line postextraction treatment to ensure chemical compatibility between the eluate medium and the initial HPLC gradient conditions. This circumvents the band-broadening effect commonly observed in conventional on-line SPE-based sample processors due to the low eluting strength of the mobile phase. The potential of the novel MSFI-BI-LOV hyphenation for online handling of complex environmental and biological samples prior to reversed-phase chromatographic separations was assessed for the expeditious determination of five acidic pharmaceutical residues (viz., ketoprofen, naproxen, bezafibrate, diclofenac, and ibuprofen) and one metabolite (viz., salicylic acid) in surface water, urban wastewater, and urine. To this end, the copolymeric divinylbenzene-co-n-vinylpyrrolidone beads (Oasis HLB) were utilized as renewable sorptive entities in the micro-machined unit. The automated analytical method features relative recovery percentages of >88%, limits of detection within the range 0.02-0.67 ng mL-1, and coefficients of variation < 11% for the column renewable mode and gives rise to a drastic reduction in operation costs (?25-fold) as compared to on-line column switching systems. © 2006 American Chemical Society.

"Sequential Flow Injection Analysis System On-line Coupled To High Intensity Focused Ultrasound: Green Methodology For Trace Analysis Applications As Demonstrated For The Determination Of Inorganic And Total Mercury In Waters And Urine By Cold Vapor Atomic"
Anal. Chem. 2006 Volume 78, Issue 8 Pages 2494-2499
C. Fernandez, Antonio C. L. Concei&ccedil;&atilde;o, R. Rial-Otero, C. Vaz, and J. L. Capelo

Abstract: A new concept is presented for green analytical applications based on coupling on-line high-intensity focused ultrasound (HIFU) with a sequential injection/flow injection analysis (SIA/FIA) system. The potential of the SIA/HIFU/FIA scheme is demonstrated by taking mercury as a model analyte. Using inorganic mercury, methylmercury, phenylmercury, and diphenylmercury, the usefulness of the proposed methodology for the determination of inorganic and total mercury in waters and urine was demonstrated. The procedure requires low sample volumes and reagents and can be further applied to all chemical reactions involving HIFU. The inherent advantages of SIA, FIA, and HIFU applications in terms of high throughput, automation, low reagent consumption, and green chemistry are accomplished together for the first time in the present work. © 2006 American Chemical Society.

"Continuous Ultrasound-assisted Extraction Coupled To Flow Injection-pervaporation, Derivatization, And Spectrophotometric Detection For The Determination Of Ammonia In Cigarettes"
Anal. Chem. 2006 Volume 78, Issue 7 Pages 2297-2301
A. Caballo-L&oacute;pez and M. D. Luque de Castro

Abstract: A dynamic system for the continuous removal of ammonia from cigarettes with ultrasound assistance and iterative change of the flow direction of the extractant through the sample cell has been developed. A 0.1-g sample of cigarette was subjected to 7 min of ultrasound-assisted extraction (application and duration of pulse 0.7 s, output amplitude 85% of the converter nominal amplitude), and 1 M NaOH solution was used both as extractant and as carrier in the dynamic system. The ultrasound-assisted extractor was coupled to a pervaporation unit through a flow injection interface in order to develop a fully automated method. In arriving at the pervaporator, the ammonia is transferred from the donor-carrier stream to an acceptor stream, where the classical Berthelot reaction takes place-thus favoring pervaporation. The blue complex formed is spectrophotometrically monitored at 655 nm. The method was applied to the determination of ammonia in a selection of 10 European cigarette brands and Kentucky Reference 2R4F cigarettes. © 2006 American Chemical Society.

"Sequential Injection Analysis System For The Sandwich Hybridization-based Detection Of Nucleic Acids"
Anal. Chem. 2006 Volume 78, Issue 6 Pages 1958-1966
Katie A. Edwards and Antje J. Baeumner

Abstract: A sequential injection analysis lab-on-valve (SIA-LOV) system was developed for the specific detection of single-stranded nucleic acid sequences via sandwich hybridization of specific DNA probes to the target sequence. One DNA probe was tagged with fluorescein; the other was biotinylated and immobilized to streptavidin-coated porous beads. The system was optimized with respect to buffer composition, length of hybridization and wash steps, and volumes and concentrations of components used. On-bead oligonucleotide hybridization was studied using UV detection at 260 nm, while a final dose response curve was quantified using fluorescence detection. A dynamic range of 1-1000 pmol was obtained for a synthetic DNA sequence that was homologous to a segment in the B. anthracis atxA mRNA. A within-day variation of 7.2% and a day-to-day variation of 9.9% was observed. Each analysis was completed within 20 min. Subsequently, the system was applied to the detection of atxA mRNA expressed in a surrogate organism and amplified using NASBA The SIA-LOV will find its application in routine laboratory-based analysis of specific single-stranded DNA/RNA sequences. Future improvements will include the integration of dye-encapsulating liposomes for signal enhancement used in lieu of the single fluorophore-labeled probe in order to lower the limit of detection. © 2006 American Chemical Society.

"Hybrid Fluorometric Flow Analyzer For Ammonia"
Anal. Chem. 2006 Volume 78, Issue 6 Pages 1890-1896
Natchanon Amornthammarong, Jaroon Jakmunee, Jianzhong Li, and Purnendu K. Dasgupta

Abstract: We describe a robust, highly sensitive instrument for the determination of ambient ammonia. The instrument uses two syringe pumps to handle three liquids. The flow configuration is a hybrid between traditional flow injection (FI) and sequential injection (SI) schemes. This hybrid flow analyzer spends ?87% of its time in the continuous flow FI mode, providing the traditional FI advantages of high baseline stability and sensitivity. The SI fluid handling operation in the remaining time makes for flexibility and robustness. Atmospheric ammonia is collected in deionized water by a porous membrane diffusion scrubber at 0.2 L/min with quantitative collection efficiency, derivatized on-line to 1-sulfonatoisoindole, and measured by fluorometry. In the typical range for ambient ammonia (0-20 ppbv), response is linear (r2 = 0.9990) with a S/N = 3 limit of detection of 135 pptv (15 nM for 500 µL of injected NH 4+(aq)) with an inexpensive light emitting diode photodiode-based detector. Automated operation in continuously repeated, 8-min cycles over 9 days shows excellent overall precision (n = 1544 p NH3 = 5 ppbv, RSD = 3%). Precision for liquid-phase injections is even better (n = 1520, [NH4+(aq)] = 2.5 ?M, RSD = 2%). The response decreases by 3.6% from 20 to 80% relative humidity. © 2006 American Chemical Society.

"Flow Injection Analysis System Equipped With A Newly Designed Electrochemiluminescent Detector And Its Application For Detection Of 2-thiouracil"
Anal. Chem. 2006 Volume 78, Issue 5 Pages 1568-1573
A. Caballo-L&oacute;pez and M. D. Luque de Castro

Abstract: A new flow injection analysis (FIA) system equipped with an electrochemiluminescent (ECL) detector has been developed and applied for the ECL detection of 2-thiouracil. The FIA-ECL system used a specially designed flow-through ECL thin-layer cell to reduce the dead volume, the IR drop across the cell, and the probability of accumulation of gas bubbles in the cell. It was thus envisioned to improve the detection limit of the FIA-ECL method. After being established, the new FIA-ECL system was used to investigate the ECL response of 2-thiouracil in the presence of the ECL of Ru(bpy)32+. It was found that 2-thiouracil could enhance the ECL of Ru(bpy)32+ over a wide pH range (pH 4.0-12.0). A highly sensitive method for detection of 2-thiouracil in biological samples was developed by the new FIA-ECL system after optimizing several experimental conditions, such as the applied potential of the working electrode, the pH value of the aqueous solution, the flow rate of carrier solution, and the concentration of Ru(bpy)32+. © 2006 American Chemical Society.

"Continuous Flow Microfluidic Demixing Of Electrolytes By Induced Charge Electrokinetics In Structured Electrode Arrays"
Anal. Chem. 2006 Volume 78, Issue 5 Pages 1425-1434
Felix C. Leinweber,* Jan C. T. Eijkel, Johan G. Bomer, and Albert van den Berg

Abstract: A continuous flow microfluidic demixing process is realized. It utilizes high external electrical fields that are applied over electrically floating noble metal electrodes in an otherwise straight microchannel. The process converts axial electrical potential gradients into lateral molecular selective transport via a structure oriented ensemble of numerous electrodes. While the individual electrodes locally modify the electrolyte distribution by nonlinear electrokinetic effects and concentration polarization, the directed orientation of the electrode array combines the individual polarization zones to a dedicated molecular enrichment against the generated concentration gradient. A homogeneously concentrated electrolyte can be separated into arbitrarily shaped laminae of increased and depleted concentration by the presented microfluidic demixer. © 2006 American Chemical Society.

"Diving Into Droplets"
Anal. Chem. 2006 Volume 78, Issue 5 Pages 1401-1404
Mukhopadhyan, R.

Abstract: Over the past decade, droplet-based microfluidics has gained strength. Rajendrani Mukhopadhyay investigates what droplet-based systems offer over continuous-flow-based devices and the challenges of manipulating samples and reagents as individual packets inside a device. © 2006 American Chemical Society.

"Design And Characterization Of Poly(dimethylsiloxane)-based Valves For Interfacing Continuous-flow Sampling To Microchip Electrophoresis"
Anal. Chem. 2006 Volume 78, Issue 4 Pages 1042-1051
Michelle W. Li, Bryan H. Huynh, Matthew K. Hulvey, Susan M. Lunte, and R. Scott Martin

Abstract: This work describes the fabrication and evaluation of a poly(dimethyl)siloxane (PDMS)-based device that enables the discrete injection of a sample plug from a continuous-flow stream into a microchannel for subsequent analysis by electrophoresis. Devices were fabricated by aligning valving and flow channel layers followed by plasma sealing the combined layers onto a glass plate that contained fittings for the introduction of liquid sample and nitrogen gas. The design incorporates a reduced-volume pneumatic valve that actuates (on the order of hundreds of milliseconds) to allow analyte from a continuously flowing sampling channel to be injected into a separation channel for electrophoresis. The injector design was optimized to include a pushback channel to flush away stagnant sample associated with the injector dead volume. The effect of the valve actuation time, the pushback voltage, and the sampling stream flow rate on the performance of the device was characterized. Using the optimized design and an injection frequency of 0.64 Hz showed that the injection process is reproducible (RSD of 1.77%, n = 15). Concentration change experiments using fluorescein as the analyte showed that the device could achieve a lag time as small as 14 s. Finally, to demonstrate the potential uses of this device, the microchip was coupled to a microdialysis probe to monitor a concentration change and sample a fluorescein dye mixture. © 2006 American Chemical Society.

"Biosensor Based On Self-assembling Acetylcholinesterase On Carbon Nanotubes For Flow Injection/amperometric Detection Of Organophosphate Pesticides And Nerve Agents"
Anal. Chem. 2006 Volume 78, Issue 3 Pages 835-843
Guodong Liu and Yuehe Lin

Abstract: A highly sensitive flow injection amperometric biosensor for organophosphate pesticides and nerve agents based on self-assembled acetylcholinesterase (AChE) on a carbon nanotube (CNT)-modified glassy carbon (GC) electrode is described. AChE is immobilized on the negatively charged CNT surface by alternatively assembling a cationic poly(diallyldimethylammonium chloride) (PDDA) layer and an AChE layer. Transmission electron microscopy images confirm the formation of layer-by-layer nanostructures on carboxyl-functionalized CNTs. Fourier transform infrared reflectance spectrum indicates the AChE was immobilized successfully on the CNT/PDDA surface. The unique sandwich-like structure (PDDA/AChE/PDDA) on the CNT surface formed by self-assembling provides a favorable microenvironment to keep the bioactivity of AChE. The electrocatalytic activity of CNT leads to a greatly improved electrochemical detection of the enzymatically generated thiocholine product, including a low oxidation overvoltage (+150 mV), higher sensitivity, and stability. The developed PDDA/AChE/PDDA/CNT/GC biosensor integrated into a flow injection system was used to monitor organophosphate pesticides and nerve agents, such as paraoxon. The sensor performance, including inhibition time and regeneration conditions, was optimized with respect to operating conditions. Under the optimal conditions, the biosensor was used to measure as low as 0.4 pM paraoxon with a 6-min inhibition time. The biosensor had excellent operational lifetime stability with no decrease in the activity of enzymes for more than 20 repeated measurements over a 1-week period. The developed biosensor system is an ideal tool for online monitoring of organophosphate pesticides and nerve agents. © 2006 American Chemical Society.

"Electrochemical Determination Of Arsenite Using A Gold Nanoparticle Modified Glassy Carbon Electrode And Flow Analysis"
Anal. Chem. 2006 Volume 78, Issue 3 Pages 762-769
Ehsan Majid, Sabahudin Hrapovic, Yali Liu, Keith B. Male, and John H. T. Luong

Abstract: A flow analysis electrochemical system has been developed, characterized, and optimized for the determination of arsenite (As(III)). Sensitivity was significantly improved by the electrochemical deposition of gold nanoparticles on a dual glassy carbon electrode, which was inserted into a cross-flow thin-layer electrochemical cell. The electrochemical system was linear up to 15 ppb with a detection limit of 0.25 ppb. Gold deposition was evident from cyclic voltammetry measurements, whereas atomic force microscopy and scanning electron microscopy revealed the size and distribution of deposited gold nanoparticles. The size and density of the nanoparticles were related to the gold salt concentration, deposition time, and potential as well as the electrode position. The response to arsenite was directly related to the frequency, increment, and amplitude of the square wave voltammetry as well as the deposition time and potential. Estimated reproducibility was ±1.1% at 95% confidence interval for 40 repeated analyzes of 8 ppb arsenite during continuous analysis. The reproducibility was far superior if the electrochemical reduction of arsenite was performed in nitric acid instead of hydrochloric or sulfuric acid. The electrochemical system was applicable for analysis of spiked arsenic in mineral water containing a significant amount of various ion elements.

"Third-generation Biosensor For Lactose Based On Newly Discovered Cellobiose Dehydrogenase"
Anal. Chem. 2006 Volume 78, Issue 2 Pages 393-398
Leonard Stoica, Roland Ludwig, Dietmar Haltrich, and Lo Gorton

Abstract: The present paper describes the principle and characteristics of a biosensor for lactose based on a third-generation design involving cellobiose dehydrogenase. As resulted from a previous comparative study (submitted manuscript), the novelty of this lactose biosensor is based on highly efficient direct electron transfer between two newly discovered cellobiose dehydrogenases (CDH), from the white rot fungi Trametes villosa and Phanerochaete sordida, and a solid spectrographic graphite electrode. CDH was immobilized on the electrode surface (0.073 cm2) by simple physical adsorption, and the CDH-modified electrode was next inserted into a wall-jet amperometric cell connected on-line to a flow injection setup (0.5 mL min-1). The P. sordida CDH-based lactose biosensor, proved to be the better one, has a detection limit for lactose of 1 ?M, a sensitivity of 1100 µA⋅mMsdot;cm-2, a response time of 4 s (the time required to obtain the maximum peak current), and a linear range from 1 to 100 µM lactose (correlation coefficient 0.998). The simplicity of construction and analytical characteristics make this CDH-based lactose biosensor an excellent alternative to previous lactose biosensors reported in the literature or commercially available. The CDH-lactose sensor was used to quantify the content of lactose in pasteurized milk, buttermilk, and low-lactose milk, using the standard addition method. No effects of the samples matrixes were observed. The operational stability of the sensor was tested for 11 h by continuous injection of 100 µM lactose (290 injections). The final signal of the sensor was maintained at 98% of its initial signal, with a low standard deviation of 1.72 (RSD 2.41%). © 2006 American Chemical Society.

"High-temperature Liquid Chromatography Coupled On-line To A Continuous-flow Biochemical Screening Assay With Electrospray Ionization Mass Spectrometric Detection"
Anal. Chem. 2005 Volume 77, Issue 24 Pages 7894-7900
Arjen R. de Boer, Juan M. Alcaide-Hidalgo, Johannes G. Krabbe, Jeffrey Kolkman, Caspar N. van Emde Boas, Wilfried M. A. Niessen, Henk Lingeman, and Hubertus Irth

Abstract: The potential of high-temperature liquid chromatography (HTLC) was investigated in an on-line combination with a screening system for bioactive compounds against the enzyme cathepsin B. Samples were separated by HTLC and subsequently analyzed by an on-line continuous-flow enzymatic assay. Detection was performed by electrospray ionization mass spectrometry, revealing both the bioactivity and the molecular mass of the bioactive compounds. Compared to conventional reversed-phase liquid chromatography, the amount of methanol necessary for separation could be decreased to only 10%, which improved the compatibility of LC with a biochemical assay. Sufficient preheating of the mobile phase prior to the separation and post-column cooling to prevent deactivation of the enzyme, even at column temperatures as high as 208°C, was achieved as indicated by the reliable peak shapes obtained. The sensitivity was comparable with previously described systems operating at ambient temperatures as similar IC50 values were obtained. Exposing the inhibitors to high temperatures did not lead to thermal decomposition. The separation of inhibitors and the subsequent biochemical assay was performed either isothermally at various temperatures or by applying various temperature gradients as well as at various flow rates. The results obtained clearly show the compatibility of HTLC with an enzymatic screening assay. © 2005 American Chemical Society.

"ATR-FT-IR Membrane-based Sensor For Integrated Microliquid-liquid Extraction And Detection"
Anal. Chem. 2005 Volume 77, Issue 22 Pages 7472-7477
Rafael Lucena, Soledad C&aacute;rdenas, Mercedes Gallego, and Miguel Valc&aacute;rcel

Abstract: A novel straightforward membrane-based sensor, which uses attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy has been developed. The flow cell designed permits the on-line microliquid-liquid extraction of the target analyte into a organic solvent layer (OSL), which was deposited on the ATR surface using a sequential injection manifold. The aqueous and organic phases are separated via a commercial hydrophobic membrane placed on the PTFE piece of the cell. The main advantage of the proposed device is that the OSL can be created and regenerated in a continuous manner using the automatic manifold without opening the cell. The analytes are enriched into the OSL after diffusion through the membrane, which excludes the typical absorption bands of water. In addition, the behavior of different organic solvents was evaluated in order to increase the applicability and versatility of the proposed system. Finally, the analytical performance of the design was established for the detection and quantitation of Triton x 100 in water. © 2005 American Chemical Society.

"Thorium Complexation By Hydroxamate Siderophores In Perturbed Multicomponent Systems Using Flow Injection Electrospray Ionization Mass Spectrometry"
Anal. Chem. 2005 Volume 77, Issue 22 Pages 7335-7341
Miranda J. Keith-Roach, Marta Vetri Buratti, and Paul J. Worsfold

Abstract: Flow injection electrospray ionization mass spectrometry has been shown to produce simple, characteristic m/z signals for Th-hydroxamate siderophore (desferrioxamine and ferrichrome) complexes, with Th complexed as a simple 4+ ion in the environmentally relevant pH range investigated (pH 5-9). All species of interest for this study were identified optimally in the positive mode; thus, multiple species were analyzed concurrently in a single spectrum. Complexation of Th by the two siderophores was rapid in 1:1 molar aqueous solution, reaching equilibrium before the first measurement was possible at 2 min. However, a significant proportion of the equimolar siderophore remained uncomplexed. Both siderophores rapidly exchanged Th for Fe when equimolar Fe(III) was added to the Th complexes, and only a small proportion of each siderophore remained complexed with Th at equilibrium (7-30 min). The results show a difference in the affinities of the two siderophores for the metals; ferrichrome has a 5-fold higher affinity than desferrioxamine for Th and a 5-fold lower affinity than desferrioxamine for Fe. Also, siderophore-complexed Th interacted strongly with a cation-exchange resin suggesting that, even when complexed by trianionic siderophores, Th mobility will be impeded by interactions with negatively charged binding sites in subsurface environmental matrixes. These results have important implications regarding siderophore-enhanced actinide(IV) mobility in the terrestrial environment. © 2005 American Chemical Society.

"Electrochemically Modulated Liquid-liquid Extraction Of Ions"
Anal. Chem. 2005 Volume 77, Issue 22 Pages 7310-7318
Alfonso Berduque, Amanda Sherburn, Mihaela Ghita, Robert A. W. Dryfe, and Damien W. M. Arrigan

Abstract: The development of ion extraction methods under electrochemical control via electrochemistry at the interface between two immiscible electrolyte solutions is discussed. A hydrodynamic flow injection system was used for the potentiostatic extraction of non-redox-active species from a flowing aqueous phase into a stationary organogel phase. The ions tetraethylammonium, 4-octylbenzenesulfonate (4-OBSA?), and p-toluenesulfonate (p-TSA?) were studied as model analytes. The extraction study comprised examination of the influence of extraction potentials, aqueous-phase flow rate, and target species concentration. The extraction process can be monitored in situ by means of the ion-transfer current, which has opposing signs for anions and cations. Hydrodynamic voltammograms were obtained from these experiments. The selective extraction of 4-OBSA?, from its mixture with p-TSA?, as well as coextraction of both anions is shown. The results demonstrate the utility of electrochemical modulation for the controlled extraction of ions from an aqueous phase into an organogel electrolyte phase. This offers potential benefits for various analytical processes including sample preparation and cleanup. © 2005 American Chemical Society.

"Universal Approach For Selective Trace Metal Determinations Via Sequential Injection-bead Injection-lab-on-valve Using Renewable Hydrophobic Bead Surfaces As Reagent Carriers"
Anal. Chem. 2005 Volume 77, Issue 18 Pages 6032-6040
Xiangbao Long, Manuel Mir&oacute;, and Elo Harald Hansen

Abstract: A new concept is presented for selective and sensitive determination of trace metals via electrothermal atomic absorption spectrometry based on the principle of bead injection (BI) with renewable reversed-phase surfaces in a sequential injection-lab-on-valve (SI-LOV) mode. The methodology involves the use of poly(styrene-divinylbenzene) beads containing pendant octadecyl moieties (C18-PS/DVB), which are preimpregnated with a selective organic metal chelating agent prior to the automatic manipulation of the beads in the microbore conduits of the LOV unit. By adapting this approach, the immobilization of the most suitable chelating agent can be effected irrespective of the kinetics involved, optimal reaction conditions can be used for implementing the chelating reaction of the target metal analyte with the immobilized reagent, and an added degree of freedom is offered in selecting the most favorable elution mode in order to attain the highest sensitivity. The potential of the SI-BI-LOV scheme is demonstrated by taking Cr(VI) as a model analyte, using a 1,5-diphenylcarbazide (DPC)-loaded bead column as the active microzone. As this reaction requires the use of high acidity, it is also shown that the bead material exhibits excellent chemical stability at low pH values. On-line pH sample adjustment prevents alteration of the original distribution of chromium species while ensuring fast rates for the DPC-Cr(VI) reaction. The proposed procedure was successfully applied to the determination of trace levels of Cr(VI) in natural waters containing high levels of dissolved salts (such as seawater and hard tap water) without requiring any dilution step. Method validation was performed by determination of total chromium in an NIST standard reference material (NIST 1640, natural water) after Cr(III) oxidation, and the results were in good agreement with the certified value. © 2005 American Chemical Society.

"Octadecyl Immobilized Surface For Precipitate Collection With A Renewable Microcolumn In A Lab-on-valve Coupled To An Electrothermal Atomic Absorption Spectrometer For Ultratrace Cadmium Determination"
Anal. Chem. 2005 Volume 77, Issue 16 Pages 5396-5401
Yang Wang, Jian-Hua Wang, and Zhao-Lun Fang

Abstract: 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 pre-concentration 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 µL 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 µL within the concentration range of 0.01-0.2 ?g L-1, achieving a precision of 2.1% RSD at the 0.05 ?g L-1 level. The enrichment factor was further enhanced to 44 by increasing the sample volume to 1200 µL. 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. © 2005 American Chemical Society.

"Atomic Absorption Spectroscopy For Mercury, Automated By Sequential Injection And Miniaturized In Lab-on-valve System"
Anal. Chem. 2005 Volume 77, Issue 16 Pages 5124-5128
Holger Erxleben and Jaromir Ruzicka

Abstract: Sodium borohydride-based hydride generation was automated by using programmable flow within the lab-on-valve module. Mercury vapor, generated in the reaction mixture, was extracted in a gas/liquid separator. The gas-expansion separator was miniaturized and compared with the performance of a novel gas separator that exploits the combination of Venturi effect and reduced pressure. Cold vapor atomic spectroscopy was used as a model system, with detection of mercury by absorption at 254 nm and limit of detection of 9 ?g of Hg/L, using 300 µL of sample and 100 µL of borohydride. This work introduces, for the first time, sequential injection technique for hydride generation, highlights advantages of using programmable flow, and outlines means for miniaturization of assays based on spectroscopy of volatile species. © 2005 American Chemical Society.

"Speciation-capable Field Instrument For The Measurement Of Arsenite And Arsenate In Water"
Anal. Chem. 2005 Volume 77, Issue 15 Pages 4765-4773
Kei Toda, Takashi Ohba, Mari Takaki, Sathrugnan Karthikeyan, Shizuko Hirata, and Purnendu K. Dasgupta

Abstract: Hydride generation to form arsine and in-line pre-concentration of the arsine into an alkaline KMnO4 receiver followed by molybdenum blue (MB) colorimetric determination of the arsenate formed is proposed for the highly sensitive and separate measurement of total inorganic As and As(III). Reduction of As to AsH3 is carried out by NaBH4; when the reduction is carried out at pH 1, all the inorganic As is reduced to AsH 3, and when carried out at pH 7, only As(III) is reduced. Reductions at the two different pH levels are carried out in two different arsine generators simultaneously using constant addition of NaBH4 with solenoid pumps. The AsH3 is collected by individual porous membrane diffusion scrubbers filled with a stationary solution of KMnO4, and the contents of the two scrubbers sequentially enter a flow analysis stream. MB is formed by merging with a ammonium molybdate-ascorbic acid reagent, passing through a heated reactor, and is then measured by a LED/photodiode-based absorbance detector. Robustness was confirmed for total As using three types of certified natural water samples. Speciation analysis data from well water samples analyzed by this method agree well with HPLC-ICPMS measurements in a different laboratory. The system has been successfully applied to field measurements of As(III) and As(V), where levels were significantly below 1 ?g/L. For a 20 mL sample, the limits of detection (LODs) for this inexpensive instrument are 0.3 ?g/L for both As(III) and total As. When an 80 mL sample is analyzed, LODs are 0.07 ?g/L As(III) and 0.09 ?g/L total As. The general approach should be applicable to many other analyte species of interest that can be isolated from the matrix by the formation of a suitable volatile compound that can be recaptured. © 2005 American Chemical Society.

"Toward Single-calibrant Quantification In HPLC. A Comparison Of Three Detection Strategies: Evaporative Light Scattering, Chemiluminescent Nitrogen, And Proton NMR"
Anal. Chem. 2005 Volume 77, Issue 14 Pages 4354-4365
Steve Lane, Bob Boughtflower, Ian Mutton, Clare Paterson, Duncan Farrant, Nick Taylor, Zoe Blaxill, Carol Carmody, and Phil Borman

Abstract: There is an urgent need for detection technologies that enable accurate and precise quantification of solutions containing small organic molecules in a manner that is rapid, cheap, non-labor-intensive, readily automated, and without a requirement for specific analyte standards. We provide a theoretical analysis that predicts that the logarithmic nature of the working domain of the evaporative light-scattering detector (ELSD) will normally bias toward underestimation of chromatographically resolved impurities, resulting in an overestimation of analyte purity. This analysis is confirmed by experiments with flow injection analysis (FIA) and gradient reversed-phase high performance liquid chromatography (RP-HPLC). Quantification is further compromised by the dependence of response parameters on the matrix composition and hence on the retention time of the analyte. Attempts were made to ameliorate these problems by using the response surface of a single compound to calibrate throughout the HPLC gradient. A chemiluminescent nitrogen detector (CLND) was also used in a similar manner, and the performance of the two techniques were compared against those of each other and that of a reference standard technique. A protocol for this purpose was developed using proton nuclear magnetic resonance ( 1H NMR) and the ERETIC method to enable quantification by integrating proton signals. The double-blind comparison exercise confirmed molar nitrogen CLND response to be sufficiently stable and robust across a methanol gradient to be used with a single external nitrogenous calibrant to quantify nitrogen-containing compounds of known molecular formula. The performance of HPLC-CLND was very similar to that of NMR, while that of HPLC-ELSD was seen to be significantly worse, showing it to be unsuitable for the purpose of single-calibrant quantification. We report details and experience of our use of RP-HPLC-CLND-MS to characterize and quantify small amounts of solutions of novel compounds at nominal levels of 10 mM in microtiter plate (MTP) format. © 2005 American Chemical Society.

"Microfiber-Directed Boundary Flow In Press-Fit Microdevices Fabricated From Self-Adhesive Hydrophobic Surfaces"
Anal. Chem. 2005 Volume 77, Issue 11 Pages 3671-3675
Tom T. Huang, David G. Taylor, Miroslav Sedlak, Nathan S. Mosier and Michael R. Ladisch

Abstract: We report a rapid microfluidic device construction technique which does not employ lithography or stamping methods. Device assembly physically combines a silicon wafer, an elastomer (poly(dimethylsiloxane) (PDMS)), and microfibers to form patterns of hydrophobic channels, wells, elbows, or orifices that direct fluid flow into controlled boundary layers. Tweezers are used to place glass microfibers in a defined pattern onto an elastomeric (PDMS) hydrophobic film. The film is then manually pressed onto a hydrophobic silicon wafer, causing it to adhere to the silicon wafer and form a liquid-tight seal around the fibers. Completed in 15 min, the technique results in an operable microdevice with micrometer-scale features of nanoliter volume. Microfiber-directed boundary flow is achieved by use of the surface wetting properties of the hydrophilic glass fiber and the hydrophobicity of surrounding surfaces. The simplicity of this technique allows quick prototyping of microfluidic components, as well as complete biosensor systems, such as we describe for the detection of pathogenic bacteria.

"Cell Stimulus And Lysis In A Microfluidic Device With Segmented Gas-Liquid Flow"
Anal. Chem. 2005 Volume 77, Issue 11 Pages 3629-3636
Jamil El-Ali, Suzanne Gaudet, Axel G&uuml;nther, Peter K. Sorger and Klavs F. Jensen

Abstract: We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks. The device uses segmented gas-liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis. Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stress-sensitive mitogen activated protein kinases observed. Jurkat E6-1 cells are stimulated in the device using α-CD3, and the resulting activations of ERK and JNK are presented for different time points. Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions.
Cell Microfluidic Air segmentation

"Application Of A Liposomal Bioluminescent Label In The Development Of A Flow Injection Immunoanalytical System"
Anal. Chem. 2005 Volume 77, Issue 11 Pages 3431-3436
Ja-an Annie Ho and Ming-Ray Huang

Abstract: A flow injection liposome immunoanalytical system was developed using biotin as the model analyte and liposomal aequorin as the label. Aequorin is a photoprotein isolated from luminescent jellyfish (notably Aequorea victoria) and other marine organisms that emits visible light in the presence of a trace of Ca2+. Because of this characteristic, the aequorin complex has been used as an intracellular Ca2+ indicator. In this study, a bioluminescent label was designed by encapsulating aequorin inside the cavity of the liposome, whose outer surface was sensitized with the analyte of interest. The analyte-tagged liposomal aequorin was employed in the development of a heterogeneous bioluminescence immunoassay for the model analyte biotin. The proposed immunoassay was based on the competition between the model biotin and aequorin-encapsulating, biotin-tagged liposomes for a limited number of anti-biotin antibody-binding sites. The anti-biotin antibodies were immobilized via protein A in a capillary immunoreactor column, and 30% MeOH was used for the regeneration of antibody-binding sites after each measurement, which allowed the immunoreactor to be used for up to 50 sequential sample injections without any loss of reactivity. The calibration curve for biotin in Tris-buffered saline solution had a linear range of 1 x 10^-11-1 x 10 -3 M. The detection limit of the assay was 50 pg (equivalent to 200-?L injection of 1 x 10^-9 M). This study demonstrates the procedures for the encapsulation of the photoprotein aequorin into the liposome, which can be used as a sensitive label in bioluminescence immunoassays for biotin or in other applications. © 2005 American Chemical Society.

"Microbioassay System For Antiallergic Drug Screening Using Suspension Cells Retaining In A Poly(dimethylsiloxane) Microfluidic Device"
Anal. Chem. 2005 Volume 77, Issue 10 Pages 3309-3314
Takahito Tokuyama, Shin-ichiro Fujii, Kiichi Sato, Mitsuru Abo and Akira Okubo

Abstract: This article describes an antiallergic drug-screening system by the detection of histamine released from mast cells (suspension cells) on a multilayer microchip. In this study, the elastmeric material, poly(dimethylsiloxane) (PDMS), was employed to fabricate microchannels and microchambers. The microchip consists of two sections: a histamine-releasing one, which has a cell chamber, and a histamine-derivatizing one. Both were laminated to one microchip. Rat peritoneal mast cells were retained in the cell chamber (1.2 µL) with a filtering system using a cellulose nitrate membrane. This filtering system could easily retain suspension cells without cell damage. Mast cells were viable for a sufficient time to conduct the assay on the cell chamber. The cells were stimulated with a chemical release compound 48/80 (C48/80), and then histamine flowed into the lower layer, where it was derivatized to the fluorescent molecules with o-phthalaldehyde and its fluorescence was detected on the microchip. This flow system could detect the time course of the histamine release, and this microchip system required only 20 min for the assay. By this integrated system, 51 pmol of histamine released from 500 cells was detected, and the number of cells required for the assay was reduced to 1% compared with conventional bulk systems. By comparing the released histamine levels with and without drugs, their effect could be evaluated. The inhibition ratio of C48/80 induced-histamine release using an antiallergic drug, disodium cromoglicate (DSCG), was related to the concentration of DSCG. This flow system was applicable for antiallergy drug screening by rapid measurement of the inhibition of histamine release from a very small amount of mast cells.

"Automated Sequential Injection-Microcolumn Approach With On-Line Flame Atomic Absorption Spectrometric Detection For Implementing Metal Fractionation Schemes Of Homogeneous And Nonhomogeneous Solid Samples Of Environmental Interest"
Anal. Chem. 2005 Volume 77, Issue 9 Pages 2270-2726
Roongrat Chomchoei, Manuel Mir&oacute;, Elo Harald Hansen and Juwadee Shiowatana

Abstract: An automated sequential injection (SI) system incorporating a dual-conical microcolumn is proposed as a versatile approach for the accommodation of both single and sequential extraction schemes for metal fractionation of solid samples of environmental concern. Coupled to flame atomic absorption spectrometric detection and used for the determination of Cu as a model analyte, the potentials of this novel hyphenated approach are demonstrated by the ability of handling up to a 300 mg sample of a nonhomogeneous sewage amended soil (viz., CRM 483). The three steps of the endorsed Standards, Measurements, and Testing sequential extraction method have been also performed in a dynamic fashion and critically compared with the conventional batchwise protocols. The ecotoxicological relevance of the data provided by both methods with different operationally defined conditions is thoroughly discussed. As compared to traditional batch systems, the developed SI assembly offers minimal risks of sample contamination, the absence of metal re-distribution/readsorption, and dramatic saving of operational times (from 16 h to 40-80 min per partitioning step). It readily facilitates the accurate manipulation of the extracting reagents into the flow network and the minute, well-defined injection of the desired leachate volume into the detector. Moreover, highly time-resolved information on the ongoing extraction is given, which is particularly relevant for monitoring fast leaching kinetics, such as those involving strong chelating agents. On-line and off-line (for Cu, Pb, and Zn) single extraction schemes are also proven to constitute attractive alternatives for fast screening of metal pollution in solid samples and for predicting the current, rather than the potential, element bioavailability by the assessment of the readily mobilizable metal forms.

"Effect Of Model Ligands On Iron Redox Speciation In Natural Waters Using Flow Injection With Luminol Chemiluminescence Detection"
Anal. Chem. 2005 Volume 77, Issue 7 Pages 1971-1978
S. J. Ussher, M. Yaqoob, E. P. Achterberg, A. Nabi, and P. J. Worsfold

Abstract: The effects of dissolved organic compounds on the determination of nanomolar concentrations of Fe(II) have been compared using two luminol-based flow injection chemiluminescence (FI-CL) methods. One used the direct injection of sample into the luminol reagent stream, and the other incorporated on-line solid-phase extraction of the analyte on an 8-hydroxyquinoline microcolumn. The CL signals from analyzes of dissolved iron species (Fe(II) and Fe(III)) with model ligands and organic compounds were examined in high-purity water and seawater. The organic compounds included natural reducing agents (e.g., ascorbic acid), nitrogen ?-donor/?-acceptor compounds (e.g., 1,4-dipyridine, protoporphyrin IX), aromatic compounds (e.g., 1,4-dihydroxybenzene), synthetic iron chelators (e.g., EDTA), and natural iron binding compounds (e.g., desferrioxamine B, ferrichrome A). Fe(II) determinations for both luminol FI-CL methods were affected by submicromolar concentrations of redox-active compounds, strong iron binding ligands (i.e., log KFeL > 6), and compounds with electron-donating functional groups in both high-purity water and seawater. This was due to reactions between organic molecules and iron species before and during analysis, rather than chemiluminescence caused by the individual organic compounds. In addition, the effects of strong ligands and size speciation on Fe(II) recoveries from seawater following acidification (pH 2) and reduction (100 ?M sodium sulfite) were investigated. © 2005 American Chemical Society.

"High-throughput Nanoliter Sample Introduction Microfluidic Chip-based Flow Injection Analysis System With Gravity-driven Flows"
Anal. Chem. 2005 Volume 77, Issue 5 Pages 1330-1337
Wen-Bin Du, Qun Fang, Qiao-Hong He, and Zhao-Lun Fang

Abstract: 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-?L 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-?m i.d., 375-?m 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 ?M 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. © 2005 American Chemical Society.

"Carrier Medium Exchange Through Ultrasonic Particle Switching In Microfluidic Channels"
Anal. Chem. 2005 Volume 77, Issue 5 Pages 1216-1221
Filip Petersson, Andreas Nilsson, Henrik J&ouml;nsson and Thomas Laurell

Abstract: This paper describes a method, utilizing acoustic force manipulation of suspended particles, in which particles in a laminar flow microchannel are continuously translated from one medium to another with virtually no mixing of the two media. During the study, 5-µm polyamide spheres suspended in distilled water, spiked (contaminated) with Evans blue, were switched over to clean distilled water. More than 95% of the polyamide spheres could be collected in the clean medium while removing up to 95% of the contaminant. Preliminary experiments to use this method to wash blood were performed. Red blood cells were switched from blood, spiked with Evans blue, to clean blood plasma. At least 95% of the red blood cells (bovine blood) could be collected in clean blood plasma while up to 98% of the contaminant was removed. The obtained results indicate that the presented method can be used as a generic method for particle washing and, more specifically, be applied for both intraoperative and postoperative blood washing.

"Miniaturized Cavity Ring-down Detection In A Liquid Flow Cell"
Anal. Chem. 2005 Volume 77, Issue 4 Pages 1188-1191
B. Bahnev, L. van der Sneppen, A. E. Wiskerke, F. Ariese, C. Gooijer, and W. Ubachs

Abstract: A novel method for applying cavity ring-down spectroscopy in the liquid phase, compatible with LC analyzes, is presented. The core of the setup is a home-built cavity ring-down flow cell (cell volume 12 µL) that is constructed using a silicon rubber spacer, which is clamped leak-tight between two high-reflectivity mirrors. The mirrors are in direct contact with the liquid flow, which provides for a small path length and short ring-down times. Inside the cavity there are no windows, reflection losses, or Brewster angles to be considered. Due to the small size of the presented cavity geometry, the setup can be implemented in conventional-size LC apparatuses. With a flow injection setup, a detection limit of 2.5 nM was obtained for Crystal Violet in ethanol, and the linear dynamic range of the system is at least 2 orders of magnitude. The method has the potential to become a powerful alternative for commercial LC UV/visible absorbance detectors.

"VUV Single-Photon Ionization Ion Trap Time-of-Flight Mass Spectrometer For On-Line, Real-Time Monitoring Of Chlorinated Organic Compounds In Waste Incineration Flue Gas"
Anal. Chem. 2005 Volume 77, Issue 4 Pages 1007-1012
Shizuma Kuribayashi, Hideo Yamakoshi, Minoru Danno, Satoshi Sakai, Shigenori Tsuruga, Hiroshi Futami and Shigeki Morii

Abstract: In this work, a sensitive and robust vacuum ultra-violet (VUV) single-photon ionization (SPI) ion trap time-of-flight mass spectrometer (VUV-SPI-IT-TOFMS) for on-line, real-time monitoring of chlorinated organic compounds in waste incineration flue gas has been newly developed. The fragment-free SPI technique with 121.6-nm VUV lamp irradiated by a microwave generator and the quadrupole ion trap to accumulate and select analyte ions were combined with a reflectron time-of-flight mass spectrometer to detect chlorinated organic compounds at trace level. This measuring system was tuned up to detect dioxins precursors with the aim at an application to monitoring trace level toxic substances in flue gases from incinerator furnaces. As a result, this technology has made it possible to analyze trichlorobenzene (T3CB), a dioxin precursor, in 18 s with a sensitivity of 80 ng/m3 N (10 pptv) using the selective accumulation of analyte substances and separation of interfering substances in the ion trap. Moreover, the first field test of the continuous monitoring T3CB in an actual waste incineration flue gas had been done for 7 months. The results show that this system has an exceeding robust performance and is able to maintain the high sensitivity in analyzing T3CB for long months of operation.

"Surface Modification Method Of Microchannels For Gas-Liquid Two-Phase Flow In Microchips"
Anal. Chem. 2005 Volume 77, Issue 3 Pages 943-947
Akihide Hibara, Shinobu Iwayama, Shinya Iwayama, Masaharu Ueno, Yoshikuni Kikutani, Manabu Tokeshi and Takehiko Kitamori

Abstract: A capillarity restricted modification method for microchannel surfaces was developed for gas-liquid microchemical operations in microchips. In this method, a microstructure combining shallow and deep microchannels and the principle of capillarity were utilized for chemical modification of a restricted area of a microchannel. A hydrophobic-hydrophilic patterning in microchannels was prepared as an example for guiding gas and liquid flows along the respective microchannels. Validity of the patterning was confirmed by measuring aqueous flow leak pressure from the hydrophilic microchannel to the hydrophobic one. The leak pressure of 7.7-1.1 kPa agreed well with that predicted theoretically from the Young-Laplace equation for the microchannel depth of 8.6-39 µm. In an experiment to demonstrate usefulness and effectiveness of the method, an air bubble was first introduced into the hydrophilic microchannel and purged from the hydrophobic-hydrophilic patterned microchannels. Next, the patterning structure was applied to remove dissolved oxygen by contacting the aqueous flow with a nitrogen flow. The concentration of dissolved oxygen decreased with contact time, and its time course agreed well with numerical simulation. These demonstrations showed that the proposed patterning method can be used in general microfluidic gas-liquid operations.

"Measurement Of The Surface Concentration For Bioassay Kinetics In Microchannels"
Anal. Chem. 2005 Volume 77, Issue 3 Pages 833-839
Aur&eacute;lien Bancaud, Gaudeline Wagner, Kevin D. Dorfman and Jean-Louis Viovy

Abstract: We present a simple and versatile method, based on fluorescence microscopy, to reliably measure the concentration of advected molecules in the vicinity of surfaces in microchannels. This tool is relevant to many microfluidic applications such as immunoassays and single-molecule experiments, where one probes the kinetics of a reaction between an immobilized target and a reactant carried by the bulk flow. The characterization of the surface concentration highlights the dominant role of transverse diffusion, which generates an apparent diffusivity at the surface 3-4 orders of magnitude greater than molecular diffusion alone, even close to the point of injection. We directly measure the effects of the longitudinal position along the channel and of the flow rate on the concentration front and develop a simple analytical model that compares well with the data. Finally, we propose a method to properly account for concentration fronts in single-molecule measurements and use it to directly access the kinetics parameters of protamine-induced condensation of DNA.

"High-throughput Screening Of Protein Surface Activity Via Flow Injection Analysis-pH Gradient-dynamic Surface Tension Detection"
Anal. Chem. 2005 Volume 77, Issue 1 Pages 250-258
Bethany A. Staggemeier, Emilia Bramanti, Chiara Allegrini, Kristen J. Skogerboe, and Robert E. Synovec

Abstract: Using flow injection analysis (FIA), a pH gradient is blended in real time with a protein sample as the pH-dependent protein surface activity is measured by a dynamic surface tension detector (FIA-pH-DSTD). This instrumental system was developed as a high-throughput method for the screening of protein surface activity at the air/liquid interface as a function of pH. This method utilizes the continuous flow, drop-based dynamic surface tension detector in combination with flow injection sample introduction and blending of a steady-state concentration of protein sample with a pH gradient ranging from pH 2.0 to pH 11.5. Dynamic surface tension is measured through the differential pressure across the air/liquid interface of repeatedly growing and detaching drops. Continuous surface tension measurement is achieved for each eluting drop of 2-s length (2 µL), providing insight into both the kinetic and thermodynamic behaviors of molecular orientation processes at the liquid/air interface. Three-dimensional data are obtained, with surface tension first converted to surface pressure, which is collected as a function of elution time versus drop time. In FIA-pH-DSTD, a commercial pH probe is used to measure pH during elution time, enabling surface pressure throughout drop time to be subsequently plotted as a function of eluting pH. An automated DSTD calibration procedure and data analysis method is applied, which allows simultaneous use of two different solvents, permitting real-time dynamic surface tension data to be obtained. The method was applied to the analysis of 14 commercial purified proteins, yielding characteristic features of surface activity as a function of pH. The reproducibility of the measurement and selectivity advantage of the DSTD was shown for the analysis of serum albumins from various mammalian sources. Several applications were also suggested and discussed in order to show the potential of the method for protein and food chemistry studies and in the study of protein-polymer interactions.

"Interfacial Scattering At Electrochemically Fabricated Atom-Scale Junctions Between Thin Gold Film Electrodes In A Microfluidic Channel"
Anal. Chem. 2005 Volume 77, Issue 1 Pages 243-249
Patrick J. Castle and Paul W. Bohn

Abstract: Atom-scale junctions were formed between two Au thin-film electrodes by a combination of lithography, microfluidics, and electrochemistry. Two Au thin-film electrodes with a small (0.25-25 µm) gap between them were lithographically defined such that the gap fell in the center of a 100-µm-wide microfluidic channel in poly(dimethylsiloxane). Directional electrodeposition between the Au thin-film electrodes, accomplished by applying a potential between the thin-film electrodes, caused Au to etch from the anode and deposit on the cathode, thereby closing the gap. Current through the gap was monitored continuously, and the directional electrodeposition was terminated when a current near that corresponding to the conductance quantum, G0 = 2e2/h, was reached. To regenerate the device, the atom-scale junction was broken with a potential sweep, the microfluidic channel was rinsed, and the junction was re-formed with a subsequent comparator-terminated directional electrodeposition. Alternating current impedance was measured while hexadecanethiol (HDT) was chemisorbed onto the atom-scale junction. The interfacial scattering from chemisorption of the Lewis base HDT on the atom-scale junction caused a normalized impedance change of 71 ± 1%, the noise level being equivalent to a population fluctuation of five HDT molecules.

"Label-Free Reading Of Microarray-Based Immunoassays With Surface Plasmon Resonance Imaging"
Anal. Chem. 2004 Volume 76, Issue 24 Pages 7257-7262
Vishal Kanda, James K. Kariuki, D. Jed Harrison and Mark T. McDermott

Abstract: A simple method is presented for patterning of protein antigens at a gold surface for use in surface plasmon resonance (SPR) imaging experiments. Microfluidic devices fabricated from poly(dimethylsiloxane) were used to flow various fluids over a gold substrate in spatially defined channels. This technique was used to pattern the surface chemistry of the gold as well as to adsorb antigens from solution to the modified substrates. The resulting antigen arrays were probed with complementary antibodies in order to demonstrate the effectiveness of the patterning for antibody capture experiments. SPR imaging was used to aid in the optimization of array fabrication and to observe the interactions of unlabeled antibodies with these microarrays. This work presents a means of fabricating microarrays with controlled surface density of antigens. SPR imaging provides both quantitative and qualitative evaluation of antibody binding in a label free format.

"On-chip Free-flow Magnetophoresis: Continuous Flow Separation Of Magnetic Particles And Agglomerates"
Anal. Chem. 2004 Volume 76, Issue 24 Pages 7250-7256
Nicole Pamme and Andreas Manz

Abstract: The separation of magnetic microparticles was achieved by on-chip free-flow magnetophoresis. In continuous flow, magnetic particles were deflected from the direction of laminar flow by a perpendicular magnetic field depending on their magnetic susceptibility and size and on the flow rate. Magnetic particles could thus be separated from each other and from nonmagnetic materials. Magnetic and nonmagnetic particles were introduced into a microfluidic separation chamber, and their deflection was studied under the microscope. The magnetic particles were 2.0 and 4.5 ?m in diameter with magnetic susceptibilities of 1.12 x 10^-4 and 1.6 x 10^-4 m3 kg -1, respectively. The 4.5-?m particles with the larger susceptibility were deflected further from the direction of laminar flow than the 2.0-?m magnetic particles. Nonmagnetic 6-?m polystyrene beads, however, were not deflected at all. Furthermore, agglomerates of magnetic particles were found to be deflected to a larger extent than single magnetic particles. The applied flow rate and the strength and gradient of the applied magnetic field were the key parameters in controlling the deflection. This separation method has a wide applicability since magnetic particles are commonly used in bioanalysis as a solid support material for antigens, antibodies, DNA, and even cells. Free-flow magnetophoretic separations could be hyphenated with other microfluidic devices for reaction and analysis steps to form a µtotal analysis system.

"Hydride Generation Interface For Speciation Analysis Coupling Capillary Electrophoresis To Inductively Coupled Plasma Mass Spectrometry"
Anal. Chem. 2004 Volume 76, Issue 23 Pages 7137-7142
Douglas D. Richardson, Sasi S. Kannamkumarath, Rodolfo G. Wuilloud and Joseph A. Caruso

Abstract: A novel hydride generation (HG) interface for coupling capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICPMS) is presented in this work. The CE-HG-ICPMS interface was applied to the separation and quantitation of common arsenic species. Lack of a commercially available HG interface for CE-ICPMS led to a three concentric tube design allowing alleviation of back pressure commonly observed in CE-HG-ICPMS. Due to the high sensitivity and element-specific detection of ICPMS, quantitative analysis of As(III), As(V), monomethylarsonic acid, and dimethylarsinic acid was achieved. Optimization of CE separation conditions resulted in the use of 20 mmol L-1 sodium borate with 2% osmotic flow modifier (pH 9.0) and -20 kV applied potential for baseline resolution of each arsenic species in the shortest time. Hydride generation conditions were optimized through multiple electrophoretic separation analyzes with 5% HCl and 3% NaBH4 (in 0.2% NaOH) determined to be the optimum conditions. After completion of system optimization, detection limits obtained for the arsenic species were less than 40 ng L-1 with electromigration time precision less than 1% within a total analysis time of 9.0 min. Finally, the interface was used for speciation analysis of arsenic in river and tap water samples.

"Determination Of Methylmercury In Environmental Matrixes By On-Line Flow Injection And Atomic Fluorescence Spectrometry"
Anal. Chem. 2004 Volume 76, Issue 23 Pages 7131-7136
Chun-Mao Tseng, Chad R. Hammerschmidt and William F. Fitzgerald

Abstract: The precision and bias of monomethylmercury (MMHg) determinations in environmental samples can be improved by directly coupling and automating the numerous steps involved with analysis of this toxic Hg species. We developed a simple and robust mercury speciation analyzer (MSA) for measurement of MMHg in environmental matrixes. This on-line hyphenated system couples the main analytical steps, including sample introduction, aqueous-phase ethylation, Tenax pre-concentration, and gas chromatographic separation, to cold vapor atomic fluorescence detection and data acquisition. Here we describe the MMHg-MSA, present results of laboratory optimization and performance tests, and compare the reproducibility between dual analytical channels. With alternating sample concentration and analysis, a dual-channel system permits six high-accuracy MMHg determinations per hour. Additional advantages compared to the traditional manual method include ease of operation and high precision (<5% relative standard deviation). The MSA is applicable to the determination of MMHg in various environmental matrixes, and it can be fully automated. This method was validated by analysis of MMHg in certified reference materials of sediment and biological tissue. Estimated detection limits for MMHg with the MSA are ~0.01 ng g-1 for a 0.1-g sample of dry sediment or fish and ~0.01 ng L-1 for 0.15 L of water.

"Phase-Shift Fiber-Loop Ring-Down Spectroscopy"
Anal. Chem. 2004 Volume 76, Issue 22 Pages 6594-6599
Zhaoguo Tong, Alexander Wright, Theresa McCormick, Runkai Li, Richard D. Oleschuk and Hans-Peter Loock

Abstract: Fiber-loop ring-down spectroscopy (FLRDS) is a recently developed absorption spectroscopic technique suitable for very small liquid samples. It is based on measurements of the optical decay constant of laser intensity in a loop made of optical waveguide material. This decay constant changes as small liquid samples containing absorbing species are introduced into the loop. In this report, it is demonstrated that one can also obtain the optical decay constant using a continuous wave laser beam that is intensity modulated and then coupled into an optical fiber loop. The inherent exponential decay in the fiber loop introduces a phase shift of the light emitted from the loop with respect to the pumping beam. By measuring this phase shift, one can readily determine the concentration of the analyte introduced between the two fiber ends and a model is established to describe the relationship. It is demonstrated that this technique, dubbed phase-shift fiber-loop ring-down spectroscopy (PS-FLRDS), is well suited as an absorption detector for any flow system in which the optical absorption path is limited by the instrument architecture. By measuring the phase angle as a function of concentration of 1,1'-diethyl-4,4'-dicarbocyanine iodide in dimethyl sulfoxide, the detection limit was determined as ~6 µM for a 30-40-µm absorption path. A temporal resolution of ~100 ms was demonstrated by a rapid displacement of the solutions between the two fiber ends. Proof-of-principle use of the PS-FLRDS detection in capillary flow systems using a commercial four-way microcross established that the alignment of the fiber and the capillary can be made simple and effective, while retaining both a low detection limit and a fast response.

"Micromixing With Linked Chains Of Paramagnetic Particles"
Anal. Chem. 2004 Volume 76, Issue 21 Pages 6448-6455
Sibani Lisa Biswal and Alice P. Gast

Abstract: Paramagnetic colloidal particles aggregate into linear chains under an applied external magnetic field. These particles can be chemically linked to create chains that can be magnetically actuated to manipulate microscopic fluid flow. The flexibility of the chain can be adjusted by varying the length of the linker molecule. In this paper, we describe the use of a suspension of linked paramagnetic chains in a rotating magnetic field to perform microscale mixing. The effect of chain rotation and flexibility on the diffusion of molecules is studied by observing the mixing of an acid and base in a microchannel. We show that, as the chain rotation frequency increases, there is marked increase in the effective mixing between fluid streams; however, a maximum frequency exists and above this frequency the chains are no longer effective in mixing. More flexible chains are more effective at mixing over a larger range of frequencies.

"On-Line Coupling Of Microdialysis Sampling With Microchip-Based Capillary Electrophoresis"
Anal. Chem. 2004 Volume 76, Issue 21 Pages 6440-6447
Bryan H. Huynh, Barbara A. Fogarty, R. Scott Martin and Susan M. Lunte

Abstract: Microdialysis sampling is a technique that has been used for in vivo and in vitro monitoring of compounds of pharmaceutical, biomedical, and environmental interest. The coupling of a commercially available microdialysis probe to a microchip-based capillary electrophoresis (CE) system is described. A continuously flowing dialysate stream from a microdialysis probe was introduced into the microchip, and discrete injections were achieved using a valveless gating approach. The effect of the applied voltage and microdialysis flow rate on device performance was investigated. It was found that the peak area varied linearly with the applied voltage. Higher voltages led to lower peak response but faster separations. Perfusion flow rates of 0.8 and 1.0 µL/min were found to provide optimal performance. The on-line microdialysis/microchip CE system was used to monitor the hydrolysis of fluorescein mono-β-D-galactopyranoside (FMG) by β-D-galactosidase. A decrease of the FMG substrate with an increase in the fluorescein product was observed. The temporal resolution of the device, which is dependent on the CE separation time, was 30 s. To the best of our knowledge, this is the first reported coupling of a microdialysis sampling probe to a microchip capillary electrophoresis device.

"Near-Simultaneous And Real-Time Detection Of Multiple Analytes In Affinity Microcolumns"
Anal. Chem. 2004 Volume 76, Issue 21 Pages 6266-6273
Menake E. Piyasena, Tione Buranda, Yang Wu, Jinman Huang, Larry A. Sklar and Gabriel P. Lopez

Abstract: A miniaturized immunoassay system based on beads in poly(dimethylsiloxane) micro-channels for analyzing multiple analytes has been developed. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer. A preliminary account of this work based on single-analyte format has been published in this journal (Buranda, T.; Huang, J.; Perez-Luna, V. H.; Schreyer, B.; Sklar, L. A.; Lopez, G. P. Anal. Chem. 2002, 74, 1149-1156). We have extended the work to a multianalyte model system composed of discrete segments of beads that bear distinct receptors. Near-simultaneous and real-time detection of diverse analytes is demonstrated. The importance of this work is established in the exploration of important factors related to the design, assessment, and utility of affinity micro-column sensors. First, beads derivatized with surface chemistry suitable for the attachment of fluorescently labeled biomolecules of interest are prepared and characterized in terms of functionality and receptor site densities by flow cytometry. Second, calibrated beads are incorporated in microfluidic channels. The analytical device that emerges replicates the basic elements of affinity chromatography with the advantages of microscale and real-time direct measurement of bound analyte on beads rather than the indirect determination from eluted sample typical of affinity chromatography. In addition, the two-compartment analysis of the assay data as demonstrated in single-analyte columns provides a template upon which the dynamics of multiple-analyte assays can be characterized using existing theoretical models and be tested experimentally. The assay can potentially detect subfemtomole quantities of protein with high signal-to-noise ratio and a large dynamic range spanning nearly four orders of magnitude in analyte concentration in microliter to submicroliter volumes of analyte fluid. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes (e.g., biomolecular indicators of deseases). Proof-of-principle analytes include FLAG peptide and carcinoembryonic antigen detected at physiologically relevant concentration levels.

"Continuous Flow Microfluidic Device For Rapid Erythrocyte Lysis"
Anal. Chem. 2004 Volume 76, Issue 21 Pages 6247-6253
Palaniappan Sethu, Melis Anahtar, Lyle L. Moldawer, Ronald G. Tompkins and Mehmet Toner

Abstract: Leukocyte isolation from whole blood to study inflammation requires the removal of contaminating erythrocytes. Leukocytes, however, are sensitive to prolonged exposure to hyper/hypoosmotic solutions, temperature changes, mechanical manipulation, and gradient centrifugation. Even though care is taken to minimize leukocyte activation and cell loss during erythrocyte lysis, it is often not possible to completely avoid it. Most procedures for removal of contaminating erythrocytes from leukocyte preparations are designed for bulk processing of blood, where the sample is manipulated for longer periods of time than necessary at the single-cell level. Ammonium chloride-mediated lysis is the most commonly used method to obtain enriched leukocyte populations but has been shown to cause some activation and selective loss of certain cell types. The leukocyte yield and subsequent activation status of residual leukocytes after NH4Cl-mediated lysis have been shown to depend on the time of exposure to the lysis buffer. We have developed a microfluidic lysis device that deals with erythrocyte removal at nearly the single-cell level. We can achieve complete lysis of erythrocytes and ~100% recovery of leukocytes where the cells are exposed to an isotonic lysis buffer for less than 40 s, after which the leukocytes are immediately returned to physiological conditions. Theoretically, this process can be made massively parallel to process several milliliterss of whole blood to obtain a pure leukocyte population in less than 15 min.

"Implantable Flow-Through Capillary-Type Microdialyzers For Continuous In Situ Monitoring Of Environmentally Relevant Parameters"
Anal. Chem. 2004 Volume 76, Issue 19 Pages 5974-5981
Manuel Mir&oacute; and Wolfgang Frenzel

Abstract: In this paper, a simple, flexible, and cost-effective flow-through microdialyzer hyphenated with a miniaturized differential potentiometric detector is proposed for continuous diffusion-controlled sampling of analytes of environmental interest. The analytical performance of the dedicated configuration involving merely a single cellulose regenerated hollow fiber is critically compared with that of commercially available concentric probes commonly exploited for in vivo monitoring of the extracellular space in living tissues and that of large dialysis-based probes furnished with flat membranes. The outstanding feature of the capillary-type design is the ability of adapting the extraction fractions (EF) to the requirements of the assays and flow-through detectors by selection of appropriate membrane length/perfusion rate ratios. Passive sampling under steady-state conditions (EF ~ 100%) has proven feasible for environmentally relevant ions, such as chloride, by perfusing a 3-cm-long capillary with water at a flow rate of 2.0 µL/min. Hence, there is no need for recalibration of the flow setup after implantation of the purpose-made probe. The effect of physical and chemical variables on the diffusive flux is discussed in detail for the various flow-through membrane separation devices assessed. Effective means to attain identical dialysate concentrations of target species under dynamic regime irrespective of the matrix ingredients are also presented. The dedicated microdialyzer features extreme tolerance to high molecular weight interfering matrix compounds ([ge]5000 mg/L humic acid) at the 5% interference level, which makes it especially suited for the interference-free potentiometric determination of ionic species in environmental samples containing high levels of organic matter. The potentials of the membrane separation unit were assessed for continuous monitoring of chemical changes in the interstitial/pore water of organic soils via stimulus-response strategies.

"Online CE-MALDI-TOF MS Using A Rotating Ball Interface"
Anal. Chem. 2004 Volume 76, Issue 19 Pages 5968-5973
Harrison K. Musyimi, Damien A. Narcisse, Xia Zhang, Wieslaw Stryjewski, Steven A. Soper and Kermit K. Murray

Abstract: We report on the construction and performance of a rotating ball interface for online coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption ionization (MALDI) mass spectrometry with a time-of-flight (TOF) mass analyzer. The interface is based on a rotating stainless steel ball that transports samples from atmospheric pressure to the high vacuum of the mass spectrometer for desorption and ionization. The sample is deposited directly from a 50-µm-i.d. separation capillary onto the 19-mm ball that is rotating at 0.03 to 0.3 rpm. The sample is mixed online with matrix flowing from a separate 50-µm-i.d. capillary. The sample deposit dries before it is rotated past a polymer gasket and into the laser ionization region. Cleaning of the interface is accomplished using solvent-saturated felt, which cleans the ball surface after it rotates out of the ionization chamber. On-line CE-MALDI is demonstrated, and the performance is evaluated with the analysis of a mixture of three peptides: [Lsy8] vasopressin, substance P, and neurotensin. The rotating ball interface to MALDI-TOF MS demonstrated mass detection limit in the high femtomole range. The interface has negligible memory effect and shows no significant electrophoretic peak broadening when operated under optimized conditions.

"Bonding Of Glass Microfluidic Chips At Room Temperatures"
Anal. Chem. 2004 Volume 76, Issue 18 Pages 5597-5602
Zhi-Jian Jia, Qun Fang, and Zhao-Lun Fang

Abstract: 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/cm2. 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.

"Preparation, Characterization, And Application Of An Enzyme-Immobilized Magnetic Microreactor For Flow Injection Analysis"
Anal. Chem. 2004 Volume 76, Issue 18 Pages 5498-5502
Akira Nomura, Shigemitsu Shin, Othman Oulad Mehdi and Jean-Michel Kauffmann

Abstract: Enzyme-immobilized magnetic microparticles (EMMP) have been prepared for use as a microreactor in flow injection analysis (FI). The microparticles were directly injected into the FI system. Their retention occurred within the flow line by small permanent magnets located near the detector. The analytical utility of this concept was illustrated by the assay of glucose using glucose oxidase (GOx), immobilized microparticles, and amperometric detection of liberated hydrogen peroxide. The microparticles were derived from silica gel (nominal pore diameter, 15-80 nm) by impregnation with a citric acid/ethanol solution and a ferric nitrate/ethanol solution and then by calcination in a nitrogen atmosphere to produce ferrimagnetic fine particles of spinel-type iron oxide (-Fe2O3) inside the pore. They were characterized by X-ray diffraction. The calibration curve of the glucose sample (2 L injected) was linear between 2.5 x 10^-6 and 5 x 10^-4 mol/L (R = 0.9995), and the detection limit was 1.0 x 10^-6 mol/L or 0.36 ng of injected glucose (S/N = 3). The repeatability for a 5 x 10^-4 mol/L glucose solution was RSD = 1.5% (n = 6). Application to the assay of glucose in a fermentation broth is illustrated. The GOx MMP were stable and active for more than eight months when kept at 10°C.

"Quantitative On-Line Monitoring Of Cellular Glucose And Lactate Metabolism In Vitro With Slow Perfusion"
Anal. Chem. 2004 Volume 76, Issue 18 Pages 5431-5435
Gea Leegsma-Vogt, Kor Venema, Nieske Brouwer, Jan Bert Gramsbergen, Sjef Copray and Jakob Korf

Abstract: An on-line in vitro perfusion technique is described that allows the continuous quantification of cellular glucose metabolism in vitro. Using biosensor technology, we measure glucose and lactate metabolism at a minute-to-minute time resolution for periods up to several days. The application of our perfusion-detection technique for in vitro monitoring is demonstrated in a wide variety of cells, including primary neuronal and astroglia cultures, yeast cells, and human lymphocytes. The method shows that variations in oxygen delivery or exposure to a noncompetitive pseudosubstrate (here 2-deoxyglucose) affects normal glucose metabolism. The innovative advantage of the present system is that, in contrast to other devices including a recently described system, metabolism per cell can be quantified. The potential of in vitro on-line monitoring is discussed for application in studying normal and abnormal metabolism, toxic and nontoxic drug effects, and human tissue biopsies.

"Microfluidic T-Form Mixer Utilizing Switching Electroosmotic Flow"
Anal. Chem. 2004 Volume 76, Issue 18 Pages 5265-5272
Che-Hsin Lin, Lung-Ming Fu and Yu-Sheng Chien

Abstract: This paper presents a microfluidic T-form mixer utilizing alternatively switching electroosmotic flow. The microfluidic device is fabricated on low-cost glass slides using a simple and reliable fabrication process. A switching DC field is used to generate an electroosmotic force which simultaneously drives and mixes the fluid samples. The proposed design eliminates the requirements for moving parts within the microfluidic device and delicate external control systems. Two operation modes, namely, a conventional switching mode and a novel pinched switching mode, are presented. Computer simulation is employed to predict the mixing performance attainable in both operation modes. The simulation results are then compared to those obtained experimentally. It is shown that a mixing performance as high as 97% can be achieved within a mixing distance of 1 mm downstream from the T-junction when a 60 V/cm driving voltage and a 2-Hz switching frequency are applied in the pinched switching operation mode. This study demonstrates how the driving voltage and switching frequency can be optimized to yield an enhanced mixing performance. The novel methods presented in this study provide a simple solution to mixing problems in the micro-total-analysis-systems field.

"Microfluidic Shear Devices For Quantitative Analysis Of Cell Adhesion"
Anal. Chem. 2004 Volume 76, Issue 18 Pages 5257-5264
Hang Lu, Lily Y. Koo, Wechung M. Wang, Douglas A. Lauffenburger, Linda G. Griffith and Klavs F. Jensen

Abstract: We describe the design, construction, and characterization of microfluidic devices for studying cell adhesion and cell mechanics. The method offers multiple advantages over previous approaches, including a wide range of distractive forces, high-throughput performance, simplicity in experimental setup and control, and potential for integration with other microanalytical modules. By manipulating the geometry and surface chemistry of the microdevices, we are able to vary the shear force and the biochemistry during an experiment. The dynamics of cell detachment under different conditions can be captured simultaneously using time-lapse videomicroscopy. We demonstrate assessment of cell adhesion to fibronectin-coated substrates as a function of the shear stress or fibronectin concentration in microchannels. Furthermore, a combined perfusion-shear device is designed to maintain cell viability for long-term culture as well as to introduce exogenous reagents for biochemical studies of cell adhesion regulation. In agreement with established literature, we show that fibroblasts cultured in the combined device reduced their adhesion strength to the substrate in response to epidermal growth factor stimulation.

"Selective Detection Of Proteins In Mixtures Using Electrospray Ionization Mass Spectrometry: Influence Of Instrumental Settings And Implications For Proteomics"
Anal. Chem. 2004 Volume 76, Issue 17 Pages 5024-5032
Seetharaman Vaidyanathan, Douglas B. Kell and Royston Goodacre

Abstract: We studied the effects of electrospray mass spectrometric instrumental settings on the relative and absolute detection of individual proteins in a five-component mixture. Conditions that were effective for a given protein could be very poor for the others, and vice versa, such that to a good approximation it was possible to find conditions for selective detection of individual proteins in a complex mixture without prior analytical separation. Some of these could be rationalized on the basis of the known biophysical properties of the individual proteins. The ability to vary the conditions of a mass spectrometric detection method on-line provides an important degree of freedom for the selective detection, and hence discrimination, of individual proteins and peptides in complex mixtures and has implications in proteomics, in particular with respect to top-down strategies for proteomic characterizations.

"Parameters Influencing Pulsed Flow Mixing In Microchannels"
Anal. Chem. 2004 Volume 76, Issue 16 Pages 4825-4832
Ian Glasgow, Samuel Lieber and Nadine Aubry

Abstract: The rapid mixing of reagents is a crucial step for on-chip chemical and biological analysis. It has been recently suggested that microfluidic mixing can be greatly enhanced by simply using time pulsing of the incoming flow rates of the two fluids to be mixed (Glasgow, I.; Aubry, N. Lab Chip 2003, 3, 114-120). This paper elaborates on the latter technique, showing through computational fluid dynamics how the mixing efficiency strongly depends on certain dimensionless parameters of the system, while remaining nearly insensitive to others. In particular, it is demonstrated that higher Strouhal numbers (ratio of flow characteristic time scale to the pulsing time period) and pulse volume ratios (ratio of the volume of fluid pulsed to the volume of inlet/outlet intersection) lead to better mixing. This paper also presents a physical device capable of mixing two reagents using pulsing, which shows improved mixing with greater values of the Strouhal number.

"Developments Toward A Microfluidic System For Long-Term Monitoring Of Dynamic Cellular Events In Immobilized Human Cells"
Anal. Chem. 2004 Volume 76, Issue 16 Pages 4715-4720
Richard Davidsson, &Aring;ke Boketoft, Jesper Bristulf, Knut Kotarsky, Bj&ouml;rn Olde, Christer Owman, Martin Bengtsson, Thomas Laurell and Jenny Emn&eacute;us

Abstract: A microfluidic system for long-term real-time monitoring of dynamic cellular events of immobilized human cells was investigated. The luciferase reporter gene activity in the reporter cell line HFF11, based on HeLa cells, was used as the model system. The cells were immobilized on silicon flow-through microchips and continuously supplied with a cell medium at 2 µL/min while maintaining the chip at 37°C. The HFF11 cell line was designed for high-throughput screening of ligands for seven-transmembrane receptors. When a ligand binds, the receptor is activated and a cascade of intracellular reactions starts, ending with the synthesis of the reporter protein Photinus luciferase. The major goal was to develop a microfluidic system for continuous long-term assaying of the intracellular reporter gene activity in real time and determine the conditions, which could minimize cells stress and hence unspecific expression of the reporter gene. In the resulting microfluidic system and assay protocol, the cell microchip could be kept and assayed for a period up to 30 h. The developed system and data outcome was compared with a corresponding microtiter plate performed with the same cell line to highlight the advantages obtained in the microfluidic format.

"Biosensor Based On Cellobiose Dehydrogenase For Detection Of Catecholamines"
Anal. Chem. 2004 Volume 76, Issue 16 Pages 4690-4696
Leonard Stoica, Annika Lindgren-Sj&ouml;lander, Tautgirdas Ruzgas and Lo Gorton

Abstract: A cellobiose dehydrogenase (CDH)-modified graphite electrode was designed for amperometric detection of catecholamines in the flow injection mode, by their recycling between the graphite electrode (+300 mV vs Ag[verbar]AgCl) and the reduced FAD cofactor of adsorbed CDH, resulting in an amplified response signal. The high efficiency of the enzyme-catecholamine reaction leads to a detection limit below 1 nM and a sensitivity of 15.8 A M-1 cm-2 (~1150 nA/µM) for noradrenaline, with a coverage of less than 2.5 µg of CDH adsorbed on the electrode surface (0.073 cm2). Working parameters such as pH, cellobiose concentration, carrier buffer, and applied potential were optimized, using hydroquinone as a model analyte. The sensitivity, linear range, and amplification factor can be modulated by the steady-state concentration of cellobiose in the flow buffer. The response of the sensor decreases only 2% when run continuously for 4 h in the flow injection mode. The response peak maximum is obtained within 6 s at a flow rate of 0.5 mL/min, representing the time of the entire sample segment to pass the electrode. CDH enzymes from Phanerochaete chrysosporium and Sclerotium rolfsii were investigated, providing different characteristics of the sensor, with sensors made with CDH from P. chrysosporium being the better ones.

"A Flow Injection Kinase Assay System Based On Time-Resolved Fluorescence Resonance Energy-Transfer Detection In The Millisecond Range"
Anal. Chem. 2004 Volume 76, Issue 15 Pages 4292-4298
Junko Hirata, Camiel F. de Jong, Maarten M. van Dongen, Joost Buijs, Freek Ariese, Hubertus Irth and Cees Gooijer

Abstract: A flow injection analysis (FIA) system for biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) in the millisecond time scale was developed. As a model system, we studied a kinase assay, measuring the phosphorylation of poly(GT)-biotin (substrate) by a receptor tyrosine kinase (epidermal growth factor receptor). A streptavidin labeled with XL665 (SA-XL665)-the acceptor-was coupled to the biotin moiety, and an antiphosphotyrosine antibody labeled with europium cryptate (Ab-EuK)-the donor-was coupled to the phosphorylated tyrosine group(s). Long-lived FRET can only occur if the substrate is successfully phosphorylated. For the time-resolved detection of such long-lived luminescence phenomena in a flow system, the repetition rate of the excitation source plays a crucial role. Good results were obtained for a small-sized commercially available quadrupled Nd:YAG laser emitting at 266 nm with a repetition rate of 7.8 kHz and a pulse width of 0.3 ns. The long-lived emissions of the donor at 625 nm and that of the acceptor at 665 nm were monitored simultaneously with two photomultipliers, using a delay time of 50 µs and a gate time of 75 µs to exclude background fluorescence interferences. In the FIA experiments, the Ab-EuK concentration was 6 nM and the substrate concentration and SA-XL665 concentrations were 7 nM. By monitoring the intensity changes at 625 and 665 nm, the inhibition of tyrosine kinase by tyrphostin AG1478 was studied and an IC50 value of 5.1 ± 0.4 nM obtained.

"Visible And Near-Infrared Spectroscopy As A Technique For Screening The Inorganic Arsenic Content In The Red Crayfish (Procambarus clarkii Girard)"
Anal. Chem. 2004 Volume 76, Issue 14 Pages 3893-3898
Rafael Font, Mercedes Del R&iacute;o-Celestino, Dinoraz V&eacute;lez, Antonio De Haro-Bail&oacute;n and Rosa Montoro

Abstract: The potential of near-infrared spectroscopy (NIRS) for screening the inorganic arsenic (i-As) content in the red crayfish (Procambarus clarkii Girard 1852) was assessed. Sixty-two samples belonging to this species were freeze-dried and scanned by NIRS. The i-As contents of the samples were obtained by acid digestion-solvent extraction followed by hydride generation atomic absorption spectrometry and were regressed against different spectral transformations by modified partial least-squares (MPLS) regression. Second derivative transformation equations of the raw optical data, previously standardized by applying standard normal variate and de-trending algorithms, resulted in a coefficient of determination in the cross-validation (1-VR) of 0.84, indicative of equations of good quantitative information. The standard error of cross-validation to standard deviation ratio, shown by the second derivative equation, was similar to those obtained for other trace metal calibrations reported in NIRS reflectance. Spectral information related to chromophores and lipids of the red crayfish tissues, and also the plant matter contained in their stomachs, were the main organic components used by MPLS for modeling the selected prediction equation. This pioneering use of NIRS to predict the i-As content in red crayfish represents an important savings in time and cost of analysis.

"Microwave-assisted Sample Treatment In A Fully Automated Flow-based Instrument: Oxidation Of Reduced Technetium Species In The Analysis Of Total Technetium-99 In Caustic Aged Nuclear Waste Samples"
Anal. Chem. 2004 Volume 76, Issue 14 Pages 3869-3877
Oleg B. Egorov, Matthew J. O'Hara, and Jay W. Grate

Abstract: An automated flow-based instrument for microwave-assisted treatment of liquid samples has been developed and characterized. The instrument utilizes a flow-through reaction vessel design that facilitates the addition of multiple reagents during sample treatment and removal of the gaseous reaction products and enables quantitative removal of liquids from the reaction vessel for carryover-free operations. Matrix modification and speciation control chemistries that are required for the radiochemical determination of total 99Tc in caustic aged nuclear waste samples have been investigated. A rapid and quantitative oxidation procedure using peroxydisulfate in acidic solution was developed to convert reduced technetium species to pertechnetate in samples with high content of reducing organics. The effectiveness of the automated sample treatment procedures has been validated in the radiochemical analysis of total 99Tc in caustic aged nuclear waste matrixes from the Hanford site.

"Attenuated Total Internal Reflectance Infrared Microspectroscopy As A Detection Technique For Capillary Electrophoresis"
Anal. Chem. 2004 Volume 76, Issue 13 Pages 3826-3832
Brian M. Patterson, Neil D. Danielson and Andr&eacute; J. Sommer

Abstract: A novel detector for capillary electrophoresis (CE) using single-bounce attenuated total internal reflectance (ATR) Fourier transform infrared (FT-IR) microspectroscopy is presented. The terminus of the CE capillary is placed ~1 µm from the internal reflectance crystal at the focus of an ATR infrared microscope. Using pressure driven flow injection, concentration and volume detection limits have been determined for 25- and 10-µm-i.d. silica capillaries. Upon injection of 820 pL of succinylcholine chloride in a 10-µm capillary, a concentration detection limit of ~0.5 parts per thousand (ppt), or 410 pg, is found. The injection volume detection limit using a 108 ppt solution is 2.0 pL (216 pg). Sample separations using a programmed series of pressure, voltage, and again pressure on 25-, 50-, and 75-µm-i.d. capillaries are shown. CE separations of citrate and nitrate, as well as succinylcholine chloride with sodium salicylate using acetone as a neutral marker, are demonstrated. Several advantages of this CE-FT-IR technique include: (1) minimization of post-column broadening as a result of a small detector volume; (2) the ability to signal average spectra of the same aliquot, thereby improving the signal-to-noise in a stopped-flow environment; and (3) simplicity of design.

"Ultrasonic Mixing In Microfluidic Channels Using Integrated Transducers"
Anal. Chem. 2004 Volume 76, Issue 13 Pages 3694-3698
Goksen G. Yaralioglu, Ira O. Wygant, Theodore C. Marentis and Butrus T. Khuri-Yakub

Abstract: This paper presents a microfluidic mixer that uses acoustic stirring created by ultrasonic waves. The ultrasound is introduced into the channel by integrated piezoelectric transducers. The transducers are made of a zinc oxide thin film, which is deposited on the bottom surface of a quartz substrate. The poly(dimethylsiloxane) channel is aligned to the transducers on the top surface of the substrate. The transducers are designed for operation around 450 MHz. The main mechanism of the mixing is the acoustic stirring of the fluid perpendicular to the flow direction. The radiation pressure that is generated by the transducer causes the stirring inside the microfluidic channel. The performance of the mixer is characterized by mixing phenolphthalein solution and sodium hydroxide dissolved in ethyl alcohol. Flow rates on the order of 1-100 µL/min are used. The transducers are driven by 1.2 Vrms sinusoidal voltages at 450 MHz.

"A Soft Lithographic Approach To Fabricate Patterned Microfluidic Channels"
Anal. Chem. 2004 Volume 76, Issue 13 Pages 3675-3681
Ali Khademhosseini, Kahp Y. Suh, Sangyong Jon, George Eng, Judy Yeh, Guan-Jong Chen and Robert Langer

Abstract: The control of surface properties and spatial presentation of functional molecules within a microfluidic channel is important for the development of diagnostic assays and microreactors and for performing fundamental studies of cell biology and fluid mechanics. Here, we present a simple technique, applicable to many soft lithographic methods, to fabricate robust microchannels with precise control over the spatial properties of the substrate. In this approach, the patterned regions were protected from oxygen plasma by controlling the dimensions of the poly(dimethylsiloxane) (PDMS) stamp and by leaving the stamp in place during the plasma treatment process. The PDMS stamp was then removed, and the microfluidic mold was irreversibly bonded to the substrate. The approach was used to pattern a nonbiofouling poly(ethylene glycol)-based copolymer or the polysaccharide hyaluronic acid within microfluidic channels. These nonbiofouling patterns were then used to fabricate arrays of fibronectin and bovine serum albumin as well as mammalian cells. In addition, further control over the deposition of multiple proteins onto multiple or individual patterns was achieved using laminar flow. Also, cells that were patterned within channels remained viable and capable of performing intracellular reactions and could be potentially lysed for analysis.

"Development Of An Automated Sample Preparation Module For Environmental Monitoring Of Biowarfare Agents"
Anal. Chem. 2004 Volume 76, Issue 13 Pages 3492-3497
Benjamin J. Hindson, Steve B. Brown, Graham D. Marshall, Mary T. McBride, Anthony J. Makarewicz, Dora M. Gutierrez, Duane K. Wolcott, Thomas R. Metz, Ramakrishna S. Madabhushi, John M. Dzenitis and Billy W. Colston, Jr.

Abstract: An automated sample preparation module, based upon sequential injection analysis (SIA), has been developed for use within an autonomous pathogen detection system. The SIA system interfaced aerosol sampling with multiplexed microsphere immunoassay-flow cytometric detection. Metering and sequestering of microspheres using SIA was found to be reproducible and reliable, over 24-h periods of autonomous operation. Four inbuilt immunoassay controls showed excellent immunoassay and system stability over five days of unattended continuous operation. Titration curves for two biological warfare agents, Bacillus anthracis and Yersinia pestis, obtained using the automated SIA procedure were shown to be similar to those generated using a manual microtiter plate procedure.

"On-Line Coupling Of High-Performance Liquid Chromatography To A Continuous-Flow Enzyme Assay Based On Electrospray Ionization Mass Spectrometry"
Anal. Chem. 2004 Volume 76, Issue 11 Pages 3155-3161
Arjen R. de Boer, Thomas Letzel, Danny A. van Elswijk, Henk Lingeman, Wilfried M. A. Niessen and Hubertus Irth

Abstract: Liquid chromatography (LC) was coupled on-line to a continuous-flow enzymatic assay using electrospray ionization mass spectrometry (ESI-MS) as readout for the screening of enzyme inhibitors in complex samples. Inhibitors were detected by changes in the concentration of the enzymatic reaction products, indicating the inhibition of enzymatic activity. The molecular masses of the inhibitors were determined with high certainty by using retention time matching and peak shape comparison. Due to the high matching accuracy, baseline separation of coeluting analytes was not necessary in order to identify the correct masses of the bioactive compounds. The continuous-flow system was successfully applied for the screening of complex samples, such as natural extracts. For a red clover extract, detection limits of 0.3-0.8 mol/L were obtained. System validation was performed by determining the IC50 values of four inhibitors in the flow-injection mode. The IC50 values were in the 0.11-5.6 mol/L range and correspond closely to data obtained by microtiter plate assays. Detection limits were in the range of 0.018-0.35 mol/L in the flow-injection mode, and 0.075-0.75 mol/L in the LC mode. These values are well below the typical compound concentrations (1-10 mol/L) used in high-throughput screening. Together with an interday precision of 12.6%, these results demonstrate the applicability of the system for bioactivity screening of complex mixtures, generating both chemical and biological information on bioactive compounds in a single run.

"Spectroelectrochemical Sensing Based On Multimode Selectivity Simultaneously Achievable In A Single Device. 17. Improvement In Detection Limits Using Ultrathin Perfluorosulfonated Ionomer Films In Conjunction With Continuous Sample Flow"
Anal. Chem. 2004 Volume 76, Issue 11 Pages 3139-3144
Sara E. Andria, John N. Richardson, Necati Kaval, Imants Zudans, Carl J. Seliskar, and William R. Heineman

Abstract: We report herein an attenuated total reflectance (ATR) absorbance-based spectroelectrochemical sensor for tris(2,2?-bipyridyl)ruthenium(II) ion [Ru(bpy)32+] that employs ultrathin (24-50 nm) Nafion films as the charge-selective layer. This film serves to sequester and pre-concentrate the analyte at the optically transparent electrode surface such that it can be efficiently detected optically via electrochemical modulation. Our studies indicate that use of ultrathin films in tandem with continuous flow of sample solution through the cell compartment leads to a 100-500-fold enhancement in detection limit (10 nM) compared to earlier absorbance-based spectroelectrochemical sensors (?1-5 ?M); markedly shorter analysis times also result. We report the dependence of the measured absorbance on sample flow rate and Nafion film thickness, and also provide calibration curves that illustrate the linear range and detection limits of the sensor using a 24 nm film at a constant sample flow rate of 0.07 mL/min.

"Microflow NMR: Concepts And Capabilities"
Anal. Chem. 2004 Volume 76, Issue 10 Pages 2966-2974
Dean L. Olson, James A. Norcross, Mark O'Neil-Johnson, Paul F. Molitor, David J. Detlefsen, Aaron G. Wilson and Timothy L. Peck

Abstract: The principles and parameters to consider when choosing an NMR probe for analysis of a volume- or mass-limited sample are identified and discussed. In particular, a capillary-based microflow probe is described which has a mass sensitivity comparable to cryoprobes (observe volume ~40 µL), but with several distinct advantages. The microflow probe has a flowcell volume of 5 µL and an observe volume of 1.5 µL and is equipped with proton and carbon observe channels, deuterium lock, and z-gradient capability. The entire flow path is fused silica; inlet and outlet capillary inner diameters are 50 µm to minimize sample dispersion, making it well-suited to volume-limited samples. An injected sample of 1 nmol of sucrose (0.34 µg in 3 µL, 0.33 mM; MW = 342 g/mol) yields a 1D proton spectrum in 10 min on a spectrometer of 500 MHz or higher. In another example, 15 µg of sucrose (in 3 µL; 15 mM, 45 nmol) is injected and parked in the probe to yield a heteronuclear multiple-quantum coherence (HMQC) spectrum in less than 15 h. The natural product muristerone A (75 µg in 3 µL, 50 mM, 150 nmol; MW = 497 g/mol) was delivered to the flow cell, and a gradient correlation spectroscopy spectrum was acquired in 7 min, a gradient HMQC in 4 h, and a gradient heteronuclear multiple-bond correlation in 11 h. Four basic modes of sample injection into the probe vary in degree of user intervention, speed, solvent consumption, and sample delivery efficiency. Manual, manual-assisted (employing a micropump), automated (using an autosampler), and capillary HPLC modes of operation are described.

"Hyphenation Of Capillary HPLC To Microcoil 1H NMR Spectroscopy For The Determination Of Tocopherol Homologues"
Anal. Chem. 2004 Volume 76, Issue 9 Pages 2623-2628
Manfred Krucker, Annette Lienau, Karsten Putzbach, Marc David Grynbaum, Paul Schuler and Klaus Albert

Abstract: Highly selective reversed phases (C30 phases) are self-packed in 250 m inner diameter fused-silica capillaries and employed for capillary HPLC separation of shape-constrained natural compounds (tocopherol homologues, vitamin E). Miniaturized hyphenated systems such as capillary HPLC-ESI-MS (positive ionization mode) and, with special emphasis, continuous-flow capillary HPLC- NMR are used for structural determination of the separated compounds. Despite the small amount of sample available (1.33 g of each tocopherol), the authors have been able to monitor the capillary HPLC separation under continuous-flow 1H NMR conditions, thus allowing an immediate peak identification. Further structural assignment was carried out in the stopped-flow NMR mode as shown, for example, by a 2D 1H,1H COSY NMR spectrum of -tocopherol. We demonstrate in this paper the considerable potential of hyphenated capillary separations coupled to MS and NMR for the investigation of restricted amounts of sample.

"Real-Time, On-Line Characterization Of Diesel Generator Air Toxic Emissions By Resonance-Enhanced Multiphoton Ionization Time-of-Flight Mass Spectrometry"
Anal. Chem. 2004 Volume 76, Issue 9 Pages 2517-2524
Lukas Oudejans, Abderrahmane Touati and Brian K. Gullett

Abstract: The laser-based resonance-enhanced multiphoton ionization time-of-flight mass spectrometry (REMPI-TOFMS) technique has been applied to the exhaust gas stream of a diesel generator to measure, in real time, concentration levels of aromatic air toxics. Volatile organic compounds, as well as several polycyclic aromatic hydrocarbons were detected in the concentration range of 10^-200 ppb in the steady-state diesel generator exhaust. The results were verified and compared with conventional extractive sampling and analytical techniques using gas chromatography/mass spectrometry (GC/MS). The high isomer selectivity of the REMPI-TOFMS instrument provided data for individual xylene isomers that are otherwise (partially) coeluting in standard GC/MS analyzes. Good agreement was observed between results for volatile and semivolatile organic compounds obtained with REMPI-TOFMS and conventional extractive sampling. Transient events, such as cold start-ups of the diesel generator, resulted in sharp (less than 15 s) peak emissions that were, for benzene, up to a factor of 90 higher than the predominately constant concentrations observed during steady-state operation; warm restarts resulted in lower peak concentrations by a factor of 2.5. These fast transient emissions are only detectable using a real-time approach (1-s resolution) as demonstrated here using REMPI-TOFMS.

"Vapor Generation By UV Irradiation For Sample Introduction With Atomic Spectrometry"
Anal. Chem. 2004 Volume 76, Issue 8 Pages 2401-2405
Xuming Guo, Ralph E. Sturgeon, Zolt&aacute;n Mester, and Graeme J. Gardner

Abstract: Volatile species of the conventional hydride-forming elements (As, Bi, Sb, Se, Sn, Pb, Cd, Te), Hg, transition metals (Ni, Co, Cu, Fe), noble metals (Ag, Au, Rh, Pd, Pt), and nonmetals (I, S) were generated following UV irradiation of their aqueous solutions to which low molecular weight carboxylic acids (formic, acetic, propionic) had been added. Free radicals arising from photodissociation of the latter provide a new and useful alternative to the common methods of chemical/electrochemical vapor generation techniques for the determination of these analytes by atomic spectrometry. Quantitative estimates of the efficiencies of these generation processes were not undertaken, although calculated signal-to-background ratios (> 1500 for 5 ng/mL As, Sb, Bi, Se, and Te; 20 for 10 ng/mL Sn, Cu, Rh, Au, Pd, Pt, and Cd; 2400 for 1 ng/mL Hg; and 1000 for Co using ICP-TOF-MS detection) do provide clear evidence of the efficacy of this approach for sample introduction. In the case of Ni and Se, the tetracarbonyl and alkylated selenium compounds have been identified, respectively.

"Electropolymerized Flavin Adenine Dinucleotide As An Advanced NADH Transducer"
Anal. Chem. 2004 Volume 76, Issue 7 Pages 2004-2009
Arkady A. Karyakin, Yulia N. Ivanova, Ksenia V. Revunova and Elena E. Karyakina

Abstract: Electropolymerizing the prosthetic group (flavin adenine dinucleotide, FAD) responsible in the active sites of dehydrogenases for NAD+[verbar]NADH regeneration, we succeeded in mimicking enzyme activity. Poly(FAD) characterized by an additional polymer-type redox reaction has been discovered as a highly effective electrocatalyst for NADH oxidation: operating at the lowest potentials reported for NADH transducers (0.00 V, pH 7.4), poly(FAD) is characterized by the electrochemical rate constant of 1.8 ± 0.6 x 10^-3 cm s-1, which is at the level of the NADH mass-transfer constant. Flow injection analysis of NADH with the poly(FAD)-modified wall-jet electrode as a detector has been characterized by a linear calibration range prolonged down to 5 x 10^-7 M and a sensitivity of 0.08 A M-1 cm-2, which taking into account the dispersion coefficient (~3), is at the diffusion-limiting value. In contrast to the low molecular weight mediators able to exhibit similar electrocatalytic properties, poly(FAD)-modified electrodes are characterized by the dramatically improved stability and, thus, can be considered as the most advantageous NADH transducers for analytical chemistry.

"Patterning Enzymes Inside Microfluidic Channels Via Photoattachment Chemistry"
Anal. Chem. 2004 Volume 76, Issue 7 Pages 1838-1843
Matthew A. Holden, Seung-Yong Jung and Paul S. Cremer

Abstract: We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be ~5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.

"A Feasibility Study Of Solid Supported Enhanced Microdialysis"
Anal. Chem. 2004 Volume 76, Issue 6 Pages 1678-1682
Andreas Pettersson, Ardeshir Amirkhani, Bj&ouml;rn Arvidsson, Karin Markides, and Jonas Bergquist

Abstract: For the first time, a solid supported enhanced microdialysis methodology for analysis of neuropeptides is described. The microdialysis samples were, in this study, subsequently collected in fractions, dissolved from the solid particles, dried, and resolved in a formic acid buffer in order to make them suitable for capillary liquid chromatography-mass spectrometry. Different microdialysis flow profiles were evaluated where air-gapped continuous flow was considered most suitable for the solid supported microdialysis mode. Six endogenous neuropeptides were initially used to investigate the feasibility of this enhanced microdialysis methodology. The improved relative recovery obtained from the solid supported enhanced microdialysis was varying from no effect to 10 times higher as compared to ordinary microdialysis. The most efficient enrichment was obtained for luteinizing hormone releasing hormone, which was the largest but also the most hydrophilic of the peptides. In contrast, no significant difference in recovery was observed for Leu-enkephalin being the smallest and the most hydrophobic peptide tested. These results indicate an increased flux and selective uptake of hydrophilic peptides across the membrane and enrichment on the particles in solid supported microdialysis.

"On-Line Solid-Phase Extraction With Surfactant Accelerated On-Column Derivatization And Micellar Liquid Chromatographic Separation As A Tool For The Determination Of Biogenic Amines In Various Food Substrates"
Anal. Chem. 2004 Volume 76, Issue 5 Pages 1289-1294
E. K. Paleologos and M. G. Kontominas

Abstract: A sensitive method was developed for the determination of biogenic amines at very low levels by combining solid phase extraction (SPE) and derivatization on-line with HPLC. The on-line derivatization and SPE were performed simultaneously on a commercially available ODS guard column, which was installed instead of the filling loop on the HPLC apparatus. Resolution of the peaks and quantification was further enhanced with micellar liquid chromatography and sensitization of the benzene ring absorption at 254 nm. Detection limits of the benzoyl derivatives of biogenic amines were in the vicinity of 0.1 µg L-1, which is even lower than those obtained by fluorescence detection and is unparallel to any other UV approach. The correlation coefficients of determinations were 0.9850-0.9998. The method was applied to the determination of Biogenic amines, that is, putrescine, cadaverine, agmatine, tyramine, tryptamine, phenylethylamine, spermine, spermidine and histamine in fish, chicken, and wine samples. Recovery of the proposed method ranged from 94 to 106%.

"Automated Enzymatic Assays In A Renewable Fashion Using The Multisyringe Flow Injection Scheme With Soluble Enzymes"
Anal. Chem. 2004 Volume 76, Issue 3 Pages 773-780
Nicolau Piz&agrave;, Manuel Mir&oacute;, Jos&eacute; Manuel Estela and V&iacute;ctor Cerd&agrave;

Abstract: In this paper, a novel flowing stream scheme based upon the multisyringe flow injection (MSFI) technique is presented as a powerful tool to perform automated enzymatic assays. The exploitation of enzymes in homogeneous phase circumvents typical drawbacks associated with the commonly used packed-bead or open tubular permanent columns, namely, malfunctions of the reactor, carryover effects, flow resistance, loss of binding sites, large reagent consumption, and use of harmful organic solvents during immobilization procedures. The proposed MSFI system is able to handle minute volumes of soluble enzymes and accommodate reactions with divergent kinetic and pH demands, as demonstrated via the indirect chemiluminescence determination of trace levels of glucose. The procedure is based on the on-line glucose oxidase-catalyzed oxidation of β-glucose in homogeneous phase to β-glucono-δ-lactone and hydrogen peroxide. Subsequently, the generated oxidant merges downstream with an alkaline slug of 3-aminopthalhydrazide and a metal-catalyst zone (viz., Co(II)) at a total flow rate as high as 72 mL/min aiming to warrant maximum light collection from the fast CL reaction. Under optimum conditions for both sequentially occurring reactions, a glucose concentration as low as 90 µg/L may be easily detected at a 1000-fold photomultiplier gain. A second-order polynomial regression equation of light emission versus substrate concentration is found over the range 90 µg/L-2.7 mg/L glucose, although a maximum concentration of 180 mg/L may be determined by suitable gain selection without requiring manifold reconfiguration. An injection throughput of 20 h-1, a repeatability better than 2.5% at the 1 mg/L level, and a 3s detection limit of 72 µg/L are the analytical features of the designed analyzer. The proposed approach was applied to the analysis of ultralow glucose content soft drinks as well as fruit juices suitable for diabetic consumers. The accuracy was assessed using the spectrophotometric batch glucose-Trinder method as an external reference methodology for the determination of the target species in parenteral solutions.
Glucose Soft drink Fruit Chemiluminescence Multisyringe

"Analysis Of The Performance Of A Flow Reactor For Use With Microcolumn HPLC"
Anal. Chem. 2004 Volume 76, Issue 3 Pages 639-645
Amy T. Beisler, Eskil Sahlin, Kathleen E. Schaefer and Stephen G. Weber

Abstract: Post-column derivatization reactions can be used to improve detector sensitivity or selectivity. The advantages of capillary chromatography for trace analysis could be augmented if there were post-column reactors suitable for micro-chromatographic systems. However, post-column derivatization is a challenge because of the small peak volumes associated with capillary columns. We have developed a post-column flow reactor from micro-channels formed in fluorinated ethylene propylene and 50-µm fused-silica tubing for use with capillary HPLC analyzes. Theoretical and experimental evidence show that the reactor, which operates in the Taylor dispersion regime, enables contact of analyte and derivatization streams purely by diffusion. Reactor lengths as short as 2 cm allow formation of copper(II)-peptide complexes that are detected electrochemically at a carbon fiber micro-electrode. The reactor has been used with 100 µm i.d. columns with insignificant effects (i.e., <3%) on peak band spreading. Theoretical calculations indicate that even smaller i.d. columns can be used with little effect on chromatographic resolution.

"Prussian Blue Based Nanoelectrode Arrays For H2O2 Detection"
Anal. Chem. 2004 Volume 76, Issue 2 Pages 474-478
Arkady A. Karyakin, Elena A. Puganova, Igor A. Budashov, Ilya N. Kurochkin, Elena E. Karyakina, Vladimir A. Levchenko, Vladimir N. Matveyenko and Sergey D. Varfolomeyev

Abstract: We propose to form nanoelectrode arrays by deposition of the electrocatalyst through lyotropic liquid crystalline templates onto inert electrode support. Whereas Prussian Blue is known to be a superior electrocatalyst in hydrogen peroxide reduction, carbon materials used as electrode support demonstrate only a minor activity. We report on the possibility for nanostructuring of Prussian Blue by its electrochemical deposition through lyotropic liquid crystalline templates, which is noticed from atomic force microscopy images of the resulting surfaces. The resulting Prussian Blue based nanoelectrode arrays in flow injection analysis mode demonstrate a sub-part-per-billion detection limit (1 x 10^-8 M) and a linear calibration range starting exactly from the detection limit and extending over 6 orders of magnitude of H2O2 concentrations (1 x 10^-8 to 1 x 10^-2 M), which are the most advantageous analytical performances in hydrogen peroxide electroanalysis.

"Volatile Analytes Formed From Arsenosugars: Determination By HPLC-HG-ICPMS And Implications For Arsenic Speciation Analyses"
Anal. Chem. 2004 Volume 76, Issue 2 Pages 418-423
Ernst Schmeisser, Walter Goessler, Norbert Kienzl and Kevin A. Francesconi

Abstract: It is generally accepted that the use of the hydride generation method to produce volatile analytes from arsenic compounds is restricted to the two inorganic forms (As(III) and As(V)) and the three simple methylated species methylarsonate (MA), dimethylarsinate (DMA), and trimethylarsine oxide. We report here that arsenosugars, major arsenic compounds in marine organisms, produce volatile analytes by the hydride generation (HG) method without a prior mineralization/oxidation step and that they can be quantitatively determined using HPLC-HG-ICPMS. The hydride generation efficiency depends on the type of hydride generation system and is influenced by the concentration of HCl and NaBH4. For the four arsenosugars investigated, the hydride generation efficiencies were ~21-28% (or 4-6%, depending on the HG system) that obtained for As(III) under conditions optimized for As(III). This hydride efficiency was less than that shown by MA (~68% relative to As(III)) and DMA (~75%) but greater than that displayed by As(V) (~18%). Analysis of two species of brown algae, Fucus serratus and Hizikia fusiforme, by HPLC-HG-ICPMS produced results comparable with those obtained from other techniques used in our laboratory (HPLC-ICPMS and LC-ESMS for F. serratus) and with results from other laboratories taking part in a round robin exercise (H. fusiforme). This study shows for the first time the quantitative determination of arsenosugars using the hydride generation method without a decomposition step and has considerable implications for analytical methods for determining inorganic arsenic based on the formation of volatile hydrides.

"Fabrication Of A Spectrophotometric Absorbance Flow Cell Using Injection-Molded Plastic"
Anal. Chem. 2004 Volume 76, Issue 1 Pages 238-243
John P. Hulme, Peter R. Fielden, and Nicholas J. Goddard

Abstract: A microfluidic device with integrated spectrophotometric elements has been injection molded in poly(methyl methacrylate) (PMMA). The strategy for producing the device combined the direct photopatterning, replica molding techniques and a rapid cooled release mechanism in conjunction with material-material transfer to form a multilayer epoxy master on an injection mold insert. An injection-molded flow cell consisting of 1800 diffractive elements patterned on 25-?m-high PMMA pillars was produced. The design incorporated a large cross sectional surround, simplifying the alignment and bonding procedure. The absorbance range of one of the devices was standardized against a commercially available visible spectrophotometer at 605 nm to increasing concentrations of Nile blue perchlorate A dye in aqueous solution. The detection limit for a new integrated device was 1.2 x 10^-6 m/Lof the mentioned dye.

"Stabilization Of High-Resistance Seals In Patch-Clamp Recordings By Laminar Flow"
Anal. Chem. 2003 Volume 75, Issue 23 Pages 6718-6722
Jon Sinclair, Jessica Olofsson, Johan Pihl and Owe Orwar

Abstract: The formation of a high-resistance electrical seal between a cell membrane and a glass micropipet tip is essential in patch-clamp experiments. We have studied the electrical properties and the mechanical stability of the seal using a microfluidic chip generating laminar flow in open volumes. We show that, by using fluid flow (1-10 mm/s) acting along the symmetry axis of the cell-pipet, seals of a higher mechanical stability with increased resistances can be achieved, allowing up to 100% longer recording times and over 40% decreased noise levels (Irms). These improved properties are beneficial for high-sensitivity patch-clamp recordings, in particular, in longtime studies of ion channel receptor systems that are relevant in biosensor applications of the technique. Furthermore, these observations support the combination of patch-clamp with microfluidic devices, for example, for rapid solution exchange around a single cell sensor for high-throughput electrophysiology and for highly resolved kinetic studies.

"Speciation Of Dimethylarsinous Acid And Trimethylarsine Oxide In Urine From Rats Fed With Dimethylarsinic Acid And Dimercaptopropane Sulfonate"
Anal. Chem. 2003 Volume 75, Issue 23 Pages 6463-6468
Xiufen Lu, Lora L. Arnold, Samuel M. Cohen, William R. Cullen and X. Chris Le

Abstract: Speciation of arsenic in urine from rats treated with dimethylarsinic acid (DMAV) alone or in combination with dimercaptopropane sulfonate (DMPS) were studied. Methods were developed for the determination of the methylarsenic metabolites, especially trace levels of dimethylarsinous acid (DMAIII) and trimethylarsine oxide (TMAO), in the presence of a large excess of DMAV. Success was achieved by using improved ion-exchange chromatographic separation combined with hydride generation atomic fluorescence detection. Micromolar concentrations of DMAIII were detected in urine of rats fed with a diet supplemented with either 100 µg/g of DMAV or a mixture of 100 µg/g of DMAV and 5600 µg/g of DMPS. No significant difference in the DMAIII concentration was observed between the two groups; however, there was a significant difference in TMAO concentrations. Urine from rats fed with the diet supplemented with DMAV alone contained 73 ± 30 µM TMAO, whereas urine from rats fed with the diet supplemented with both DMAV and DMPS contained only 2.8 ± 1.4 µM TMAO. Solutions containing mixtures of 100 µg/L DMAV or TMAO and 5600 µg/L DMPS did not show reduction of DMAV and TMAO. The significant decrease (p < 0.001) of the TMAO concentration in rats administered with both DMAV and DMPS suggests that DMPS inhibits the biomethylation of arsenic.

"Digital Pulse AC Voltammetry For The Simultaneous Analysis Of Electroactive And Electrosorptive Species In Flow Systems"
Anal. Chem. 2003 Volume 75, Issue 22 Pages 6368-6373
Chur-Min Chang and Hsuan-Jung Huang

Abstract: A digital two-step and three-step pulse potential ac voltammetry system was proposed and applied for the simultaneous analysis of electrosorptive and electroactive species in flow systems. To perform the ac polarography function, a PC was interfaced to a potentiostat to mimic all the necessary hardware functions of an analog ac polarograph. From the measurement of the change of phase-selective charging current and the zero-order current, I3-, Br- specifically adsorbed and Cd2+, Pb2+ reduced at a hanging mercury drop electrode can be determined simultaneously in a FIA and IC system. With the digital pulse ac voltammetry-coupled IC, detection limits as low as 5.0 µM and linear dynamic ranges from 5.0 to 100 or 200 µM with linear correlation coefficients better than 0.9990 were found for the analysis of I3-, Br-, and S2O32-.

"Flow Injection Analysis Of An Ultratrace Amount Of Arsenite Using A Prussian Blue-Modified Screen-Printed Electrode"
Anal. Chem. 2003 Volume 75, Issue 21 Pages 6017-6022
Jyh-Myng Zen, Pei-Yan Chen and Annamalai Senthil Kumar

Abstract: We report here a new electrochemical method for the selective detection of ultratrace amount of arsenite (AsO2-, As3+) using a Prussian blue-modified screen-printed electrode (designated as PBSPE) by flow injection analysis (FIA) in 0.1 M, pH 4 KCl/HCl carrier solution. The Prussian yellow/Prussian blue redox couple of the PBSPE was found to mediate the As3+ oxidation. Various factors influencing the determination of As3+ were thoroughly investigated in this study. Under the optimized FIA conditions, a linear calibration plot in the range of 50 nM-300 µM with a detection limit (S/N = 3) of 25 nM (i.e., 64.9 pg in 20 µL loop) was observed at an operation potential of +0.6 V vs Ag/AgCl. The sensitivity was good enough to detect arsenite at levels lower than the current EPA standard. This modified electrode showed good resistance to interference from common ions, especially Cl-, which is generally considered as a major interference in the determination of As3+ by ICPMS. The practical utility of the PBSPE to detect As3+ was demonstrated in 'blackfoot' disease endemic village groundwater from the southwestern coast area of Taiwan (Pei-Men).

"Capillary Electrophoresis Coupled On-Line With Ultraviolet Resonance Raman Spectroscopy"
Anal. Chem. 2003 Volume 75, Issue 21 Pages 5697-5702
Reyer J. Dijkstra, Evtim V. Efremov, Freek Ariese, Udo A. Th. Brinkman and Cees Gooijer

Abstract: Capillary electrophoresis (CE) and resonance Raman spectroscopy (RRS) with excitation in the deep ultraviolet (UV) region (λex: 244 or 257 nm) were coupled on-line. The potential of this hyphenated technique, denoted as CE-UV-RRS, for analyte confirmation/identification purposes was explored with aromatic sulfonic acids and nucleotides as test compounds. Good-quality UV-RRS spectra could be recorded on-the-fly. Identification limits for the nucleotides were in the 10^-125 µg/mL range. The RRS spectra showed sufficient characteristic features to enable analyte confirmation. In addition, the identification power of UV-RRS was studied with substituted pyrenes as model compounds. The compounds were distinguishable on the basis of their RRS spectra at 244 nm.

"A Surface Acoustic Wave Biosensor Concept With Low Flow Cell Volumes For Label-Free Detection"
Anal. Chem. 2003 Volume 75, Issue 20 Pages 5561-5566
Kerstin L&auml;nge, Florian Bender, Achim Voigt, Hui Gao and Michael Rapp

Abstract: Special surface acoustic wave (SAW) devices using horizontally polarized surface shear waves can be operated in water. They allow an easy detection of molecules with biological relevance (e.g., proteins) via direct detection of the adsorbed mass. The transducer structures of conventional SAW devices are usually connected to the electronics by bond wires. In consequence, flow cell volumes can hardly be designed smaller than 50 µL. A new type of SAW device that is coupled capacitively with the electronics enables the reduction of flow cell volumes down to 60 nL, which decreases sample consumption and reduces the length of the measurement cycles down to a few minutes. To create an immunosensor, the SAW devices first are coated with a thin parylene layer for creating a sensor surface that is chemically homogeneous. Then OptoDex, a dextran containing both photoactive and functional groups is immobilized photochemically. Finally, antibodies are coupled via conventional EDC/NHS chemistry. The technique has been used to monitor urease binding at anti-urease-coated SAW devices in real time and with good resolution. Because of the simple sensor handling and the economical sample use, the new SAW device is particularly suitable for the design of an array.

"Chip-Based Solid-Phase Extraction Pretreatment For Direct Electrospray Mass Spectrometry Analysis Using An Array Of Monolithic Columns In A Polymeric Substrate"
Anal. Chem. 2003 Volume 75, Issue 20 Pages 5504-5511
Aimin Tan, Salete Benetton and Jack D. Henion

Abstract: An array of eight porous monolithic columns, prepared in a Zeonor polymeric chip by UV-initiated polymerization of butyl methacrylate and ethylene dimethacrylate, was tested for solid-phase extraction (SPE) cleanup of biological samples prior to directly coupled electrospray mass spectrometry (ESI-MS). The chip, fabricated by hot embossing and thermal bonding, consists of eight parallel channels (10 mm long, 360 µm i.d.) connected via external fused-silica capillaries. The monomer mixture was aspirated simultaneously into the eight channels using a homemade vacuum manifold device and polymerized in parallel for 20 min under UV irradiation. The porous monolithic columns were then characterized by scanning electron microscopy and evaluated by ESI-MS applications with respect to sample capacity, recovery, reproducibility of peak area or peak height ratios, and linearity between peak height ratio and concentration using imipramine as a pharmaceutical test compound. The average sample capacity was estimated to be 0.30 µg with a relative standard deviation (RSD) of 26.5% for the eight monolithic columns on the same polymeric chip. For two chips prepared using the same monomer mixture, the difference in average sample capacity was 7.0%. The average recovery for the eight monolithic SPE columns on the same chip was 79.1% with an RSD of 7.9%. Using imipramine-d3 as an internal standard, the RSD of peak height ratios for the eight different columns was 2.0% for a standard solution containing 1 µg/mL imipramine. A linear calibration curve (R2 = 0.9995) was obtained for standard aqueous solutions of imipramine in the range from 0.025 to 10 µg/mL. To demonstrate the analytical potential of the chip-based SPE system, two different types of real-world samples including human urine sample and P450 drug metabolism incubation mixture were tested. Similar to standard aqueous solution, a linear correlation (R2 = 0.9995) was also found for human urine sample spiked with imipramine in the range of 0.025-10 µg/mL. When aliquots of a human urine sample spiked with 1 µg/mL imipramine were loaded onto eight different monolithic columns, the RSD of peak height ratios was 3.8%. For a P450-imipramine incubation mixture, the formation of the N-demethylated metabolite (m/z 267.2) and the monohydroxylated metabolite (m/z 297.2) of imipramine was observed following chip-based monolithic SPE sample cleanup and pre-concentration.

"Development Of A Poly(dimethylsiloxane) Interface For On-Line Capillary Column Liquid Chromatography-Capillary Electrophoresis Coupled To Sheathless Electrospray Ionization Time-of-Flight Mass Spectrometry"
Anal. Chem. 2003 Volume 75, Issue 20 Pages 5461-5467
Sara K. Bergstr&ouml;m, Jenny Samskog and Karin E. Markides

Abstract: An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI-MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC-CE-TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 µL LC injection of 25 µg/mL peptide standard.

"Robust Interconnects And Packaging For Microfluidic Elastomeric Chips"
Anal. Chem. 2003 Volume 75, Issue 19 Pages 5287-5291
Hao Chen, Dhruv Acharya, Arivalagan Gajraj and Jens-Christian Meiners

Abstract: A stable, rugged, and easy-to-use microfluidic platform has been developed and evaluated. The system is based on multilayer silicone elastomer chips with integrated flow channels and active components, such as pressure-actuated valves. The silicone chips and stainless steel interconnect tubes are embedded in a block of an epoxy resin to give the chip and the interconnects outstanding mechanical stability. Full optical accessibility of the chip for demanding optical detection and manipulation applications, such as fluorescence and bright-field microscopy and optical tweezing, is achieved through the use of an optically transparent epoxy resin and microscope cover glasses as the packaging materials. Furthermore, this packaging technique uses a purely mechanical seal between the elastomer and cover glass, enabling applications that use chemically functionalized glass surfaces as the bottom of the flow channels. In addition, a socket was developed in which the microfluidic chips can be plugged in to provide all external connections for reagent delivery and pressure control of the integrated valves. The utility of these devices is demonstrated by showing that single DNA molecules can be attached to protein-coated walls of the flow channels and manipulated with optical tweezers.

"Fluidic Preconcentrator Device For Capillary Electrophoresis Of Proteins"
Anal. Chem. 2003 Volume 75, Issue 19 Pages 5207-5212
Juan Astorga-Wells and Harold Swerdlow

Abstract: A new pre-concentration device was developed for analysis of proteins by capillary electrophoresis (CE). The microfluidic device uses an electric field to capture proteins that pass through the system. The capture zone is maintained in the flow stream by the interaction between hydrodynamic and electrical forces. The device consists of a flow channel made of PEEK tubing with two electrical junctions, each of which is covered with a conductive membrane. A syringe pump provides the flow stream and also allows the injection of up to 13.5 µL of a dilute sample. The system can be easily connected to a CE device post-capture for off-line pre-concentration of proteins. For the proteins used in this study, pre-concentration factors up to 40-fold can be achieved. CE detection limits for bovine carbonic anhydrase, -lactalbumin and [beta2]-lactoglobulins A and B were in the nanomolar range using UV detection at 200 nm. Preconcentration is dependent on both time and initial protein concentration. We show the possibility of using an off-line fluidic pre-concentrator device employing counterflow capillary electrophoresis with minimum sample manipulation, achieving detection limits similar to on-line approaches.

"An Amperometric Detector Formed Of Highly Dispersed Ni Nanoparticles Embedded In A Graphite-like Carbon Film Electrode For Sugar Determination"
Anal. Chem. 2003 Volume 75, Issue 19 Pages 5191-5196
Tianyan You, Osamu Niwa, Zilin Chen, Katsuyoshi Hayashi, Masato Tomita and Shigeru Hirono

Abstract: We achieved improved detection limits for sugars by developing a novel thin film containing 0.8% highly dispersed Ni nanoparticles in disordered graphite-like carbon (Ni-NDC) as a detection electrode for high-performance liquid chromatography. The Ni-NDC film was prepared in one step by a simple radio frequency (rf) sputtering method at a temperature below 200°C. We characterized the film by XPS, TEM, and AFM analysis and found that the average Ni nanoparticle size was 3 nm and that the film consisted of a mixture of Ni, NiO, Ni2O3, and Ni(OH)2. We studied the electrochemical detection of sugars using the 0.8% Ni-NDC film electrode. The film electrode had excellent electrocatalytic ability and good stability compared with a Ni-bulk electrode with regard to the electrooxidation of sugars. We employed the Ni-NDC film as an HPLC detection electrode. We achieved a good separation of four sugars (glucose, fructose, sucrose, lactose) at a relatively low constant detection potential (0.40 V vs Ag/AgCl) and a linearity of over 3 orders of magnitude. We obtained improved detection limits for the investigated sugars, namely, 20, 25, 50, and 37 nM for glucose, fructose, sucrose, and lactose, respectively. This is at least 1 order of magnitude lower than the detection limits obtained with a Ni-bulk electrode with the same measurement condition. The Ni-NDC film electrode also showed good reproducibility with a relative standard deviation of 1.75% for 40 consecutive injections of glucose in a flow system.

"Simultaneous Quantitative Analysis Of Anionic, Cationic, And Nonionic Surfactants In Water By Electrospray Ionization Mass Spectrometry With Flow Injection Analysis"
Anal. Chem. 2003 Volume 75, Issue 19 Pages 5129-5136
M&oacute;nica Barco, Carles Planas, Oscar Palacios, Francesc Ventura, Josep Rivera and Josep Caixach

Abstract: A rapid method is described for the quantitative analysis of anionic, cationic, and nonionic surfactants in water samples by flow injection analysis coupled to electrospray ionization mass spectrometry (FIA/ESI-MS). All surfactants were isolated by liquid-liquid extraction and quantified using labeled triethoxylated nonylphenol ([13C6]-NP3EO) and sodium dibutylnaphthalenesulfonate as internal standards. FIA/ESI-MS was performed by alternating both positive and negative ionization modes, which allows simultaneous analysis of most common surfactants in a short time. Quality parameters of the method, such as linear range, repeatability, reproducibility, and limits of detection were studied. This method was applied to the analysis of wastewater treatment plant effluents from Catalonia (NE Spain).

"High-Throughput Nuclear Magnetic Resonance Analysis Using A Multiple Coil Flow Probe"
Anal. Chem. 2003 Volume 75, Issue 19 Pages 5116-5123
Megan A. Macnaughtan, Ting Hou, Jun Xu and Daniel Raftery

Abstract: An automated method for high-throughput nuclear magnetic resonance (NMR) spectroscopy has been developed using a four-coil Multiplex NMR probe. The probe is constructed with solenoidal microcoils optimized for detection of small volume, mass-limited samples and a flow-through design. Four samples can be simultaneously injected into the Multiplex probe with a robotics liquid handler and then analyzed in rapid succession using a selective excitation experiment. Due to the simultaneous injection of four samples and the reduced analysis time with rapid selective excitation, the analysis rate achieved thus far is as low as 1 sample/34 s for 1D 1H NMR.

"Total Protein Determinations By Particle Beam/Hollow Cathode Optical Emission Spectroscopy"
Anal. Chem. 2003 Volume 75, Issue 18 Pages 4801-4810
Fuxia Jin, Keith Lenghaus, James Hickman and R. Kenneth Marcus

Abstract: A novel method for quantitative total protein determinations is presented. Total protein content is determined by particle beam/hollow cathode optical emission spectroscopy (PB/HC-OES) through monitoring of carbon atomic emission. The PB/HC-OES offers such advantages as ease of operation, exclusion of labor-intensive sample pretreatment processes, rapid analysis, high sensitivity, and low detection limit. The method could also be adapted to be integrated to current microfluidics devices. Parametric optimization for sample introduction, nebulization, desolvation, and hollow cathode source conditions is performed for the analysis of aqueous bovine serum albumin solutions. Response curves of C (I) 193.0-nm emission were obtained under the optimized conditions with both 10% HCl and 100 µg/mL KCl added to the sample matrix as potential carriers. The detection limit for triplicate injections of bovine serum albumin standards was found to be on the single-nanogram level with 200 µL injections. The addition of KCl significantly improved the sensitivity, supporting the proposed 'carrier effect' of chloride salts in the particle transport process. Results obtained here suggest a range of applications for the use of the PB/HC-OES source for total protein determinations; emphasis here is future use in assessing protein quantification in microfluidic systems.

"Quantitative Analysis Of Hydroperoxyl Radical Using Flow Injection Analysis With Chemiluminescence Detection"
Anal. Chem. 2003 Volume 75, Issue 17 Pages 4696-4700
Jun Zheng, Stephen. R. Springston and Judith Weinstein-Lloyd

Abstract: The hydroperoxyl radical (HO2) is one of the most abundant free radicals in the atmosphere, where it participates in a series of photochemical reactions that determine the fate of natural and anthropogenic emissions. In addition, HO2 is found in droplets and surface water as a result of photochemical formation and gas-phase scavenging. We describe a quantitative method for determining trace concentrations of HO2 radicals that exploits the chemiluminescence produced upon reaction with a synthetic analogue of luciferin from the crustacean Cypridina. The technique is linear at least up to 1 µM HO2(aq) and has a minimum detection limit of 0.1 nM. A unique feature of this analysis is a calibration method using stable aqueous HO2 standards produced in submicromolar concentrations using 60Co γ-radiolysis. The advantage of this method in comparison to others in consideration of field deployment is its simplicity, low cost, and minimal size and power requirements. One intended application of this technique is the measurement of atmospheric HO2 radicals following collection into aqueous solution.

"Liquid Chromatography Coupled On-Line To Flow Cytometry For Postcolumn Homogeneous Biochemical Detection"
Anal. Chem. 2003 Volume 75, Issue 16 Pages 4272-4278
T. Schenk, A. Molendijk, H. Irth, U. R. Tjaden and J. van der Greef

Abstract: The feasibility of flow cytometry as read-out principle for homogeneous cell- or bead-based assays coupled on-line to LC is demonstrated using digoxin-coated beads (Dig-Beads) and fluorescent-labeled anti-digoxin (AD-FITC) as model system. The assay is carried out in a post-column continuous-flow reaction detection system where the AD-FITC and Dig-Beads are simultaneously added to the eluate of an LC separation column. Binding of AD-FITC to Dig-Beads results in a constant amount of fluorescence associated with the beads, which is detected by the flow cytometer. The presence of active compounds, such as digoxin and its analogues, in the sample will results in a decrease of the AD-FITC-Dig-Bead complex and, consequently, in the bead-associated fluorescence. Hence, the bead-associated fluorescence detected is inversely related to the digoxin concentration. A data-handling algorithm was developed in-house for adequate analysis of raw data output from the flow cytometer. Various conditions that influence the performance of this novel LC-biochemical detection (LC-BCD) system were investigated to determine the optimal settings of the bead-based biochemical interaction. The optimized flow injection bead-based assay was capable of detecting very low concentrations of digoxigenin (0.5 nmol/L), digoxin (0.1 nmol/L), and gitoxigenin (50 nmol/L). The applicability of LC coupled on-line to flow cytometry was demonstrated by the individual detection of digoxin, digoxigenin, and gitoxigenin in a single LC analysis. The successful coupling of LC on-line to flow cytometry principally enables the use of a wide range of new homogeneous assay formats in LC-BCD, such as membrane-bound receptor assays, cell-binding assays, and functional cell-based assays. Next to the ability to use insoluble targets, and also multiplexing assays, i.e., performing a number of assays simultaneously, using color- or size-coded beads becomes at hand in LC-BCD.

"Advantages Of Soft Versus Hard Constraints In Self-Modeling Curve Resolution Problems. Alternating Least Squares With Penalty Functions"
Anal. Chem. 2003 Volume 75, Issue 16 Pages 4236-4243
Paul J. Gemperline and Eric Cash

Abstract: A new algorithm for self-modeling curve resolution (SMCR) that yields improved results by incorporating soft constraints is described. The method uses least squares penalty functions to implement constraints in an alternating least squares algorithm, including nonnegativity, unimodality, equality, and closure constraints. By using least squares penalty functions, soft constraints are formulated rather than hard constraints. Significant benefits are obtained using soft constraints, especially in the form of fewer distortions due to noise in resolved profiles. Soft equality constraints can also be used to introduce incomplete or partial reference information into SMCR solutions. Four different examples demonstrating application of the new method are presented, including resolution of overlapped HPLC-DAD peaks, flow injection analysis data, and batch reaction data measured by UV/visible and near-infrared spectroscopy (NIR). Each example was selected to show one aspect of the significant advantages of soft constraints over traditionally used hard constraints. Incomplete or partial reference information into self-modeling curve resolution models is described. The method offers a substantial improvement in the ability to resolve time-dependent concentration profiles from mixture spectra recorded as a function of time.

"Trace Gas Measurement With An Integrated Porous Tube Collector/long-path Absorbance Detector"
Anal. Chem. 2003 Volume 75, Issue 16 Pages 4050-4056
Kei Toda, Ken-Ichi Yoshioka, Shin-Ichi Ohira, Jianzhong Li, and Purnendu K. Dasgupta

Abstract: Porous membrane tubes filled with an absorbing solution that change colors upon selective reactions with specific gases provide high sensitivity inexpensive gas sensors. These can be routinely used for ambient monitoring in a fully automated manner. We consider both stopped and continuous flow operations and show the superiority of the stopped flow mode theoretically and experimentally. Light throughput through various membrane tubes is presented, and superior performance of such tubes over Teflon AF is shown. Sensors for NO2 and for O3 were based on Griess-Saltzman and indigotrisulfonate chemistries, respectively. A computer-controlled two-LED absorbance measurement system (one wavelength monitors the signal, the other references the system) that also governs automated reagent refilling was implemented. Sub-parts-per-billion-volume detection limits are attainable within a few minutes for both gases. Comparative data with a commercial UV-photometry-based ozone monitor showed excellent agreement with the response pattern of the present instrument. Low cost, ready applicability to the measurement of different gases by merely changing the light source and chemistry, and high sensitivity makes this instrument attractive for both pedagogic and practical purposes.

"A Nanoinjector For Microanalysis"
Anal. Chem. 2003 Volume 75, Issue 15 Pages 3919-3923
Valeri Gorbounov, Petr Kuban, Purnendu K. Dasgupta and Henryk Temkin

Abstract: We describe a simple miniature injection device that can be used for introduction of nanoliter sample volumes in microfluidic systems. The hybrid microstructure consists of two hydraulically connected parts, a pulse micropump, and a multilevel cross-flow injector. Sample injection is accomplished by creating a transient pressure pulse in the sample line by means of the solenoid-based micropump. The sample line is aligned at right angles to the main carrier flow line. The two flow channels are located in two different parallel planes. The cross section of the two channels is defined by a self-sealing aperture in an elastomer. During the pressure pulse, the sample is introduced through this aperture directly into the main flow stream. Fast impulse-based injection causes rapid mixing of the injected sample with the main flow stream. This permits simple single-line manifold µflow injection (MFI) systems. The deformation/relaxation of the elastomer is fast and repeatable; as such, rapid serial actuations essentially result in a larger injected sample volume without significantly affecting the peak shape. In the present form, 2-40 nL samples are easily injected by single injection, and the injected volume can be chosen by system parameters. The injection repeatability as observed by a photometric detector is better than 1.2% (n = 100).

"A Continuous-Flow System For High-Precision Kinetics Using Small Volumes"
Anal. Chem. 2003 Volume 75, Issue 15 Pages 3681-3687
Xianzhi Zhou, Rohit Medhekar and Michael D. Toney

Abstract: A generally applicable continuous-flow kinetic analysis system that gives data of a precision high enough to measure small kinetic isotope effects for enzymatic and nonenzymatic reactions is described. It employs commercially available components that are readily assembled into an apparatus that is easy to use. It operates under laminar flow conditions, which requires that the time between the initiation of the reaction in the mixer and the observation be long enough that molecular diffusion can effect a symmetrization of the concentration profile that results from a thin plug of reagents introduced at the mixer. The analysis of a second-order irreversible reaction under pseudo-first-order conditions is presented. The Yersinia pestis protein tyrosine phosphatase catalyzed hydrolysis of p-nitrophenyl phosphate is characterized with the system, and a proton inventory on kcat is presented.

"Miniaturized Tris(2,2'-bipyridyl)ruthenium(II) Electrochemiluminescence Detection Cell For Capillary Electrophoresis And Flow Injection Analysis"
Anal. Chem. 2003 Volume 75, Issue 14 Pages 3637-3642
Jifeng Liu, Jilin Yan, Xiurong Yang and Erkang Wang

Abstract: The design and performance of a miniaturized chip-type tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)32+] electrochemiluminescence (ECL) detection cell suitable for both capillary electrophoresis (CE) and flow injection (FI) analysis are described. The cell was fabricated from two pieces of glass (20 x 15 x 1.7 mm), and the 0.5-mm-diameter platinum disk was used as working electrode held at +1.15 V (vs silver wire quasi-reference), the stainless steel guide tubing as counter electrode, and the silver wire as quasi-reference electrode. The performance traits of the cell in both CE and FI modes were evaluated using tripropylamine, proline, and oxalate and compared favorably to those reported for CE and FI detection cells. The advantages of versatility, sensitivity, and accuracy make the device attractive for the routine analysis of amine-containing species or oxalate by CE and FI with Ru(bpy)32+ ECL detection.

"Microfluidic Device For Single-Cell Analysis"
Anal. Chem. 2003 Volume 75, Issue 14 Pages 3581-3586
Aaron R. Wheeler, William R. Throndset, Rebecca J. Whelan, Andrew M. Leach, Richard N. Zare, Yish Hann Liao, Kevin Farrell, Ian D. Manger and Antoine Daridon

Abstract: We have developed a novel microfluidic device constructed from poly(dimethylsiloxane) using multilayer soft lithography technology for the analysis of single cells. The microfluidic network enables the passive and gentle separation of a single cell from the bulk cell suspension, and integrated valves and pumps enable the precise delivery of nanoliter volumes of reagents to that cell. Various applications are demonstrated, including cell viability assays, ionophore-mediated intracellular Ca2+ flux measurements, and multistep receptor-mediated Ca2+ measurements. These assays, and others, are achieved with significant improvements in reagent consumption, analysis time, and temporal resolution over macroscale alternatives.

"Vertically Stratified Flows In Microchannels. Computational Simulations And Applications To Solvent Extraction And Ion Exchange"
Anal. Chem. 2003 Volume 75, Issue 14 Pages 3549-3556
Petr Kuban, Jordan Berg and Purnedu K. Dasgupta

Abstract: In this paper, we describe the conditions under which two immiscible fluids flow atop one another (viewed perpendicular to the plane on which the channel is inscribed) in a shallow microfluidic channel. First, we predict the behavior of a two-phase system using fluid dynamic simulations with water-butanol and water-chloroform as model systems. We numerically model the effect of various physical parameters, such as interfacial surface tension, density, viscosity, wall contact angle, and flow velocity on the type of flow observed and find that interfacial surface tension and viscosity are the parameters responsible for formation of vertically stratified, side-by-side, or segmented flows. As predicted by numerical simulations, a water-chloroform system never assumes a vertically stratified configuration, but a water-butanol system does when the two liquids flow at sufficiently high flow velocities. In actual experiments, we test conditions under which potentially useful two-phase systems form stable vertically stratified flows. We also demonstrate that compared to side-by-side flow schemes, shorter diffusion paths are achievable, and thus, the system can be used at higher flow rates to obtain the same performance. We then apply such findings to practical analytical problems, such as solvent extraction and ion exchange.

"A Reusable Flow-Through Polymerase Chain Reaction Instrument For The Continuous Monitoring Of Infectious Biological Agents"
Anal. Chem. 2003 Volume 75, Issue 14 Pages 3446-3450
Phillip Belgrader, Christopher J. Elkin, Steven B. Brown, Shanavaz N. Nasarabadi, Richard G. Langlois, Fred P. Milanovich, Bill W. Colston, Jr. and Graham D. Marshall

Abstract: Continuous monitoring of the environment for infectious diseases and related biowarfare agents requires the implementation of practical cost-effective methodologies that are highly sensitive and specific. One compatible method employed in clinical diagnostics is real-time polymerase chain reaction (PCR) analysis. The utility of this technique for environmental monitoring is limited, however, by the utilization of single-use consumables in commercial PCR instruments. This greatly increases mechanical complexity, because sophisticated robotic mechanisms must replenish the disposable elements. An alternative strategy develops an autonomous monitoring system consisting of reusable modules that readily interface with fluidic circuitry in a flow-through scheme. The reduced complexity should increase reliability while decreasing operating costs. In this report, we describe a reusable, flow-through PCR module that functions as one component in such a system. This module was rigorously evaluated with Bacillus anthracis genomic DNA and demonstrated high repeatability, sensitivity, and efficiency, with no evidence of sample-to-sample carryover.

"Dual Electrospray Ionization Source For Confident Generation Of Accurate Mass Tags Using Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry"
Anal. Chem. 2003 Volume 75, Issue 14 Pages 3411-3418
Angelito I. Nepomuceno, David C. Muddiman, H. Robert Bergen, III, James R. Craighead, Michael J. Burke, Patrick E. Caskey and Jonathan A. Allan

Abstract: Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has rapidly established a prominent role in proteomics because of its unparalleled resolving power, sensitivity and ability to achieve high mass measurement accuracy (MMA) simultaneously. However, space-charge effects must be quantitatively, routinely, and confidently corrected because they are known to profoundly influence MMA. We argue that the most effective way to account for space-charge effects is to introduce an internal mass calibrant (IMC) using a dual electrospray ionization (ESI) source where the IMC is added from a separate ESI emitter. The major disadvantage of our initial dual ESI source to achieve high MMA, and arguably the only one, was the time required to switch between the analyte emitter and IMC emitter (i.e., >300 ms). While this 'switching time' was acceptable for direct infusion experiments, it did not lend itself to high-throughput applications or when conducting on-line liquid separations. In this report, we completely redesigned the dual ESI source and demonstrate several key attributes. First, the new design allows for facile alignment of ESI emitters, undetectable vibration, and the ability to extend to multiple emitters. Second, the switching time was reduced to <50 ms, which allowed the analyte and IMC to be accumulated 'simultaneously' in the external ion reservoir and injected as a single ion packet into the ion cyclotron resonance cell, eliminating the need for a separate accumulation and ion injection event for the IMC. Third, by using a high concentration of the IMC, the residence time on this emitter could be reduced to ~80 ms, allowing for more time spent accumulating analyte ions of significantly lower concentration. Fourth, multiplexed on-line separations can be carried out providing increased throughput. Specifically, the new dual ESI source has demonstrated its ability to produce a stable ion current over a 45-min time period at 7 T resulting in mass accuracies of 1.08 ppm ± 0.11 ppm (mean ± confidence interval of the mean at 95% confidence; N = 160). In addition, the analysis of a tryptic digest of apomyoglobin by nanoLC-dual ESI-FT-ICR afforded an average MMA of -1.09 versus -74.5 ppm for externally calibrated data. Furthermore, we demonstrate that the amplitude of a peptide being electrosprayed at 25 nM can be linearly increased, ultimately allowing for dynamic analyte/IMC abundance modulation. Finally, we demonstrate that this source can reliably be used for multiplexing measurements from two (eventually more) flow streams.

"On-Line Continuous-Flow, Multi-Protein Biochemical Assays For The Characterization Of Bioaffinity Compounds Using Electrospray Quadrupole Time-of-Flight Mass Spectrometry"
Anal. Chem. 2003 Volume 75, Issue 14 Pages 3376-3384
R. J. E. Derks, A. C. Hogenboom, G. van der Zwan and H. Irth

Abstract: The applicability of a homogeneous on-line continuous-flow, multi-protein biochemical assay was demonstrated for the interaction between fluorescein-biotin and streptavidin and for digoxin and anti-digoxigenin using electrospray quadrupole time-of-flight mass spectrometry (Q-TOF MS). In the on-line continuous-flow biochemical MS-based system several receptors (e.g., streptavidin and anti-digoxigenin, respectively) were allowed to react with corresponding reporter ligands (e.g.,fluorescein-biotin and digoxin, respectively). The methodology presented allows the simultaneous measurement of affinity and molecular mass of an active compound. By using automated MS and MS-MS switching functions of the Q-TOF, structure information is obtained allowing the characterization of bioactive compounds. No cross-reactivities were observed between the two model systems fluorescein-biotin/streptavidin and digoxin/anti-digoxigenin.

"Using Speciated Isotope Dilution With GC-Inductively Coupled Plasma MS To Determine And Unravel The Artificial Formation Of Monomethylmercury In Certified Reference Sediments"
Anal. Chem. 2003 Volume 75, Issue 13 Pages 3202-3211
R. C. Rodr&iacute;guez Mart&iacute;n-Doimeadios, M. Monperrus, E. Krupp, D. Amouroux and O. F. X. Donard

Abstract: Speciated isotope-dilution mass spectrometry (SID-MS) is claimed to be an absolute method; however, it has been found to be affected by artifact monomethylmercury (MMHg) formation in sediments. The determination of MMHg in sediments was carried out by SID-MS after open-focused microwave extraction. The extracted mercury species were then ethylated and separated by capillary gas chromatography (CGC). Isotope ratios (peak area ratios at different masses) were measured by on-line ICP-MS detection of the CGC-separated compounds. Reproducibility of 202Hg/201Hg isotope ratio measurements were 0.60% for MeEtHg and 0.69% for Et2Hg; for 202Hg/199Hg, 0.43 and 0.46%, respectively, were determined. The absolute detection limits for CGC-ICPMS measurements were better than 26 fg for 202Hg, 20 fg for 201Hg, and 24 fg for 199Hg. For the direct determination of MMHg in sediment reference materials (CRM 580, IAEA 356, and IAEA 405), higher values than the certified were always found. Systematic experiments were carried out to localize the sources of the unintentional abiotic methylmercury formation during analysis. Different spiking and derivatization procedures (either ethylation, propylation, or derivatization by Grignard reagents) were tested. In addition, isotopically enriched inorganic mercury was spiked. The amount of inorganic mercury initially present in the sample was found to be the critical factor that should be known and carefully controlled. A simple solvent extraction technique involving no critical cleanup steps was applied in order to reduce high Hg2+ amounts. The method was applied to the determination of MMHg in sediment reference material IAEA-405 with satisfactory results after organic solvent extraction. The limitations of applicability of the proposed method are evaluated as related to inorganic mercury, organic carbon, and sulfur contents. The results obtained confirmed that available sediment reference materials are adequate to achieve traceable mercury speciation analysis and to detect potential sources of MMHg artifact formation.

"Measurement Of Enzyme Kinetics Using A Continuous-Flow Microfluidic System"
Anal. Chem. 2003 Volume 75, Issue 13 Pages 3161-3167
Gi Hun Seong, Jinseok Heo and Richard M. Crooks

Abstract: This paper describes a microanalytical method for determining enzyme kinetics using a continuous-flow microfluidic system. The analysis is carried out by immobilizing the enzyme on microbeads, packing the microbeads into a chip-based microreactor (volume ~1.0 nL), and flowing the substrate over the packed bed. Data were analyzed using the Lilly-Hornby equation and compared to values obtained from conventional measurements based on the Michaelis-Menten equation. The two different enzyme-catalyzed reactions studied were chosen so that the substrate would be nonfluorescent and the product fluorescent. The first reaction involved the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and N-acetyl-3,7-dihydroxyphenoxazine (amplex red) to yield fluorescent resorufin, and the second the β-galactosidase-catalyzed reaction of nonfluorescent resorufin-β-D-galactopyranoside to yield D-galactose and fluorescent resorufin. In both cases. the microfluidics-based method yielded the same result obtained from the standard Michaelis-Menten treatment. The continuous-flow method required ~10 µL of substrate solution and 10^9 enzyme molecules. This approach provides a new means for rapid determination of enzyme kinetics in microfluidic systems, which may be useful for clinical diagnostics, and drug discovery and screening.

"Development Of A Liquid Chromatography-Electrospray-Tandem Mass Spectrometry Method For The Quantitative Determination Of Benzoxazinone Derivatives In Plants"
Anal. Chem. 2003 Volume 75, Issue 13 Pages 3128-3136
Lea Bonnington, Ethel Eljarrat, Miriam Guillam&oacute;n, Peter Eichhorn, Andreu Taberner and Dami&agrave; Barcel&oacute;

Abstract: A new method for the quantification of benzoxazinone derivatives in extracts of wheat foliage and root samples using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) is described. Using this method, the characterization, separation, and quantitative detection of a mixture of six naturally occurring 1,4-benzoxazin-3(4H)-one derivatives, including the hydroxamic acids (DIMBOA, DIBOA), lactams (HBOA, and HMBOA), benzoxazilinones (BOA, MBOA), and two synthetic methoxylated variations of DIBOA and HBOA, was achieved. The application of a novel, highly modified reversed-phase LC column, the dodecyl (C12) TMS end-capped Synergi MAX-RP, enhanced the on-line chromatographic separation through improvements to component resolution, analyte stability and peak shape and also to the column lifetime. The complete ESI-MS-MS precursor-product ion fragmentation pathways for the benzoxazinone derivatives are described for the first time and used to deduce a generic fragmentation pattern for the compound class. Characteristic transitions for the benzoxazinones were thus used in the developed analytical method enabling reliable quantification with simultaneous screening for other potentially present derivatives, while eliminating interferences from other coeluting contaminants from the complex plant extract matrix. Quantitative analysis was done in the multiple reaction monitoring mode, using two specific combinations of a precursor-product ion transitions for each compound. The ESI-MS-MS detection method offered improvements to the sensitivity and selectivity, as compared with previously applied LC methods, with detection limits down to 0.002-0.023 ng/µL. The developed method was demonstrated by analyzing foliages and roots of six different wheat cultivars using pressurized liquid extraction-solid-phase extraction cleanup-LC-ESI-MS-MS. The analytes were detected in the range of 0.7-207 µg/g of dry weight.

"A Smart Microfluidic Affinity Chromatography Matrix Composed Of Poly(N-isopropylacrylamide)-Coated Beads"
Anal. Chem. 2003 Volume 75, Issue 13 Pages 2943-2949
Noah Malmstadt, Paul Yager, Allan S. Hoffman and Patrick S. Stayton

Abstract: The efficient upstream processing of complex biological or environmental samples for subsequent biochemical analysis remains a challenge in many analytical systems. New microfluidic platforms that provide multidiagnostic capabilities on single chips face a similar challenge in getting specific analytes purified or contaminants removed in different fluid streams. Here, stimuli-responsive polymers have been used to construct 'smart' beads that can be reversibly immobilized on microfluidic channel walls to capture and release targets. The 100-nm latex beads were surface-modified with the temperature-sensitive polymer poly(N-isopropylacrylamide) (PNIPAAm). At room temperature, a suspension of these beads flows through a microfluidic channel constructed of poly(ethylene terephthalate). However, when the temperature in the channel is raised above the lower critical solution temperature (LCST) of PNIPAAm, the beads aggregate and adhere to the walls of the channel. The adhered beads are stable for long durations on the channel walls (demonstrated up to 70 min) in the presence of flow. The beads were further modified with the affinity moiety biotin, which tightly binds streptavidin. The dual-modified beads were adhered to the channel walls and functioned as a chromatographic affinity separation matrix, capable of binding streptavidin that was flowed through the microfluidic channel. Upon the reverse thermal stimulation to below the PNIPAAm LCST, the beads and captured streptavidin were observed to quickly dissolve and elute from the channel walls. This temperature-responsive affinity chromatography matrix can thus be flowed into a column and aggregated via temperature change, followed by the controlled release of affinity-captured targets back into the microfluidic flow stream.

"Automated Flow Injection Analyzer With On-Line Solid-Phase Extraction And Chemiluminescence Detection For The Determination Of Dodecylamine In Diesel Fuels"
Anal. Chem. 2003 Volume 75, Issue 11 Pages 2618-2625
Philip J. Fletcher, Kevin N. Andrew, Stuart Forbes and Paul J. Worsfold

Abstract: This paper describes the development of a portable, automated flow injection-chemiluminescence (FI-CL) analyzer incorporating on-line solid-phase extraction (SPE) for the determination of dodecylamine (detergent) in diesel fuels. The method is based on the peroxyoxalate/sulforhodamine 101 chemiluminescence reaction, with SPE required to remove indigenous compounds within the diesel fuel matrix that interfere with the CL response. The automated analyzer achieved a detection limit of 2.9 mg L-1 and a linear range of 2.9-50 mg L-1, which was suitable for determinations of dodecylamine at levels typically present in fully formulated diesel fuels (40 mg L-1). Analyses of base fuels from five different sources demonstrated that an automated FI-CL-SPE system could provide a portable instrument for monitoring the presence/absence of dodecylamine in diesel fuels.

"An Autonomous Nutrient Analyzer For Oceanic Long-Term In Situ Biogeochemical Monitoring"
Anal. Chem. 2003 Volume 75, Issue 11 Pages 2601-2609
Dani&eagrave;le Thouron, Renaud Vuillemin, Xavier Philippon, Antonio Louren&ccedil;o, Christine Provost, Antonio Cruzado and V&eacute;ronique Gar&ccedil;on

Abstract: An autonomous nutrient analyzer in situ (ANAIS) has been developed to monitor nitrate, silicate, and phosphate concentrations while deployed at sea at pressure (down to 1000 m). Detection is made by spectrophotometry. The instrument uses solenoid-driven diaphragm pumps to propel the sample, the standards, and the reagents through a microconduit, flow injection-style thermostated manifold. The analyzers are placed in an equipressure container filled with oil. The analyzers operate until a pressure of 100 bar and show a linear response up to 40 µM nitrate,150 µM silicate, and 5 µM phosphate with a detection limit less than 0.1, 0.5, and 0.1 µM and an accuracy of 1, 1, and 3% for nitrate, silicate, and phosphate, respectively. The measurement protocol includes three steps over 13 min: rinsing with the sample stream, reagents introduction, and absorbance detection. Field tests comprise ANAIS nitrate, silicate, and phosphate testing alone in the surface ocean. Phosphate results are not yet fully satisfactory. The instrument implemented on top of a YOYO vertical eulerian profiler was then deployed successfully in the northwestern Mediterranean Sea acquiring 30 nitrate profiles between 200 and 1100 m over a 15-day period. This chemical analyzer can be a valuable observing asset adapted on any type of oceanographic platform.

"Chemical Vapor Generation Atomic Spectrometry Using Amineboranes And Cyanotrihydroborate(III) Reagents"
Anal. Chem. 2003 Volume 75, Issue 11 Pages 2591-2600
Alessandro D'Ulivo, Valeria Loreti, Massimo Onor, Emanuela Pitzalis and Roberto Zamboni

Abstract: Amineboranes of the type L-BH3 (L = NH3; tert-BuNH2; Me2NH; Me3N) and sodium cyanotrihydroborate(III) (NaBH3CN) have been tested as derivatization reagents in the generation of volatile hydrides and elemental mercury following aqueous phase reaction with ionic species of Hg(II), As(III), As(V), Sb(V), Sb(III), Bi(III), Se(IV), Se(VI), Te(IV), and Te(VI). Continuous flow generation atomic absorption spectrometry coupled with a flameless quartz tube atomizer (T = 25°C) and a miniature argon-hydrogen diffusion flame atomizer were employed for the detection of mercury vapors and volatile hydrides, respectively. All of the reductants were able to reduce Hg(II) to the elemental state, giving sensitivities comparable to NaBH4 reduction. Under reaction conditions giving maximum sensitivity for hydride generation with NaBH4, only some amineboranes are able to produce volatile hydrides from all the elements. No evidence of hydride formation was observed from the Se(VI) and Te(VI). In general, the reducing power decreased in the order NaBH4 > H3N-BH3 > tert-BuNH2-BH3 > NaBH3CN [ge] Me2HN-BH3 > Me3N-BH3. In comparison with THB, amineboranes and NaBH3CN allowed, in general, a better control of interference effects of Fe(III), Ni(II), Co(II), and Cu(II). Application to determination of mercury in certified reference material is reported. The most likely mechanism of reaction of borane complexes in chemical vapor generation is based on the direct action of hydrogen bound to boron.

"Separation And Quantitation Of The Stereoisomers Of Ephedra Alkaloids In Natural Health Products Using Flow Injection-Electrospray Ionization-High Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry"
Anal. Chem. 2003 Volume 75, Issue 11 Pages 2538-2542
Margaret McCooeye, Luyi Ding, Graeme J. Gardner, Catharine A. Fraser, Joe Lam, Ralph E. Sturgeon and Zolt&aacute;n Mester

Abstract: A method is described for the determination of ephedrine (E) and pseudoephedrine (PE) and their metabolites norephedrine (NE), norpseudoephedrine (NPE), methylephedrine (ME), and methylpseudoephedrine (MPE) alkaloids in natural health products by flow injection-electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry (FI-ESI-FAIMS-MS). The determination of the six alkaloids requires the separation of diastereomic pairs of E-PE, NE-NPE, and ME-MPE. FAIMS was able to resolve/separate these isomeric pairs based on their gas-phase ion mobility differences. The FAIMS-based separation and detection approach has been tested on over-the-counter diet pills. Following the extraction of the tablets, either by pressurized fluid extraction developed in-house or with sonication, the ephedra alkaloids were quantified using a modified isotope dilution approach. Detection limits for the alkaloids ranged from 0.1 to 3 ng/mL, and a linear range of at least 2 orders of magnitude was observed for the six analytes. The throughput of the current configuration of the FI-ESI-FAIMS-MS system is 2 min/sample, which is significantly higher than conventional chromatographic approaches. The developed FI-ESI-FAIMS-MS method has been compared with a conventional LC-UV analysis, and good agreement has been found for the major alkaloids.

"Anodic Stripping Voltammetry Combined On-Line With Inductively Coupled Plasma-MS Via A Direct-Injection High-Efficiency Nebulizer"
Anal. Chem. 2003 Volume 75, Issue 10 Pages 2507-2511
Alfred J. Baca, Ana B. De La Ree, Feimeng Zhou and Andrew Z. Mason

Abstract: A direct-injection high-efficiency nebulizer (DIHEN) is used to couple a thin-layer electrochemical flow cell on-line with an ICP-mass spectrometer to perform anodic stripping voltammetry (ASV) at a thin mercury film followed by subsequent ICPMS measurements for the stripped metal analytes. The resultant hyphenated technique (ASV-DIHEN-ICPMS) is capable of analyzing select heavy metals present at ultratrace levels (down to low-ppt to sub-ppt levels) that are lower than the detection limits obtained by conventional ICPMS. In addition to its good analytical performance, the technique offers other attractive features such as the ability to eliminate detrimental matrix effects that can compromise ICPMS analyzes and the possibility of probing electrode reactions involving trace amounts metal species with ICPMS. For conducting ASV on-line with ICPMS, the DIHEN was found to be more advantageous than the microconcentric nebulizer in terms of minimizing memory effects and potential artifacts caused by the erosion of the Hg film into the flowing solution stream. Compared to a direct injection nebulizer (DIN), the DIHEN was easier to operate. Moreover, its simpler design and the lack of back pressure from the DIHEN capillary made it more compatible with coupling to the thin-layer electrochemical cell than a DIN system.

"Interfaces To Connect Thin-Layer Chromatography With Electrospray Ionization Mass Spectrometry"
Anal. Chem. 2003 Volume 75, Issue 10 Pages 2493-2498
Feng-Liu Hsu, Chun-Hsien Chen, Cheng-Hui Yuan and Jentaie Shiea

Abstract: This study has developed two interfaces to connect small-size thin-layer chromatography (TLC) with electrospray ionization mass spectrometry (ES-MS) for the continuous analysis of organic mixtures. The interfaces are (1) two bound optical fibers inserted into the C18-bonded particles at the exit of a small TLC channel and (2) a small commercial TLC strip with a sharpened tip. A reservoir continuously supplied a makeup solution to the tip of the TLC channel. The high voltage required for electrospray ionization was introduced into the makeup solution or mobile phase through a Pt wire, and electrospray was generated at the tip of the bonded optical fibers and at the sharp end of the TLC strip. Since small-size TLC channels were used, the elution time was short and less than 0.2 µL of the sample solution and 200 µL of the eluting solvent were required. Organic mixtures were separated successfully and detected on-line using the TLC/ES-MS techniques.

"Continuous Extraction Of Trapped Air From Bubble Ice Or Water For On-Line Determination Of Isotope Ratios"
Anal. Chem. 2003 Volume 75, Issue 10 Pages 2324-2332
Christof Huber, Markus Leuenberger and Oliver Zumbrunnen

Abstract: We describe a new continuous extraction system for trapped air from bubble ice or water for on-line determination of the isotopic composition of the main air components nitrogen and oxygen (δ15N, δ18O, and δ17O). Studies of the composition of air from bubbles trapped in polar ice are providing fundamental information about ancient atmospheric composition and, therefore, are an important tool to learn more about Earth's climate. The new system proved to work reliably for standard air admixed and subsequently removed from a water stream. The precision (1 SD) of standard measurements is ~0.04" for δ15N, ~0.1" for δ18O, and ~0.15" for δ17O. Ice measurements with the new on-line system are promising. Continuous measurements of nitrogen as well as oxygen isotope ratios can be performed with a spatial resolution of ~3 cm and nearly the same precision as for the standards. However, the measured <δ values of ice are generally lower, as compared to ice measured with conventional techniques, as a result of a time-dependent dissolution process of air in water associated with kinetic fractionation, which affects standard and sample differently. By modeling the dynamics of the this dissolution process, we found a reason for the lack of accuracy and propose an improvement of the system that will lead to a better accuracy of the ice measurements.

"Selective Measurement Of Ultratrace Methylmercury In Fish By Flow Injection On-Line Microcolumn Displacement Sorption Preconcentration And Separation Coupled With Electrothermal Atomic Absorption Spectrometry"
Anal. Chem. 2003 Volume 75, Issue 10 Pages 2251-2255
Xiu-Ping Yan, Yan Li and Yan Jiang

Abstract: A novel nonchromatographic speciation technique for ultratrace methylmercury in biological materials was developed by flow injection microcolumn displacement sorption pre-concentration and separation coupled on-line with electrothermal atomic absorption spectrometry (ETAAS). In the developed technique, Cu(II) was first on-line complexed with diethyldithiocarbamate (DDTC), and the resultant Cu-DDTC was presorbed onto a microcolumn packed with the sorbent from a cigarette filter. Selective pre-concentration of methylmercury (MeHg) in the presence of Hg(II), ethylmercury (EtHg), and phenylmercury (PhHg) was achieved at pH 6.8 through loading the sample solution onto the microcolumn due to a displacement reaction between MeHg and the presorbed Cu-DDTC. The retained MeHg was subsequently eluted with 50 µL of ethanol and on-line determined by ETAAS. Interferences from coexisting heavy metal ions with lower stability of their DDTC complexes relative to Cu-DDTC were minimized without the need of any masking reagents. No interferences from 5.5 mg L-1 Cu(II), 4.5 mg L-1 Cd(II), 2.5 mg L-1 Cr(III), 3 mg L-1 Fe(III), 10 mg L-1 Ni(II), 10 mg L-1 Pb(II), and at least 25 mg L-1 Zn(II) were observed for the determination of MeHg at the 50 ng L-1 level (as Hg). With the consumption of only 3.4 mL of sample solution, an enhancement factor of 75, a detection limit of 6.8 ng L-1 (as Hg) in the digest (corresponding to 3.4 ng g-1 in original solid sample for a final 50 mL of digest of 0.1 g of solid material), and a precision (RSD, n = 13) of 2.3% for the determination of methylmercury at the 50 ng L-1 (as Hg) level were achieved at a sample throughput of 30 samples h-1. The recoveries of methylmercury spike in real fish samples ranged from 97 to 108%. The developed technique was validated by determination of methylmercury in a certified reference material (DORM-2, dogfish muscle), and was shown to be useful for the determination of methylmercury in real fish samples.

"Nanocapillary Array Interconnects For Gated Analyte Injections And Electrophoretic Separations In Multilayer Microfluidic Architectures"
Anal. Chem. 2003 Volume 75, Issue 10 Pages 2224-2230
Donald M. Cannon, Jr., Tzu-Chi Kuo, Paul W. Bohn and Jonathan V. Sweedler

Abstract: An electrokinetic injection technique is described which uses a nuclear track-etched nanocapillary array to inject sample plugs from one layer of a microfluidic device into another vertically separated layer for electrophoretic separations. Gated injection protocols for analyte separations, reported here, establish nanocapillary array interconnects as a route to multilevel microfluidic analytical designs. The hybrid nanofluidic/microfluidic gated injection protocol allows sample preparation and separation to be implemented in separate horizontal planes, thereby achieving multilayer integration. Repeated injections and separations of FITC-labeled arginine and tryptophan, using 200-nm pore-diameter capillary array injectors in place of traditional cross injectors are used to demonstrate gated injection with a bias configuration that uses relay switching of a single high-voltage source. Injection times as rapid as 0.3 s along with separation reproducibilities as low as 1% for FITC-labeled arginine exemplify the capability for fast, serial separations and analyzes. Impedance analysis of the micro-/nanofluidic network is used to gain further insight into the mechanism by which this actively controlled nanofluidic-interconnect injection method works. Gated sample introduction via a nanocapillary array interconnect allows the injection and separation protocols to be optimized independently, thus realizing the versatility needed for real-world implementation of rapid, serial microchip analyzes.

"On-Line Monitoring Of Airborne Chemistry In Levitated Nanodroplets: In Situ Synthesis And Application Of SERS-Active Ag-Sols For Trace Analysis By FT-Raman Spectroscopy"
Anal. Chem. 2003 Volume 75, Issue 9 Pages 2166-2171
Nicolae Leopold, Michael Haberkorn, Thomas Laurell, Johan Nilsson, Josefa R. Baena, Johannes Frank and Bernhard Lendl

Abstract: We report a new strategy for on-line monitoring of chemical reactions in ultrasonically levitated, nanoliter-sized droplets by Raman spectroscopy. A flow-through microdispenser connected to an automated flow injection system was used to dose picoliter droplets into the node of an ultrasonic trap. Taking advantage of the flow-through characteristics of the microdispenser and the versatility of the automated flow system, a well-defined sequence of reagents could be injected via the microdispenser into the levitated droplet placed in the focus of the collection optics of the Fourier transform Raman spectrometer. In that way, chemical reactions could be carried out and monitored on-line. The developed system was used for fast, reproducible, in situ synthesis of a highly active surface enhanced Raman scattering (SERS) sol resulting from the reduction of silver nitrate with hydroxylamine hydrochloride in basic conditions. With this chemical system, SERS substrate preparation could be achieved at room temperature and in short time. The in situ prepared silver sol was used for trace analysis of several organic test molecules that were injected into the levitated SERS-active droplet again using the microdispenser. The concentration dependence of the SERS spectra was studied using 9-aminoacridine, revealing that down to the femtogram region high-quality SERS spectra could be obtained. Additionally, SERS spectra of 6-mercaptopurine, thiamine, and acridine were recorded in the levitated drop as well.

"Slurry Sampling-Microwave Assisted Leaching Prior To Hydride Generation-Pervaporation-Atomic Fluorescence Detection For The Determination Of Extractable Arsenic In Soil"
Anal. Chem. 2003 Volume 75, Issue 9 Pages 2011-2017
A. Caballo-L&oacute;pez and M. D. Luque de Castro

Abstract: A flow injection-pervaporation method, where the sample was introduced as slurry, has been developed for the continuous derivatization and determination of arsenic in soil by hydride generation-atomic fluorescence spectrometry. The removal of arsenic is achieved with the help of a microwave digestor, which facilitates an on-line leaching in the flow injection manifold. Slurries, prepared by mixing the soil (particle size <65 µm) with 6 mol L-1 HCl, were magnetically stirred for 3 min, and while stirring, the pump aspirated the aliquot and filled the loop (500 µL) of the injection valve. An industrial soil and five types of soil (sandy, clayey, slimy, limy, organic) were selected for the optimization of the leaching and determination steps of arsenic, respectively. The results obtained from three certified reference materials [stream sediment GBW 07311 (188 µg/mL As), river sediment CRM 320 (76.7 µg/mL As), and soil GBW 07405 (412 µg/mL As)] using direct calibration against aqueous standards demonstrate the reliability of the method. The relative standard deviation for within-laboratory reproducibility was 4.5%.

"Application Of Flow NMR To An Open-Access Pharmaceutical Environment"
Anal. Chem. 2003 Volume 75, Issue 8 Pages 1954-1957
Gregory C. Leo, Aaron Krikava and Gary W. Caldwell

Abstract: We present the application of flow NMR in an automated, open-access environment. The adjustment of parameters affecting the selection of the correct sample size, the elimination of carry-over, and the optimization of sample recovery are addressed. Advantages of this method include ease of use, elimination of NMR tubes, and elimination of handling errors that can result in the contamination of the probe. Sample throughput is similar to instruments using a conventional autosampler (for Bruker instruments, a BACS) although the time used for shimming the sample can be eliminated when the sample solvent does not change. The key feature of our methodology is that only one push solvent is used. This has major advantages over methods that switch solvents because there is no extensive flushing required between solvents and the deuterated push solvent, deuterium oxide, is economical. The disadvantage is the need to maintain the push and transfer solvents and usually the loss of the exchangeable protons that the sample may have. The protocol we present, using a single push solvent, contributes to the application of flow NMR in hands-on medicinal chemistry environments.

"Passively Driven Integrated Microfluidic System For Separation Of Motile Sperm"
Anal. Chem. 2003 Volume 75, Issue 7 Pages 1671-1675
Brenda S. Cho, Timothy G. Schuster, Xiaoyue Zhu, David Chang, Gary D. Smith and Shuichi Takayama

Abstract: This paper describes a self-contained integrated microfluidic system that can separate motile sperm from small samples that are difficult to handle using conventional sperm-sorting techniques. The device isolates motile sperm from nonmotile sperm and other cellular debris, based on the ability of motile sperm to cross streamlines in a laminar fluid stream. The device is small, simple, and disposable yet is an integrated system complete with sample inlets, outlets, sorting channel, and a novel passively driven pumping system that provides a steady flow of liquid; it requires no external power source or controls. The device fulfills a need in clinical settings where small amounts of sperm need to be sorted. It also opens the way for convenient bioassays based on sperm motility including at-home motile sperm tests.

"Direct Electrochemical Oxidation Of Disulfides At Anodically Pretreated Boron-Doped Diamond Electrodes"
Anal. Chem. 2003 Volume 75, Issue 7 Pages 1564-1572
C. Terashima, Tata N. Rao, B. V. Sarada, Y. Kubota and A. Fujishima

Abstract: Anodically oxidized diamond electrodes have been used to oxidize disulfides, thiols, and methionine in aqueous acidic media and tested for amperometric detection of these compounds after chromatographic separation. Cyclic voltammetric signals for 1 mM glutathione disulfide (GSSG) were observed at 1.39 and 1.84 V vs SCE, the values being less positive than those of its as-deposited counterpart as well as glassy carbon electrode. The voltammetric and chronocoulometric results have indicated the high stability of the electrode with negligible adsorption. A positive shift in the peak potential with increasing pH indicated the attractive electrostatic interaction between the anodically oxidized diamond surface and the positively charged GSSG in acidic media that promoted its analytical performance. The results of the electrolysis experiments of disulfides and thiols showed that the oxidation reaction mechanism of glutathione (GSH) and GSSG involves oxygen transfer. Following separation by liquid chromatography (LC), the determination of both GSH and GSSG in rat whole blood was achieved at a constant potential (1.50 V vs Ag/AgCl), and the limits of detection for GSH and GSSG were found to be 1.4 nM (0.028 pmol) and 1.9 nM (0.037 pmol) with a linear calibration range up to 0.25 mM. These detection limits were much lower than those reported for the amperometry using Bi-PbO2 electrodes and LC-mass spectrometry, and the LC method using diamond electrodes were comparable with enzymatic assay in real sample analysis. The high response stability and reproducibility together with the possibility of regeneration of the electrode surface by on-line anodic treatment at 3 V for 30 min further support the applicability of anodically pretreated diamond for amperometric detection of disulfides.

"Surface Plasmon Resonance Detection For Capillary Electrophoresis Separations"
Anal. Chem. 2003 Volume 75, Issue 6 Pages 1542-1547
Rebecca J. Whelan and Richard N. Zare

Abstract: A miniaturized surface plasmon resonance sensor has been used as an on-line detector for capillary electrophoresis separations. The capillary was modified slightly to shield the sensor electronics from the high voltages applied during the separation. A three-component mixture of high refractive index materials was separated and detected at the millimolar level by an untreated gold-sensing surface. A simple protein immobilization procedure was used to functionalize the surface for selective protein detection. A hybrid buffer system was developed, in which both the deposition of immobilized protein layers and the electrophoretic delivery of protein analytes were optimized. The detection system has a reproducibility of 15%, a dynamic range of 3 orders of magnitude, and a detection limit for IgG of 2 fmol.

"Advancing NMR Sensitivity For LC-NMR-MS Using A Cryoflow Probe: Application To The Analysis Of Acetaminophen Metabolites In Urine"
Anal. Chem. 2003 Volume 75, Issue 6 Pages 1536-1541
M. Spraul, A. S. Freund, R. E. Nast, R. S. Withers, W. E. Maas and O. Corcoran

Abstract: Cryogenic cooling of the NMR radio frequency coils and electronics to give greatly enhanced sensitivity is arguably the most significant recent advance in NMR spectroscopy. Here we report the first cryogenic probe built in flow configuration and demonstrate the application to LC-NMR-MS studies. This probe provides superior sensitivity over conventional noncryogenic flow NMR probes, allowing the use of 100 µL of untreated urine (40% less material than previous studies that required pre-concentration) and yet revealing drug metabolites hitherto undetected by LC-NMR-MS at 500 MHz. Besides the known sulfate and glucuronide metabolites, previously undetected metabolites of acetaminophen were directly observable in a 15-min on-flow experiment. Simultaneous MS data also provided knowledge on the NMR-silent functional moieties. Further, stop-flow LC-NMR-MS experiments were conducted for greater signal-to-noise ratios on minor metabolites. The cryoflow probe enables the NMR analysis of lower concentrations of metabolites than was previously possible for untreated biofluids. This strategy is generally applicable for samples containing mass-limited analytes, such as those from drug metabolism studies, biomarker and toxicity profiling, impurity analysis, and natural product analysis.

"Autoanalyzer For Milk Quality Control Based On The Lactose, Fat, And Total Protein Contents"
Anal. Chem. 2003 Volume 75, Issue 6 Pages 1425-1429
Rafael Lucena, Mercedes Gallego, Soledad C&aacute;rdenas and Miguel Valc&aacute;rcel

Abstract: A novel autoanalyzer was developed to assess the quality of milk samples according to the percentage of lactose, fat, and total protein they contain. The module comprises two pumps (one of high pressure), an injection valve, a filter, and an evaporative light-scattering detector. A volume of 15 µL of dilute milk was injected in an ethanol-water (50% v/v) stream for precipitation/retention of protein/fat, being the lactose content determined in the filtrate. The fat fraction was calculated using an ethanol stream, and total protein was finally dissolved by means of a 1.7 mol/L acetic acid solution. The simplicity of the proposed automatic module lies in the universal response of the detector, which permits the determination of the three macrocomponents in milk. In addition, the flow injection method allows their sequential analysis in the same injected sample by using selective reagents for each compound. The proposed method was validated with an SRM milk sample as well as by comparison of the results obtained with those provided by the IR method. In addition, the proposed analyzer is cheaper than its counterpart that is based on infrared technique.

"Improved Electrodeposited Iridium Oxide PH Sensor Fabricated On Etched Titanium Substrates"
Anal. Chem. 2003 Volume 75, Issue 6 Pages 1258-1266
Sayed A. M. Marzouk

Abstract: In the present paper, the preparation and characterization of an improved solid-state pH sensor are described. The sensor is based on anodically electrodeposited iridium oxide film, as a pH-sensing layer. Merits of the present sensor include (i) excellent adhesion of the pH sensitive layer to the substrate, (ii) excellent reproducibility of sensor fabrication, (iii) faster preparation procedure, and (iv) low cost of the titanium substrate. These advantages are realized by combining acid-etched titanium as the electrode substrate with an optimized electrodeposition solution consisting of IrCl4 as an iridium source, hydrogen peroxide, potassium oxalate, and potassium carbonate. Heating the electrodeposition solution to 90°C reduced the time required for solution development from ~3 days to 10 min. The pH-sensing layer is protected with a layer of Nafion and a microporous polyester membrane. The improved sensor showed a super-Nernstian response (-73.7 ± 1.2 mV/pH unit) in the pH range of 1.5-11.5. The present pH sensor, fabricated in a tubular form, is used as a detector in a flow injection analysis (FIA) system for pH measurements. Optimization of the FIA experimental parameters resulted in a linear dependence of peak heights on the pH of the injected samples in the pH range of 2-11.

"A Multichannel Microfluidic Sensor That Detects Anodic Redox Reactions Indirectly Using Anodic Electrogenerated Chemiluminescence"
Anal. Chem. 2003 Volume 75, Issue 6 Pages 1233-1238
Wei Zhan, Julio Alvarez, Li Sun and Richard M. Crooks

Abstract: Here, we describe a new approach for detecting redox-active targets by electrochemical oxidation and reporting their presence by electrogenerated chemiluminescence (ECL) based on electrochemical oxidation of Ru(bpy)32+ (bpy = 2,2'-bipyridine) and tripropylamine (TPA). This new strategy, which complements our previous reports of using ECL to signal the presence of targets undergoing electrochemical reduction, takes advantage of many of the attractive attributes of microfluidic-based electrochemical cells. These attributes include close proximity of multiple flow channels and electrodes, ability to move reagents through channels under laminar flow conditions, and the capacity to precisely place device components relative to one another using photolithography. Specifically, the microfluidic electrochemical sensor described here consists of three channels. The analyte and ECL reporting cocktail flow through separate channels, but they share a common anode. The cathode resides in a channel containing a sacrificial reductant. In this configuration, the target analyte competes with Ru(bpy)32+ and TPA to provide electrons for the reductant. Accordingly, in this competitive assay approach, the presence of the analyte is signaled as a lowering of the ECL intensity. In this report, the device performance characteristics are reported, and the detection of both ferrocyanide and dopamine is demonstrated at micromolar concentrations.

"Miniaturized Electrochemical Flow Cells"
Anal. Chem. 2003 Volume 75, Issue 4 Pages 1031-1036
Eskil Sahlin, Alexandra ter Halle, Kathleen Schaefer, Jeffery Horn, Matthew Then and Stephen G. Weber

Abstract: Several novel types of miniaturized electrochemical flow cells are described. The flow cells are fabricated in fluorinated ethylene propylene using a novel technique where channels with inner diameters down to 13 µm are integrated with electrodes. The channel is formed by shrinking and simultaneous melting of a heat shrink/melt tubing around a channel template (a tungsten wire) and electrodes followed by removal of the channel template. The technique allows incorporation of different electrode materials of different sizes. The electrode configuration consists of one or two working electrodes inside the channel and a counter electrode located in the channel outlet reservoir. Electrode configurations with different channel and working electrode sizes, different electrode materials including carbon fibers, glassy carbon rods, poly(tetrafluoroethylene)/carbon composite material, and platinum wires, and different arrangements have been assembled. Hydrodynamic voltammograms in dual-electrode (generator-collector) experiments indicate good potential control for cells with 25-µm channels, while there is some iR drop in cells with 13-µm channels. Cells prepared with a cylindrical working electrode tangent and perpendicular to a flow channel show a flow rate dependence consistent with thin-layer cell behavior. Electrode areas can be made in the range of 10^-10-10^-8 m2.

"Combined Electrospray Ionization-Atmospheric Pressure Chemical Ionization Source For Use In High-Throughput LC-MS Applications"
Anal. Chem. 2003 Volume 75, Issue 4 Pages 973-977
Richard T. Gallagher, Michael P. Balogh, Paul Davey, Mike R. Jackson, Ian Sinclair and Lisa J. Southern

Abstract: Fast and accurate analytical methods are essential to keep pace with sample libraries produced from combinational chemistry and high-throughput biological screening. Many laboratories now use a combination of ionization techniques for the characterization of these samples, including atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and photoionization (PI). Data are shown here from the analysis of a compound collection plate containing a variety of sample structures. ESI will normally analyze around 80% of these samples, necessitating a source change to analyze a further 10%. In this work, we have developed a new combined ESI-APCI source (ESCi) for use in on-line HPLC applications. The combined source allows alternate on-line ESI and APCI scans with polarity switching within a single analysis. The ESCi source has been designed to be a simple replacement for the existing mass spectrometer interfaces. Each ionization method is optimized independently using separate tuning parameters. Instrument electronics can readily switch between the two ionization methods and polarities within normal interscan time periods. The new source has reduced the analysis time of sample plates by eliminating the need for a source hardware change, source optimization, and repeat analyzes.

"Flow Injection Analysis In A Microfluidic Format"
Anal. Chem. 2003 Volume 75, Issue 4 Pages 967-972
Andrew M. Leach, Aaron R. Wheeler and Richard N. Zare

Abstract: A microfluidic flow injection analysis system has been designed and evaluated. The system incorporates within a single two-layer poly(dimethylsiloxane) monolith multiple pneumatically driven peristaltic pumps, an injection loop, a mixing column, and a transparent window for fluorescence detection. Central to this device is an injection system that mimics the operation of a standard six-port, two-way valve used in conventional liquid chromatography and flow injection experiments. Analyte and carrier solutions continuously flow through this injection system allowing for measurements and sample changes to be performed rapidly and simultaneously. Injection volumes of 1.25 nL generated peak area reproducibility of better than 3% relative standard deviation. The flow injection device was evaluated with fluorescent dyes and demonstrated a detection limit of 400 zmol for fluorescein. A rudimentary sample selection system allowed calibration curves to be rapidly produced, often in less than 10 min. The hydrolysis of fluorescein diphosphate by alkaline phosphatase demonstrates that chemical assays can be carried out with this device in a manner characterized by short analysis times and low sample consumption.

"Micromixer-Based Time-Resolved NMR: Applications To Ubiquitin Protein Conformation"
Anal. Chem. 2003 Volume 75, Issue 4 Pages 956-960
Masaya Kakuta, Dimuthu A. Jayawickrama, Andrew M. Wolters, Andreas Manz and Jonathan V. Sweedler

Abstract: Time-resolved NMR spectroscopy is used to study changes in protein conformation based on the elapsed time after a change in the solvent composition of a protein solution. The use of a micromixer and a continuous-flow method is described where the contents of two capillary flows are mixed rapidly, and then the NMR spectra of the combined flow are recorded at precise time points. The distance after mixing the two fluids and flow rates define the solvent-protein interaction time; this method allows the measurement of NMR spectra at precise mixing time points independent of spectral acquisition time. Integration of a micromixer and a microcoil NMR probe enables low µL volumes to be used without losing significant sensitivity in the NMR measurement. Ubiquitin, the model compound, changes its conformation from native to A-state at low pH and in 40% or higher methanol/water solvents. Proton NMR resonances of the His-68 and the Tyr-59 of ubiquitin are used to probe the conformational changes. Mixing ubiquitin and methanol solutions under low pH at microliter per minute flow rates yields both native and A-states. As the flow rate decreases, yielding longer reaction times, the population of the A-state increases. The micromixer-NMR system can probe reaction kinetics on a time scale of seconds.

"Development Of A Flow Amperometric Enzymatic Method For The Determination Of Total Glucosinolates In Real Samples"
Anal. Chem. 2003 Volume 75, Issue 4 Pages 927-934
Constantinos G. Tsiafoulis, Mamas I. Prodromidis and Miltiades I. Karayannis

Abstract: The first amperometric flow analyzer, based on the biosensor concept, capable of determining total glucosinolates in real samples, is described. Myrosinase was immobilized on aminopropyl-modified controlled pore glass, which was then used for the construction of a packed-bed reactor. Myrosinase catalyzes the hydrolysis of glucosinolates (sinigrin) to glucose (among the other products), which is then oxidized by the action of glucose oxidase to produce hydrogen peroxide. The glucose enzyme electrode is based on a multimembrane architecture and was mounted on an amperometric flow cell (hydrogen peroxide detection at a platinum anode poised at +0.65 V vs Ag/AgCl/3 M KCl). Different membrane types and different activation procedures were tested. The system was optimized to various working parameters, either as a glucose electrode or as a glucosinolate analyzer. The interference effect of various compounds was also investigated. Application of the method to real samples was carried out using glucose/glucose, hydrolyzed sinigrin and glucose/sinigrin solution as calibrators of the glucose electrode and the glucosinolate analyzer. Deviations due to the enantioselectivity of glucose oxidase to the [beta2]-glucose anomer were observed, and a data elaboration protocol is proposed. The possibility of the simultaneous determination of glucose and glucosinolates is also demonstrated.

"Liquid Chromatography With On-Line Electrochemical Derivatization And Fluorescence Detection For The Determination Of Phenols"
Anal. Chem. 2003 Volume 75, Issue 4 Pages 922-926
J&ouml;rg Meyer, Andr&eacute; Liesener, Sebastian G&ouml;tz, Heiko Hayen and Uwe Karst

Abstract: A new methodological approach for the determination of monosubstituted phenols is described. After liquid chromatographic separation of the analytes, an on-line electrochemical derivatization is carried out and the reaction products are detected fluorometrically. Phenols are oxidized in the electrochemical cell to form fluorescent dimers and higher oligomers, which were identified by on-line electrochemistry/mass spectrometry. Major advantages of the proposed method include enhanced selectivity and sensitivity. Without prior enrichment of the analytes, limits of detection down to 2 x 10^-9 M (20 fmol) may be reached for selected phenols, e.g., for 4-octylphenol, 4-ethylphenol, and 4-(1-indanyl)phenol. Only readily available instrumentation is required for these measurements.

"Hydrodynamic Study Of Ion Transfer At The Liquid/Liquid Interface: The Channel Flow Cell"
Anal. Chem. 2003 Volume 75, Issue 3 Pages 486-493
Simon S. Hill, Robert A. W. Dryfe, Edward P. L. Roberts, Adrian C. Fisher and Kamran Yunus

Abstract: A hydrodynamic system based on the channel flow cell for voltammetric detection of ions at the liquid/liquid interface is reported. The current response for tetraethylammonium ion transfer across a membrane-supported liquid/liquid interface is shown to be consistent with existing theory for both the flow rate and voltage scan rate dependence of such processes, with no calibration factors or other adjustable parameters required. The analytical utility of such a device is discussed with specific regard to in situ measurements in flow systems.

"The SLIM Spectrometer"
Anal. Chem. 2003 Volume 75, Issue 1 Pages 27-35
Kevin M. Cantrell and James D. Ingle, Jr.

Abstract: A new spectrometer, here denoted the SLIM (simple, low-power, inexpensive, microcontroller-based) spectrometer, was developed that exploits the small size and low cost of solid-state electronic devices. In this device, light-emitting diodes (LED), single-chip integrated circuit photodetectors, embedded microcontrollers, and batteries replace traditional optoelectronic components, computers, and power supplies. This approach results in complete customizable spectrometers that are considerably less expensive and smaller than traditional instrumentation. The performance of the SLIM spectrometer, configured with a flow cell, was evaluated and compared to that of a commercial spectrophotometer. Thionine was the analyte, and the detection limit was ~0.2 µM with a 1.5-mm-path length flow cell. Nonlinearity due to the broad emission profile of the LED light sources is discussed.

"A Microfluidic Bioreactor Based On Hydrogel-Entrapped E. Coli: Cell Viability, Lysis, And Intracellular Enzyme Reactions"
Anal. Chem. 2003 Volume 75, Issue 1 Pages 22-26
Jinseok Heo, K. Joseph Thomas, Gi Hun Seong and Richard M. Crooks

Abstract: Viable E. coli cells were entrapped in hydrogel micropatches photopolymerized within microfluidic systems. The microfluidic channels and the micropatches have sizes on the order of 100-500 µm. Small molecules, such as dyes and surfactants, present in the solution surrounding the hydrogel, are able to diffuse into the gel and encounter the cells, but the cells are sufficiently large to be retained. For example, sodium dodecyl sulfate is a lysis agent that is able to penetrate the hydrogel and disrupt the cellular membrane. Entrapment of viable cells within hydrogels, followed by lysis, could provide a convenient means for preparing biocatalysts without the need for enzyme extraction and purification. Hydrogel-immobilized cells are able to carry out chemical reactions within microfluidic channels. Specifically, a nonfluorescent dye, BCECF-AM, is able to penetrate both the hydrogel and the bacterial membrane and be converted into a fluorescent form (BCECF) by the interior cellular machinery. These results suggest that cells immobilized within microfluidic channels can act as sensors for small molecules and as bioreactors for carrying out reactions.

"Continuous Segmented-Flow Polymerase Chain Reaction For High-Throughput Miniaturized DNA Amplification"
Anal. Chem. 2003 Volume 75, Issue 1 Pages 1-7
Mario Curcio and Johan Roeraade

Abstract: A continuous segmented-flow method for sequential DNA amplification is described in order to provide a basis for high-throughput genetic analysis. The approach allows an immediate distinction between amplified and nonamplified products. A mixture of sample and reagents are loaded in the form of small segments one after another in a 15-m-long narrow-bore Teflon tube, coiled such as to be repeatedly exposed to three different temperature zones. After having passed the heated zones, the samples are mixed with an intercalating dye by flow injection and sequentially detected on-line by laser-induced fluorescence. The aqueous samples travel as separate segments in a continuous flow of an immiscible, organic liquid. Perfluorodecalin was shown to be particularly suitable due to its hydrophobicity and inert properties. To reduce carryover between samples, an intermediate water plug between two consecutive samples was required. Selected regions from human genomic DNA were successfully amplified in 300 nL volumes after 30 passes through the heated zones. The total reaction time was ~45 min, and the detection interval between individual samples was 1 min. Automation and the possibility to further reduce sample volumes, as well as to employ many reaction columns simultaneously, should provide a platform for an extremely high throughput.

"Microfluidic Actuation Using Electrochemically Generated Bubbles"
Anal. Chem. 2002 Volume 74, Issue 24 Pages 6392-6396
Susan Z. Hua, Frederick Sachs, David X. Yang and Harsh Deep Chopra

Abstract: Bubble-based actuation in microfluidic applications is attractive owing to elementary microfabrication requirements. In the present study, the mechanical and chemical characteristics of electrochemically generated bubble valves were studied. By generating electrochemical bubbles as valves directly inside the channel, valves could be closed and opened in milliseconds. Whereas bubble inflation (or valve closing) rate increases with applied voltage, small microfluidic dimensions accelerate bubble deflation rates. It is found that bubbles need not collapse fully to restore full flow, and the channel opens when its hydraulic resistance equals that between the bubble and the wall-a process requiring only milliseconds. Since only picomoles of salt are needed to generate bubbles, pH gradients that are invariably associated with electrochemical reactions were readily suppressed by using a small amount of buffer, as visualized by a pH-sensitive fluorescent dye. A range of common laboratory reagents and electrolytes in varying concentrations, including weak to strong acids and bases, as well as nonaqueous/aqueous mixtures were successfully tested. Using such bubble valves, an eight-way multiplexer was fabricated and tested.

"Electrochemical Flow Injection Analysis Study Of Ion Partitioning At High Surface Area Carbon Fiber Electrodes"
Anal. Chem. 2002 Volume 74, Issue 24 Pages 6364-6369
Richard S. Kelly, Brian D. Coleman, Tina Huang, Prachak Inkaew, and Theodore Kuwana

Abstract: Charge-selective electrochemistry was previously shown to occur at high surface area carbon fibers that were produced by fracturing the outer periphery with anodic current or positive potential. The cyclic voltammetric behavior of electroactive species observed at these fibers exhibited a distinct pH dependence related to the protonation/deprotonation of oxygen-containing functional groups at the surface of the carbon fiber. In this paper, electrochemical flow injection analysis (EC-FIA) is used to probe ion partitioning in to and out of the interior microstructure of the treated carbon fiber, for both electroactive and electroinactive species. It was found that the extent of partitioning was the result of both ion charge and hydrated ionic radius, in addition to the level of fracture. It was further observed that the direction of movement for an injected ionic species could be controlled relative to the ion concentration, the pH of the carrier solution, or both. EC-FIA allowed the simultaneous observation of current due to ion movement and that due to electron transfer to a redox-active species. The results presented are consistent with a model in which fixed negatively charged sites in the interior of fractured fibers govern ion partitioning with positively charged ions in the carrier solution, with counterions located in the interior free volume.

"Multiple Open-Channel Electroosmotic Pumping System For Microfluidic Sample Handling"
Anal. Chem. 2002 Volume 74, Issue 24 Pages 6259-6268
Iulia M. Lazar and Barry L. Karger

Abstract: 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 µm 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 (µ-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.

"Finite Element Simulation Of Pinched Pressure-driven Flow Injection In Microchannels"
Anal. Chem. 2002 Volume 74, Issue 24 Pages 6205-6215
Xiaoxia Bai, Jacques Josserand, Henrik Jensen, Jo&euml;l S. Rossier, and Hubert H. Girault

Abstract: A pinched pressure-driven flow injection on a microchip is numerically simulated in order to optimize the relative values of the operational parameters. The geometry studied is a two-dimensional rectangular channel featuring a cross-junction with a large depth-over-width ratio. The hydrodynamic and convection-diffusion equations are solved for the two steps of the process: first, the sample solution is pinched into the transversal channel (injection channel), and then it is injected into the longitudinal one (separation channel), where the time evolution of the concentration is analyzed for different types of the detectors. Electroosmotic flow calculations have also been performed and have shown a good agreement with literature. The results for pressure-driven flow point out that the shape of the detection signal is strongly dependent on the velocity in the separation channel and on the position of the detection probes. The so-called double-humped peak, caused by the parabolic flow profile at high driving flow rate is analyzed. A tight pinch greatly decreases the amount of injected sample and, consequently, the signal sensitivity without increasing its quality. A proper pullback of the sample during the separation process can decrease the tailing due to the sample leakage from the injection channel. Although a high sample pullback causes a considerable decrease in the signal sensitivity, it also greatly enhances the peak resolution. Finally, it is shown that a wider injection channel with high sample pullback ensures an improved signal sensitivity with good resolution.

"Autonomous Microfluidic Capillary System"
Anal. Chem. 2002 Volume 74, Issue 24 Pages 6139-6144
David Juncker, Heinz Schmid, Ute Drechsler, Heiko Wolf, Marc Wolf, Bruno Michel, Nico de Rooij and Emmanuel Delamarche

Abstract: The transport of minute amounts of liquids using microfluidic systems has opened avenues for higher throughput and parallelization of miniaturized bio/chemical processes combined with a great economy of reagents. In this report, we present a microfluidic capillary system (CS) that autonomously transports aliquots of different liquids in sequence: liquids pipetted into the service port of the CS flow unidirectionally through the various sections of the CS, which comprises a 15 pL reaction chamber, into the capillary pump. A CS can thus be operated by simply delivering the different samples to its service port. The liquid transport concept presented here is advantageous because the pumping and valving functions are integrated into the device by means of capillary phenomena, and it therefore does not require any external power supply or control device. Thus, arrays of CSs can easily be formed by cloning a functional CS. Alternatively, the flow of liquids in CSs can also be interactively tuned if desired by (i) forcing the evaporating of liquid out of the capillary pumps and (ii) by contacting a secondary, removable capillary pump to the embedded ones. We illustrate the possibilities of CSs by conducting a surface immunoassay for a cardiac marker, within 25 min, on an area of 100 x 100 µm2, using 16 sequential filling steps.

"A Cell-Based Bar Code Reader For High-Throughput Screening Of Ion Channel-Ligand Interactions"
Anal. Chem. 2002 Volume 74, Issue 24 Pages 6133-6138
Jon Sinclair, Johan Pihl, Jessica Olofsson, Mattias Karlsson, Kent Jardemark, Daniel T. Chiu and Owe Orwar

Abstract: This paper presents a microfluidics-patch clamp platform for performing high-throughput screening and rapid characterization of weak-affinity ion channel-ligand interactions. This platform integrates a microfluidic chip consisting of multiple channels entering an open volume with standard patch clamp equipment. The microfluidic chip is placed on a motorized scanning stage and the method relies on the ability to scan rapidly, on the order of milliseconds, a patch-clamped cell across discrete zones of different solutions created in the open volume. Under ideal conditions, this method has the capacity to obtain kinetically resolved patch clamp measurements and dose-response curves of up to 10^3 ligand solutions in a single day.

"Photoelectrochernical Oxygen Copper-plated Screen-printed Sensor Using Carbon Electrodes"
Anal. Chem. 2002 Volume 74, Issue 23 Pages 6126-6130
Jyh-Myng Zen, Yue-Shian Song, Hsieh-Hsun Chung, Cheng-Teng Hsu, and Annamalai Senthil Kumar

Abstract: We report here an efficient photocatalytic amperometric sensor for the determination of dissolved oxygen (DO) in phosphate buffer solution using a disposable copper-plated screen-printed carbon electrode (CuSPE). The photoelectrochemical activity toward DO of the CuSPE was related to the formation of a p-type semiconductor (Cu2O)-O-I The solution pH and biased potential (E-bias) were systematically optimized as pH 8 PBS and -0.7 V vs Ag/ AgCl, respectively. Under optimized conditions, the calibration plot was linear in the range of 1-8 ppm with sensitivity and regression coefficient of 23.51 µA-1 cm-2 ppm-1 and 0.9982, respectively. The reproducibility of the system was good with seven successive measurements of DO yielding a RSD value of 1.87%. Real sample assays for groundwater and tap water were also consistent with those measured by a commercial DO meter. The principle used in DO measurement has an opportunity to extend into various research fields.

"Segmented Flow Injection, UV Digestion, And Amperometric Detection For The Determination Of Total Cyanide In Wastewater Treatment Plant Effluents"
Anal. Chem. 2002 Volume 74, Issue 23 Pages 6055-6063
Howard S. Weinberg and Steven J. Cook

Abstract: The currently approved method for the analysis of total cyanide (TCN) in wastewaters has remained virtually unchanged in the 25 years since its initial use; this despite its subjection to a number of interferences, many of which provide a positive bias in cyanide measurements, including the formation of TCN during sample processing and some of which remain undocumented to this day. In particular, many municipal wastewater treatment plant chlorinated effluents throughout North America have often been cited for permit violations on the levels of total cyanide in their effluents measured using this methodology. A recently developed procedure for the analysis of TCN in various matrixes that utilizes segmented flow injection for sample transport and reaction, on-line acidic UV digestion for conversion of complexed cyanide to HCN, and amperometric detection achieved within 4 min of sample injection is demonstrated on chlorinated effluents discharged from municipal wastewater treatment plants. Through a systematic evaluation of the chemistry of the processes involved in this method and an understanding of the complexity of the wastewater matrix, an application was developed that showed consistent reproducibility in measuring TCN in a variety of effluents. The method was employed initially on synthetic solutions containing controlled levels of constituents identified in the literature as interfering with the measurement of TCN through the traditional distillation/colorimetric approach. In its application to the analysis of TCN in chlorinated waste-waters, an approach was evolved that ensured that dechlorination carried out during sample processing did not bias the results of analysis.

"A Disposable, Reagentless, Third-generation Glucose Biosensor Based On Overoxidized Poly(pyrrole)/tetrathiafulvalene -Tetracyanoquinodimethane Composite"
Anal. Chem. 2002 Volume 74, Issue 23 Pages 5913-5918
Francesco Palmisano, Pier Giorgio Zambonin, Diego Centonze, and Maurizio Quinto

Abstract: A disposable glucose biosensor based on glucose oxidase immobilized on tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) conducting organic salt synthesized in situ onto an overoxidized poly(pyrrole) (PPYox) film is described. The TTF-TCNQ crystals grow through the nonconducting polypyrrole film (ensuring electrical connection to the underlying Pt electrode) and emerge from the film forming a treelike structure. The PPYox film prevents the interfering substances from reaching the electrode surface. The sensor behavior can be modeled by assuming a direct reoxidation of the enzyme at the surface of the TTF-TCNQ crystals. A heterogeneous rate constant around 10^-6-10-7 cm s-1 has been estimated. The biosensor is nearly oxygen- and interference-free and when integrated in a flow injection system displays a remarkable sensitivity (70 nA/mM) and stability.

"Fiber Introduction Mass Spectrometry: Fully Direct Coupling Of Solid-phase Microextraction With Mass Spectrometry"
Anal. Chem. 2002 Volume 74, Issue 21 Pages 5688-5692
Eduardo C. Meurer, Daniela M. Tomazela, Rogerio C. Silva, Fabio Augusto, and Marcos N. Eberlin

Abstract: This work describes the first fully direct coupling of solid-phase microextraction (SPME) with mass spectrometry. An inlet system using a septum as the only interface between the ambient and the high-vacuum mass spectrometer was constructed to allow the introduction of the SPME needle directly into the ionization region of a mass spectrometer. The PDMS-coated fiber was then placed and exposed exactly between the two ionization filaments. Uniform heating of the fiber, efficient thermal desorption, and electron ionization of the analytes were achieved. Using this new analytical technique, here termed fiber introduction mass spectrometry (FIMS), we have been able to detect and quantitate several volatile (VOC) and semivolatile (SVOC) organic chemicals (carbon tetrachloride, benzene, toluene, xylenes, γ-terpinene, diisoamyl ether, chlorobenzene, and many PAHs) and two herbicides (Sylvex and its methyl ether) from aqueous solutions at low-ppb to ppt levels using either SPME headspace or solution extraction. FIMS shows high sensitivity (ng/L), good reproducibility, and accuracy, providing therefore a simple and effective approach to rapid analysis of VOC and SVOC in various matrixes.

"Fluorescent Ion-Selective Optode Membranes Incorporated Onto A Centrifugal Microfluidics Platform"
Anal. Chem. 2002 Volume 74, Issue 21 Pages 5569-5575
Ibrahim H. A. Badr, R. Daniel Johnson, Marc J. Madou and Leonidas G. Bachas

Abstract: The development of an integrated analysis system for small ions based on ion-selective optodes and centrifugal microfluidics is reported. The performance of this system was evaluated through five-point calibration plots for two types of optode membranes, one being cation-selective and the other anion-selective, which were incorporated into a microfluidics platform on which fluid motion is induced via angular rotation. Additionally, the application of the microfluidic platform to ion analysis is studied via a two-point calibration protocol used to quantify an unknown sample. Calibrant solutions are delivered from reservoirs fabricated onto the platform to a measuring area that contains the optode membrane, with a change in membrane fluorescence being monitored. This work demonstrates the first instance of a microfluidic-based analysis system with detection based on ion-selective optode membranes monitored with fluorescence transduction. Furthermore, in addition to employing a standard excitation source where a fiber-optic probe is coupled to a tungsten-halogen lamp, laser diodes such as those employed in portable CD/DVD players were studied as excitation sources to enhance the observed fluorescence signals.

"Breaking The 10(-7) Barrier For RI Measurements In Nanoliter Volumes"
Anal. Chem. 2002 Volume 74, Issue 20 Pages 5438-5441
Dmitry Markov, Deepak Begari, and Darryl J. Bornhop

Abstract: Refractive index (RI) detection is a common technique used in chemical and biochemical analysis. It can be employed to perform universal solute detection in muHPLC and CE, as well as temperature measurements. However, accurate RI measurements in nanoliter volumes still present a significant challenge. Here we present an alternative method to extract RI information encoded in spatial distribution of the backscattered fringes produced by a microinterferometric backscatter detector (MIBD) based on spatial Fourier analysis. By monitoring the phase in the Fourier domain, we were able to obtain detection limits of 7 x 10^-8 RIU. It was also shown that such calculations could be performed in real time, thus making MIBD with Fourier analysis compatible with muHPLC, CE, and FIA.
Laser

"Detection Of Flowing Fluorescent Particles In A Microcapillary Using Fluorescence Correlation Spectroscopy"
Anal. Chem. 2002 Volume 74, Issue 20 Pages 5350-5357
Beno H. Kunst, Arjen Schots and Antonie J. W. G. Visser

Abstract: Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule. It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam.

"Three-Dimensional Microfluidic Confinement For Efficient Sample Delivery To Biosensor Surfaces. Application To Immunoassays On Planar Optical Waveguides"
Anal. Chem. 2002 Volume 74, Issue 20 Pages 5243-5250
Oliver Hofmann, Guy Voirin, Philippe Niedermann and Andreas Manz

Abstract: A microchip-based flow confinement method for rapid delivery of small sample volumes to sensor surfaces is described. For flow confinement, a sample flow is joined with a perpendicular makeup flow of water or sample medium. Under laminar flow conditions, the makeup flow confines the sample into a thin layer above the sensing area and increases its velocity. This can benefit mass transport limited processes such as DNA hybridization or heterogeneous immunoassays. For proof of concept, this method was applied to a high-affinity immunoassay with excess capture antibody. Rabbit IgG was immobilized onto a silicon nitride waveguide. Cy5-labeled anti-rabbit IgG was hydrodynamically pumped over the immobilized zone through an attached 3D-PDMS flow cell with 20-µm-deep microchannels. The degree of confinement was adjusted through the volume flow rate of the confining flow. Evanescent field-based fluorescence detection enabled monitoring of the binding event. Assays were allowed to reach equilibrium to enable sensorgram normalization for inter-run comparison. The corresponding assay completion times could be reduced from 55 min for static drop conditions to 13 min for 25:1 flow confinement (ratio of confining to sample flow). For typical analytical applications, where equilibrium formation is not required, the faster response should translate to very short analysis times. Concurrently with the faster binding, sample consumption was reduced by 96% compared to conventional whole-channel sample delivery. Diffusional loss of analyte into the confining layer was identified as the main limitation of flow confinement, particularly for long sensing pads.

"Reusable Platforms For High-Throughput On-Chip Temperature Gradient Assays"
Anal. Chem. 2002 Volume 74, Issue 19 Pages 5071-5075
Hanbin Mao, Matthew A. Holden, Min You and Paul S. Cremer

Abstract: This paper describes a reusable platform that can apply a linear temperature gradient to a lab-on-a-chip device. When a planar microfluidic device with a series of microchannels is placed on top of the platform with the channels perpendicular to the gradient, each channel is held at a discrete temperature. This allows temperature-dependent data for chemical or biochemical species flowed into the device to be obtained in a concurrent fashion. As a demonstration, a melting curve for dsDNA is performed by collecting all the data simultaneously. The gradient is stable enough to easily distinguish between 30-mers where the complement strand contains a single C-A mismatch or a single T-G mismatch or is a perfect match. On the other hand, a temperature gradient can be formed parallel to the direction of flow of the microchannels. This allows the temperature in each channel to vary continuously as the liquid flows downstream. If each microchannel in the array contains a distinct pH value, ionic strength, species concentration, or chemical composition, then a high-throughput two-variable experiment can be performed. We demonstrate this mode of data collection by measuring the fluorescence yield of fluorescein dye molecules in aqueous solution simultaneously as a function of concentration and temperature.

"Integration Of Enzymes And Electrodes: Spectroscopic And Electrochemical Studies Of Chitosan-enzyme Films"
Anal. Chem. 2002 Volume 74, Issue 19 Pages 5039-5046
Xin Wei, Juan Cruz, and Waldemar Gorski

Abstract: A new film-forming solution was developed for the efficient immobilization of enzymes on solid substrates. The solution consisted of a biopolymer, chitosan (CHIT), that was chemically modified with a permeability-controlling agent, Acetyl Yellow 9 (AY9), using glutaric dialdehyde (GDI) as a molecular tether. A model enzyme, glucose oxidase (GOx), was mixed with the CHIT-GDI-AY9 solution and cast on the surface of platinum electrodes to form robust CHIT-GDI-AY9-GOx films for glucose biosensing. UV-visible and infrared spectroscopies were used to determine the composition of the films. The optimized films contained on average 1 molecule of AY9/3 glucosamine units of chitosan and 25 free GDI tethers/1 molecule of GOx. The electrochemical assays of the films indicated both a very high efficiency of enzyme immobilization (similar to99%) and large enzyme activity (60 units cm-2). The latter translated into a high sensitivity (42 mA M-1 cm-2) of the Pt/CHIT-GDI-AY9-GOx biosensor toward glucose. The biosensor operated at 0.450 V, had a fast response time (t(90%) less than or equal to 3 s), and was free of typical interferences, and its dynamic range covered 3 orders of magnitude of glucose concentrations. The lowest actually detectable concentration was 10 muM glucose. In addition, the biosensor displayed a practical shelf life and excellent operational stability; e.g. its response was stable during 24-h testing under continuous polarization and continuous flow of 5.0 mM glucose solution. The proposed approach to enzyme immobilization is simple, efficient, and cost-effective and should be of importance in the development of biosensors based on other enzymes that are more expensive than glucose oxidase.
Immobilized enzyme Polymer Graphite

"Electrochemically Controlled Solid-phase Microextraction Based On Conductive Polypyrrole Films"
Anal. Chem. 2002 Volume 74, Issue 18 Pages 4855-4859
Jingcun Wu, W. M. Mullett, and Janusz Pawliszyn

Abstract: Solid-phase microextraction (SPME) fiber coatings based on conductive polypyrrole films were prepared for the electrochemical extraction and desorption of ionic analytes. Simple preparation of each of the PPY extraction coatings on a platinum wire was possible with a constant potential method, but more importantly, cycling of the film between oxidation and reduction potentials facilitated the extraction and desorption of ionic analytes. The analytes were desorbed into a sample aliquot of water and were determined by flow injection analysis using a mass spectrometer. The fiber coatings and the developed electrochemical SPME method were found to be stable and reproducible (RSD < 5%; N = 5) and could be extended to several cations and anions, confirming the versatility of the approach. Preconcentration of the analyte on the fiber was also possible by repeating the processes to increase the amount of analyte extracted.
Resin

"Sampling BIAS At Channel Junctions In Gated Flow Injection On Chips"
Anal. Chem. 2002 Volume 74, Issue 18 Pages 4835-4840
Benjamin E. Slentz, Natalia A. Penner, and Fred Regnier

Abstract: The commonly used gated injection scheme was examined and found to suffer from multiple levels of electrokinetic sampling bias, including a new type based on transradial electrokinetic selection (TREKS). TREKS occurs as analytes of differing electrophoretic mobilities migrate around the corner at a channel junction in a microchip. The overall sample bias in gated injection was shown to be time-dependent and resulted in a larger sample bias against components of negative electrophoretic mobility. A new injection procedure for microchip devices based on interstream diffusion at zero potential is proposed. Diffusion of molecules into the separation channel is the main driving force for this type of injection. The new scheme is shown to be useful for injection of complex samples with multiply charged components, such as peptide mixtures. This procedure allows sampling of volumes from 12 to 45 pL, reproducible retention times (RSD < 1.5%), and reproducible peak areas (RSD < 2.3%). [Journal Article; In English; United States]

"Automated Affinity Capture - Release Of Biotin-containing Conjugates Using A Lab-on-valve Apparatus Coupled To UV/visible And Electrospray Ionization Mass Spectrometry"
Anal. Chem. 2002 Volume 74, Issue 18 Pages 4702-4708
Yuko Ogata, Louis Scampavia, Jarom&iacute;r Ruzicka, C. Ronald Scott, Michael H. Gelb, and Frantiek Tureek

Abstract: We report a new method for automated affinity capture and release of biotin-containing conjugates on immobilized streptavidin using a lab-on-valve (LOV) bead injection apparatus. The apparatus is also coupled to UV/ visible and electrospray ionization mass spectrometry (ESI-MS) for monitoring the captured and released biotin-containing conjugates. Dissociation rate constants for release from streptavidin of two chromophore-tagged biotin conjugates were measured by UV/visible spectrometry and the dissociation was simultaneously monitored by ESI-MS. The LOV-ESI-MS instrument was also used for repetitive assays of lysosomal ?-galactosidase in human cell homogenates. Fast analysis in 4.5 min/full cycle and robust operation in 60 repetitive analyzes are demonstrated that are promising for transfer of the LOV-ESI- MS technology into clinical practice.

"Hydrogel-Based Microreactors As A Functional Component Of Microfluidic Systems"
Anal. Chem. 2002 Volume 74, Issue 18 Pages 4647-4652
Wei Zhan, Gi Hun Seong and Richard M. Crooks

Abstract: A simple two-step method for fabricating poly(ethylene glycol) (PEG) hydrogel-based microreactors and microsensors within microfluidic channels is described. The intrachannel micropatches contain either a dye, which can report the pH of a solution within a fluidic channel, or enzymes that are able to selectively catalyze specific reactions. Analytes present within the microfluidic channel are able to diffuse into the micropatches, encounter the enzymes, and undergo conversion to products, and then the products interact with the coencapsulated dye to signal the presence of the original substrate. The micropatches are prepared by photopolymerizing the PEG precursor within the channel of a microfluidic system consisting of a poly(dimethylsiloxane) mold and a glass plate. Exposure takes place through a slit mask oriented perpendicular to the channel, so the size of the resulting micropatch is defined by the channel dimensions and the width of the slit mask. Following polymerization, the mold is removed, leaving behind the micropatch(es) atop the glass substrate. The final microfluidic device is assembled by irreversibly binding the hydrogel-patterned glass slide to a second PDMS mold that contains a larger channel. Multiple micropatches containing the same or different enzymes can be fabricated within a single channel. The viability of this approach is demonstrated by sensing glucose using micropatches copolymerized with glucose oxidase, horseradish peroxidase, and a pH-sensitive dye.

"A Microscale-Molecular Weight Sensor: Probing Molecular Diffusion Between Adjacent Laminar Flows By Refractive Index Gradient Detection"
Anal. Chem. 2002 Volume 74, Issue 17 Pages 4558-4565
Colin D. Costin and Robert E. Synovec

Abstract: A detection scheme that measures the refractive index gradient (RIG) between adjacent laminar flows in a microfluidic device has been used to develop a microscale-molecular weight sensor. The behavior of low Reynolds number flows has been well documented and shows that molecular transport (mixing) between adjacent laminar flows occurs by molecular diffusion between flow boundaries. A diode laser beam, incident upon and illuminating the entire width of a microchannel, measured the transverse concentration gradient at two different positions along a microchannel. The concentration gradient is impacted by the transverse diffusion from a flow with analyte into a flow initially without analyte. The RIG that forms as analyte diffuses from one adjacent flow to the other causes the laser beam, impinging orthogonal to the RIG through the microchannel, to be deflected. The angle of deflection is then monitored on a position-sensitive detector (PSD) at two different positions along the axis of flow to provide a measurement of analyte diffusion. The two positions are just after the flow initially without analyte merges with the flow initially containing all of the analyte (upstream) and then after the two streams have had more time to diffuse together (downstream). The ratio of the PSD signals obtained at the two positions along the flow, downstream signal divided by the upstream signal, is readily correlated to the analyte diffusion coefficient and, thus, the analyte molecular weight for a given class of compounds. The device was evaluated as a molecular weight sensor for poly(ethylene glycol) (PEG) solutions over a molar mass range from 106 to 22 800 g/mol. The ratio signal was found to be both independent of PEG concentration and sensitive to molecular weight changes for samples ranging from 960 to 22 800 g/mol. Independence of concentration is important for obtaining a reliable molecular weight measurement. The limit of detection for 11 840 g/mol PEG measured at the upstream detection position was determined to be 56 ppm, equivalent to 4.5 x 10^-6 RI (3s). This technique provides a much needed universal detection method, without requiring analyte derivatization chemistry (e.g., fluorescence), for microfluidic analyzes that are becoming increasingly useful in monitoring chemical systems such as continuous-flow reactors or batch polymerization processes. Thus, the molecular weight determination capability is potentially applicable to other compound classes, such as DNA or proteins.

"Simultaneous Multiple Substrate Tag Detection With ESI-ion Trap MS For In Vivo Bacterial Enzyme Activity Profiling"
Anal. Chem. 2002 Volume 74, Issue 16 Pages 4290-4293
Franco Basile, Imma Ferrer, Edward T. Furlong, and Kent J. Voorhees

Abstract: A bacterial identification method in which multiple enzyme activities are measured simultaneously and in vivo with electrospray ionization-mass spectrometry (ESI-MS) is described. Whole-cell bacteria are immobilized onto a filter support and incubated with a mixture of substrates. Each substrate is chosen to measure a specific enzyme activity of a targeted bacterium and to produce a tag of unique molecular weight. After A predetermined incubation time, the solution is filtered, and the supernatant consisting of a mixture of released tags and unhydrolyzed substrates is directly analyzed, without chromatographic separation, by ESI-MS. Bacteria remain viable on the filter for further analyzes. The method was tested by measuring the aminopeptidase activity. of the bacteria Escherichia coli, Bacillus subtilis, Bacillus cereus, and Pseudomonas aeruginosa. The resulting,aminopeptidase enzyme profiles allowed the differentiation between the four bacteria tested. The method is rapid, since a multiplex advantage is realized when assaying for multiple enzymes, and it is amenable to, automation via a flow injection analysis setup.

"Principles Of Surface-Directed Liquid Flow In Microfluidic Channels"
Anal. Chem. 2002 Volume 74, Issue 16 Pages 4259-4268
Bin Zhao, Jeffrey S. Moore and David J. Beebe

Abstract: To direct liquid flow inside microchannels, surface free energies were patterned by use of self-assembled monolayers (SAMs) in combination with either multistream laminar flow or photolithography. For the photolithographic method, two photocleavable SAMs were designed and synthesized. Carboxylic acid-terminated monolayers were obtained by photodeprotection, which was confirmed by contact angle and X-ray photoelectron spectroscopy. Using either of these patterning methods, we show that aqueous liquids flow only along the hydrophilic pathways when the pressure is maintained below a critical value; the liquids are referred to as being confined by virtual walls. Several principles of liquid flow in surface-patterned channels were derived analytically and verified experimentally. These principles include the maximum pressure that virtual walls can withstand, the critical width of the hydrophilic pathway that can support spontaneous flow, the smallest width of the liquid streams under an external pressure, the critical radius of curvature of turns that can be introduced into the hydrophilic pathway without liquid crossing the hydrophilic-hydrophobic boundary, and the minimal distance for two liquid streams to remain separated under the maximum pressure. Experimental results are in good agreement with the analytical predictions.

"Pressurized Hot Water Extraction With On-line Fluorescence Monitoring: A Comparison Of The Static, Dynamic, And Static-dynamic Modes For The Removal Of Polycyclic Aromatic Hydrocarbons From Environmental Solid Samples"
Anal. Chem. 2002 Volume 74, Issue 16 Pages 4213-4219
S. Morales-Mu&ntilde;oz, J. L. Luque-Garc&iacute;a, and M. D. Luque de Castro

Abstract: A comparison of the feasibility of the three operational modes of pressurized hot solvent extraction (PHSE) (namely, static, where a fixed extractant volume is used; dynamic, where the extractant continually flows through the sample; and static-dynamic mode, which consists of a combination of the two previous modes) for the extraction of polycyclic aromatic hydrocarbons (PAHs) from environmental solid samples (such as soil, sediment, trout, and sardine) has been performed. In all cases, a sodium dodecyl sulfate (SDS) aqueous solution was used as leaching agent. The use of a flow injection manifold between the extractor and a molecular fluorescence detector allowed real-time on-line fluorescence monitoring of the PAHs extracted from the samples, thus working as a screening system and providing qualitative and semiquantitative information on the target analytes extracted from both natural and spiked samples. The on-line monitoring option allowed the extraction kinetics to be monitored and the end of the leaching step to be determined independently of the sample matrix, thereby reducing extraction times. Efficiencies close to 100% have been provided by the three modes, which differ in the extraction time required for total removal of the target compounds. The time needed for the dynamic mode was shorter than that for the static mode. However, the establishment of a static extraction step prior to dynamic extraction was the key to shorten the time required for complete extraction. The method has been applied to a certified reference material (CRM 524, BCR, industrial soil/organics) for quality assurance/validation. [Journal Article; In English; United States]

"Selective Preconcentration And Determination Of Chromium(VI) Using A Flat Sheet Polymer Inclusion Sorbent: Potential Application For Cr(VI) Determination In Real Samples"
Anal. Chem. 2002 Volume 74, Issue 16 Pages 4204-4212
Y. M. Scindia, A. K. Pandey, A. V. R. Reddy, and S. B. Manohar

Abstract: A method for sorption pre-concentration of Cr(VI) from aqueous samples was developed using a polymer inclusion sorbent (PIS)6 The PIS used in this method was prepared. by physical inclusion of Aliquat-336 in the matrix,formed by cellulose triacetate and 2-nitrophenyl octyl ether. This sorbent was found to. be stable, cost-effective, efficient for pre-concentration of Cr(VI) present in the aqueous samples, and amenable to direct quantitative analysis of U(VI) held in, Why neutron activation analysis and spectrophotometry. The quantifying of Cr(VI) in PIS by spectrophotometry was carried out by developing color directly on the PIS after reacting it with 1,5-diphenylcarbazide. The distinct color developed on the PIS even at very low concentrations of Cr(VI) suggests its possible use for field determination of Cr(VI). The composition of PIS was optimized to obtain maximum uptake of Cr(VI) without sacrificing uniformity in terms of thickness and distribution of ion-exchange sites, stability, and time required for quantitative sorption of Cr(VI) from aqueous samples. The Cr(VI) species held in the PIS, mainly HCrO4- and CrO42-, were found to vary as a function of pH of the aqueous samples from which Cr(VI) was pre-concentrated. A close agreement was found in the abundances of Cr(VI) species held in the PIS with those reported in the literature for aqueous solutions at different pH. The variation of Cr(VI) species as a function of pH was found to have a significant impact on the tolerance to anions on the uptake of Cr(Vi) in the PIS. The high selectivity of PIS toward Cr(VI) from aqueous solution at pH = 2 was explained on the basis of hydration of anions. The uptake of Cr(VI) was found to be fairly constant (88±3%) up to nearly complete exchange of counterions present in the PIS. The method developed in the present work was successfully used for the pre-concentration of Cr(VI) from tap water and seawater samples containing low levels of Cr(VI).
Speciation

"Cell Docking And On-Chip Monitoring Of Cellular Reactions With A Controlled Concentration Gradient On A Microfluidic Device"
Anal. Chem. 2002 Volume 74, Issue 16 Pages 3991-4001
Mengsu Yang, Cheuk-Wing Li and Jun Yang

Abstract: We have developed a microfluidic device for on-chip monitoring of cellular reactions. The device consists of two primary analytical functions: control of cell transport and immobilization, and dilution of an analyte solution to generate a concentration gradient. In this device, a dam structure in parallel to the fluid flow was constructed for docking and alignment of biological cells, which allows the fragile cells to move in the microfluidic channels and to be immobilized with controllable numbers in desired locations. The cells docked on the parallel dam structure are exposed to minimal stress caused by fluidic pressure. Additionally, a network of microfluidic channels was designed to generate a concentration gradient by controlled fluid distribution and diffusive mixing. An analyte solution could be diluted to different gradients as a function of distance along the dam. We used the ATP-dependent calcium uptake reaction of HL-60 cells as a model for on-chip measurement of the threshold ATP concentration that induces significant intracellular calcium signal. The results have demonstrated the feasibility of using the microchip for real-time monitoring of cellular processes upon treatment of a concentration gradient of a test solution. The integration of cell manipulation and solution manipulation on a microchip allows the measurement of concentration-dependent biological responses within a confined microscale feature.

"Integrated Microfluidic System Enabling (bio)chemical Reactions With On-line MALDI-TOF Mass Spectrometry"
Anal. Chem. 2002 Volume 74, Issue 16 Pages 3972-3976
Monica Brivio, Roel H. Fokkens, Willem Verboom, David N. Reinhoudt, Niels R. Tas, Martijn Goedbloed, and Albert van den BergMonica Brivio, Roel H. Fokkens, Willem Verboom, David N. Reinhoudt, Niels R. Tas, Martijn Goedbloed, and Albert van den Berg

Abstract: A continuous flow µtotal analysis system (µ-TAS) consisting of an on-chip microfluidic device connected to a matrix assisted laser desorption ionization [MALDI] time-of-flight [TOF] mass spectrometer (MS) as an analytical screening system is presented. Reaction microchannels and inlet/outlet reservoirs were fabricated by powderblasting on glass wafers that were then bonded to silicon substrates. The novel lab-on-a-chip was realized by integrating the microdevice with a MALDI-TOFMS standard sample plate used as carrier to get the microfluidic device in the MALDI instrument. A novel pressure-driven pumping mechanism using the vacuum of the instrument as a driving force induces flow in the reaction microchannel in a self-activating way. Organic syntheses as well as biochemical reactions are carried out entirely inside the MALDI-MS ionization vacuum chamber and analyzed on-line by MALDI-TOFMS in real time. The effectiveness of the µ-TAS system has been successfully demonstrated with several examples of (bio)chemical reactions.

"Inductively Coupled Plasma Mass Spectrometry With On-line Leaching: A Method To Assess The Mobility And Fractionation Of Elements"
Anal. Chem. 2002 Volume 74, Issue 15 Pages 3924-3928
Diane Beauchemin, Kurt Kyser, and Don Chipley

Abstract: A new technique has been developed to assess the mobility and site of specific elements in complex natural materials such as rocks. Concentration profiles during leaching were obtained by pumping reagents (water, 1% HNO3, 10% HNO3, 30% HNO3), either continuously or with flow injection, through a microcolumn of sample while continuously monitoring analyte signals by inductively coupled plasma mass spectrometry (ICPMS). Compared to batch extraction procedures normally used, the approach involves minimal sample preparation and reduced contamination since the leaching is performed in a closed system. Continuous on-line monitoring also allows a greater resolution of the various phases reacting with given reagent. Compared to continuous leaching, flow injection increased the resolution of the various phases using discrete injections of reagents while reducing reagent consumption and minimizing etching of the MS interface. Furthermore, sensitivity was preserved by injecting into air instead of an aqueous carrier. Whether in the continuous or flow injection modes, the proposed approach provides real-time data on what phases are breaking down and what metals are released. It can therefore be used to design effective leaching strategies and to trace isotopic compositions. However, the resulting spectra are complex and the correct determination of some elements requires high-resolution ICPMS. [Journal Article; In English; United States]

"Flow Injection Analysis: Quietly Pushing Ahead"
Anal. Chem. 2002 Volume 74, Issue 13 Pages 385A-388A
James P. Smith and Vicki Hinson-Smith

Abstract: James Smith and Vicki Hinson-Smith report that advances have facilitated the development of new techniques.

"A Flow Method With Photocatalytic Oxidation Of Dissolved Organic Matter Using A Solid-phase (TiO2) Reactor Followed By Amperometric Detection Of Consumed Oxygen"
Anal. Chem. 2002 Volume 74, Issue 15 Pages 3858-3864
Yoon-Chang Kim, Satoshi Sasaki, Kazuyoshi Yano, Kazunori Ikebukuro, Kazuhito Hashimoto, and Isao Karube

Abstract: A photocatalytic sensor for the determination of chemical oxygen demand (COD) using titanium dioxide, based on the use of a pair of oxygen electrodes and flow injection analysis, is described. The measuring principle is based on the direct determination of the oxygen concentration change resulting from photocatalytic oxidation of organic compounds. One of the two oxygen electrodes, the reference oxygen electrode, was utilized to measure the reference signal responding only to oxygen present in the injected samples. Oxygen consumption due to the TiO2-catalyzed photochemical oxidation of organic compounds in samples was monitored with the working oxygen electrode. The COD value of this sensor was calculated as the difference of the currents at reference and working oxygen electrodes, respectively. The operation characteristics of the sensor are demonstrated using artificially treated wastewater as a substrate. The sensor was also applied to the determination of COD in real water samples from dam reservoirs (n = 20) all over Japan. The results were in good agreement with those from the conventional COD methods (i.e., permanganate and dichromate methods).

"Direct Determination Of Methylmercury And Inorganic Mercury In Biological Materials By Solid Sampling-electrothermal Vaporization-inductively Coupled Plasma-isotope Dilution-mass Spectrometry"
Anal. Chem. 2002 Volume 74, Issue 15 Pages 3833-3842
I. Gelaude, R. Dams, M. Resano, F. Vanhaecke, and L. Moens

Abstract: This paper reports on the use of solid sampling-electrothermal vaporization-inductively. coupled plasma mass spectrometry (SS-ETV-ICPMS) for the direct and simultaneous determination of methylmercury and inorganic mercury in biological materials. The main advantage of this fast and sensitive method is that no sample preparation is required. In this way, the sample throughput can be considerably increased, problems of contamination and analyte losses are kept to a minimum and, even more important, the original chemical form of the different analyte species in the solid samples is preserved. To achieve this goal, a solid sample is inserted into a graphite furnace of the boat-in-tube type, and is subsequently submitted to an appropriate temperature program, leading to the separate vaporization of methylmercury and inorganic mercury, which are transported into the ICP by means of an argon carrier gas. The separation was accomplished within 75 s. For the quantification of the two peaks, species-unspecific isotope dilution was used. For this purpose, a stable flow of argon loaded with gaseous Hg isotopically enriched in Hg-200 was generated using a permeation tube that was constructed in-house. Its emission rate was determined by collecting the mercury released during a given time interval on a gold-coated silica absorber, after which the amount, collected was released by heating of the absorber and determined by cold vapor atomic absorption spectrometry (CVAAS) and cold vapor atomic fluorescence spectrometry (CVAFS). A reference material from the Canadian National. Research Council (NRC) (TORT-2) was used to assess the accuracy of the,method. For.,the application of the method to samples with diverse mercury contents, the spike/sample ratio can be optimized by varying the emission rate of the permeation tube simply by adapting its temperature. To prove the feasibility of this approach, two reference materials (BCR 463 and DORM-2) with a methylmercury content more than 10 times higher than that of TORT-2 were also analyzed. The detection limits obtained for 1 mg of sample (2 ng g-1 and 6 ng g-1 for methylmercury and inorganic mercury, respectively) were found to be sufficiently low for this kind of application and are competitive when compared to other techniques.
Speciation

"Determination Of Pu Isotopes In Eawater By An On-line Sequential Injection Technique With Sector Field Inductively Coupled Plasma Mass Spectrometry"
Anal. Chem. 2002 Volume 74, Issue 15 Pages 3824-3832
Cheol-Su Kim, Chang-Kyu Kim, and Kun Jai Lee

Abstract: An on-line sequential injection (SI) system combined with sector field inductively coupled plasma mass spectrometry was developed for the determination of ultratrace level Pu-239 and Pu-240 in seawater. The potential of this method is the substantial reduction of a sample volume and rapidity in the determination of Pu isotopes. A chemical purification and pre-concentration of Pu isotopes were accomplished by the on-line S1 system with two micro-columns of solid-phase extraction resins, Sr-Spec and TEVA-Spec. The MCN-6000 microconcentric nebulizer was used as a sample introduction system because of low interference effect and good sample utilization. With this method, it was possible to analyze ultratrace levels of Pu isotopes in only 5 L of surface seawater with an analysis speed of 4 h/sample. The precision of the measurement for the Pu-239 and Pu-240 was less than 3.4 (n = 7) and 5% (n = 7) for 5 L of seawater. The detection limits for Pu-239 and Pu-240 were 0.64 (1.5 muBq/mL) and 0.19 fg/mL (1.6 muBq/mL), respectively. The accuracy of this method was verified by using the reference seawater (IAEA-381) as well as by the comparison with the α-spectrometry.
Preconcentration

"On-line Coupling Of Capillary Electrophoresis To Hydride Generation Atomic Fluorescence Spectrometry For Arsenic Speciation Analysis"
Anal. Chem. 2002 Volume 74, Issue 15 Pages 3720-3725
Xue-Bo Yin, Xiu-Ping Yan, Yan Jiang, and Xi-Wen He

Abstract: A novel hyphenated technique, on-line coupling of capillary electrophoresis (CE) to atomic fluorescence spectrometry (AFS), was developed for speciation analysis of four environmentally significant and toxic forms of arsenic: arsenite, arsenate, monomethylarsenic acid, and dimethylarsenic acid. Baseline separation of the four arsenic species was achieved by capillary electrophoresis in a 50 cm x 100 mum W. fused-silica capillary at 20 kV and using a 20 mmol L-1 phosphate buffer (pH 6.5). A hydride generation (HG) technique was employed to convert the arsenic species from the CE effluent into their respective hydrides. The CE-AFS interface was constructed on the basis of a cross design for introducing a sheath flow around the CE capillary and a Pt electrode, which provided an electrical connection for stable electrophoretic separations and allowed on-line volatile hydride formation. A laboratory-made gas-liquid separator was used to isolate the generated volatile species from the reaction mixture solution, and an argon flow was used to transport the volatile hydrides into the atomizer of AFS for on-line detection. The precision (RSD, n = 7) ranged from 2.1 to 3.1% for migration time, from 2.8 to 4.2% for peak area response, and from 2.0 to 4.1% for peak height response for the arsenic species at the 1 mg L-1 (as As) level. The detection limits were in the range of 9-18 µg L-1 (as As). The recoveries of the four arsenic species in locally collected water samples and urine sample ranged from 91 to 115%. The developed technique was successfully applied to the speciation of the water-methanol extractable arsenic in a sediment sample.

"Continuous On-line Measurement Of Cerebral Hydrogen Peroxide Using Enzyme-modified Ring-disk Plastic Carbon Film Electrode"
Anal. Chem. 2002 Volume 74, Issue 15 Pages 3684-3689
Lanqun Mao, Peter G. Osborne, Katsunobu Yamamoto, and Takeshi Kato

Abstract: An amperometric method suitable for the continuous online measurement of cerebral hydrogen peroxide from a microdialysate has been successfully performed for the first time by using an enzyme-modified ring-disk plastic carbon film electrode (PCFE) in a thin-layer radial flow cell. PCFE consists of a ring electrode modified with horseradish peroxidase to detect H2O2 at 0.0 V (vs Ag/ AgCl) and a disk electrode coated with ascorbate oxidase (AOx) to preoxidize ascorbic acid (AA) and thus suppress interference via direct oxidation. Analytes in solution (brain dialysates or standards) are mixed on-line with a phosphate-buffered solution containing dissolved oxygen and chelating agent, EDTA. The buffered solution is used to provide the O2 necessary for the AOx catalytic reaction, stabilize the changes in dialysate pH that are associated with the in vivo formation of H2O2, and remove heavy metal ion impurities and thus suppress reactions between AA and H2O2. This procedure enables trace levels of H2O2 to be readily monitored, virtually interference-free from physiological levels of AA, uric acid, electroactive neurotransmitters and their principle metabolites, in a continuous-flow system.

"Electrostretching DNA Molecules Using Polymer- Enhanced Media Within Microfabricated Devices"
Anal. Chem. 2002 Volume 74, Issue 14 Pages 3378-3385
Vijay Namasivayam, Ronald G. Larson, David T. Burke and Mark A. Burns

Abstract: In this paper, we demonstrate immobilization and stretching of single λ-phage DNA molecules within microfluidic systems using ac fields. We present a novel 'thiol-on-gold'-based immobilization technique for fixing one specific end (3' end) of a DNA molecule onto a gold electrode. A polymer-enhanced medium (~3.75 wt % linear polyacrylamide in Tris-HCl) is used to obtain fully stretched configurations (21 µm) of fluorescently stained λ-DNA molecules. We also present an optimized microelectrode design with pointed electrodes and an electrode spacing of 20 µm for stretching DNA molecules with an ac field (1 MHz, 3 x 10^5 V/m). Finally, using these techniques, we immobilize a single DNA molecule at one electrode edge, stretch the molecule, and fix the other end at an adjacent electrode edge, forming a bridge between two electrodes within a microfabricated device.

"Improved Integrated Waveguide Absorbance Optodes For Ion-selective Sensing"
Anal. Chem. 2002 Volume 74, Issue 14 Pages 3354-3361
Mar Puyol, &Iacute;&ntilde;igo Salinas, Ignacio Garc&eacute;s, Francisco Villuendas, Andreu Llobera, Carlos Dom&iacute;nguez, and Juli&aacute;n Alonso

Abstract: The first prototype of a technologically improved integrated waveguide absorbance optode (IWAO) was developed and tested with a membrane based on a new H+-selective ketocyanine dye and a cadmium ionophore. It was designed with curved instead of rectilinear planar waveguides. Results demonstrated the suitability of the new IWAOs to be employed as sensing platforms, which confer versatility, robustness, and mass production capabilities besides high sensitivity on conventional bulk optodes, as well as the usefulness of such dyes in developing ion-selective membranes in combination with a selective ionophore. The sensor integration as a detector in a flow injection system (FIA) was proposed to obtain an automated, simple, and sufficiently reproducible (RSD < 5%) analytical methodology with a sample throughput of 55 h-1. Very sensitive optodes were obtained, and detection limits on the order of 20 ppb were achieved. Because of the ionophore employed, the optode system showed excellent selectivity over alkali and alkaline-earth metals,with the exception of samples containing lead and cadmium ions, where the membrane responded to both analytes. The proposed procedure combines an the advantages of the FIA systems, the simplicity of optical detection, ion recognition selectivity, and sensitivity of ketocyanine dyes, and the features achieved using the integrated device, which comprise an improved sensitivity and short response times as well as robustness, easy handling, and mass production.
Detection limit

"Self-contained Microelectrochemical Immunoassay For Small Volumes Using Mouse IgG As A Model System"
Anal. Chem. 2002 Volume 74, Issue 14 Pages 3321-3329
Zoraida P. Aguilar, Walter R. Vandaveer, IV, and Ingrid Fritsch

Abstract: A self-contained, microelectrochemical immunoassay on the smallest volumes reported to date (1 µL for the antigen, 1 µL for the secondary antibody-enzyme conjugate, and 200 nL for the electrochemically detected species) has been developed using mouse IgG as a model system in a sandwich-type enzyme-linked immunosorbent assay, which takes less than 30 min to both complete the assembly of immunoassay components onto the antibody-modified surface and detect enzymatically generated species (excluding time for electrochemical cleaning of electrodes). These studies demonstrate the advantage of the close proximity of electrodes to modified surfaces and their application in the analysis of small volumes. Using a 50 mum diameter x 8 mum deep cavity with individually addressable electrodes on a microfabricated chip, the primary antibody was selectively and covalently attached at a gold, recessed microdisk (RMD) at the bottom of the microcavity to the free end of SAMs of either 11-mercaptoundecanoic acid or 11-mercapto-1-undecanol using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. Nonspecific adsorption to the surrounding material, polyimide, of the microcavity device was eliminated. Electrochemical desorption was used to confine the immunoassay activity at the RMD. Enzymatic conversion of the substrate p-aminophenyl phosphate to p-aminophenol is detectable in less than 30 s using cyclic voltammetry at a gold, tubular nanoband electrode, which is on the wall of the microcavity and immediately adjacent to the modified RMD. A third electrode, also within the region of the microcavity, served as the pseudoreference/auxiliary electrode. Calibration curves obtained for 1 µL solutions of 5-100 ng/mL of IgG and for 200 nL-solutions of 5 muM to 4 mM of PAP(R) gave detection limits of 4.4 nM (6.4 ng/mL) or 880 fmol (129 pg) for PAPR and 56 fM (9 pg/mL) or 56 zmol (9 fg) for IgG. It is expected that the device may be suitable for analysis with volumes down to tens of picoliters.
Adsorption Desorption

"Quantitative Analysis Of Mitoxantrone By Surface-enhanced Resonance Raman Scattering"
Anal. Chem. 2002 Volume 74, Issue 13 Pages 3160-3167
Clare McLaughlin, Donald MacMillan, Colin McCardle, and W. Ewen Smith

Abstract: Mitoxantrone is an anticancer agent for which it is important to know the concentration in blood during therapy. Current methods of analysis are cumbersome, requiring a pretreatment stage. A method based on surface-enhanced resonance Raman scattering (SERRS) has been developed using a flow cell and silver colloid as the SERRS substrate. It is simple, sensitive, fast, and reliable. Both blood plasma and serum can be analyzed directly, but fresh serum is preferred here due to reduced fluorescence in the clinical samples available. Fluorescence is reduced further by the dilution of the serum in the flow cell and by quenching by the silver of surface-adsorbed material. The effectiveness of the latter process is dependent on the contact time between the serum and the silver. The linear range encompasses the range of concentrations detected previously in patient samples using HPLC methods. In a comparative study of a series of samples taken from a patient at different times, there is good agreement between the results obtained by HPLC and SERRS with no significant difference between them at the 95% limit. The limit of detection in serum using the final optimized procedure for SERRS was 4.0 x 10^-11 M (0.02 ng/mL) mitoxantrone. The ease with which the SERRS analysis can be carried out makes it the preferred choice of technique for mitoxantrone analysis.
Detector

"Selection Of Optimal Process Analyzers For Plant-wide Monitoring"
Anal. Chem. 2002 Volume 74, Issue 13 Pages 3105-3111
Frans W. J. van den Berg, Huub C. J. Hoefsloot, and Age K. Smilde

Abstract: In this paper, the effect of process analyzer selection and positioning on plant-wide process monitoring is investigated. A fundamental problem in process analytical chemistry is the incomparability of different instrument characteristics. A fast but imprecise instrument is incomparable to a slow but precise instrument. Theory is developed to overcome this problem by using an abstract definition of a process analyzer. This, definition allows us to. put all instrument characteristics for a particular monitoring task on an equal footing. This results in a measurability factor M that expresses monitoring performance of any process measurement by combining instrument characteristics such as precision, sampling rate, grab size, response correlation, and delay time. Both the choice of location and the performance characteristics of different process analyzers can be evaluated busing the measurability factor. The unifying nature of the measurability factor allows for a rational decision between completely different process analyzers and locations (Smilde et at., in this issue). The theory is illustrated and validated with an experiment. A tubular reactor for free-radical bulk polymerization of styrene is monitored by in-line short-wave near-infrared spectroscopy at different positions. Alternatively, product samples are collected for at-linle near-infrared analysis. Both analyzers measure styrene monomer concentration. The analysis results are used, to predict conversion as well as number and weight average molecular mass of the polystyrene reactor product. The theoretical measurability factors for this, case study correspond well with the experimental findings. An ever-increasing number of process analyzers are implemented in the chemical in uistr. At the same time, the diversity in techniques suitable for harsh process conditions-e.g., chromatography (near)-infrared, and Raman or (low-field) nuclear magnetic resonance spectroscopy, mass spectrometry, flow injection analysis, an ultrasonic analysis, to name just a few grows steadily. The implementation and operation of analytical in process measurements is, however, still relatively expensive.

"Species-specific Isotope Dilution-based Calibration For Trace Element Speciation And Its Combined Uncertainty Evaluation: Determination Of Tributyltin In Sediment By HPLC-ICPMS"
Anal. Chem. 2002 Volume 74, Issue 13 Pages 2968-2976
Lu Yang, Zolt&aacute;n Mester, and Ralph E. Sturgeon

Abstract: method is described for the determination of tributyltin (TBT) in NRCC sediment CRM PACS-2 by isotope dilution (ID) analysis using HPLC-ICPMS. Reverse spike ID analysis was performed to determine the accurate concentration of a Sn-117-enriched TBT spike using a well-characterized natural abundance TBT standard. The accuracy of the latter is critical for obtaining reliable results. A unique approach, using hydride generation GC/MS, was developed to quantify the inorganic Sn and dibutyltin impurities in the natural abundance TBT standard. The true natural abundance TBT standard concentration was obtained following correction for these impurities. The total Sn concentration in the natural abundance TBT standard was determined by ID analysis using an enriched inorganic Sn-117 following closed vessel mixed-acid digestion. Calibration of the enriched inorganic 117Sn standard was achieved by reverse ID analysis against a natural abundance inorganic tin standard prepared from the high-purity metal. An overall uncertainty associated with the present method was estimated, to which the uncertainties arising from measurement of the natural abundance TBT concentration, from the measurement of the isotope ratio in the spiked sample and in the reverse ID calibration solutions, and from estimation of the extraction efficiency were the main contributors. A concentration of 1.018± 0.054 mg kg-1 (expanded uncertainty, k = 2) as tin was obtained for TBT in PACS-2 using the present method, in excellent agreement with the certified value of 0.98 0.13 mg kg-1 (95% confidence interval). A TBT concentration of 0.97±0.11 mg kg-1 (expanded uncertainty, k = 2) as tin in PACS-2 was determined using the standard additions technique. Much smaller expanded uncertainty was obtained with ID, clearly demonstrating its superiority in providing more accurate and precise results over the method of additions. A detection limit (3s) of 0.02 mg kg-1 for TBT, based on a 0.5-g subsample, was obtained.
Extraction

"Development And Testing Of A Detection Method For Liquid Chromatography Based On Aerosol Charging"
Anal. Chem. 2002 Volume 74, Issue 13 Pages 2930-2937
Roy W. Dixon and Dominic S. Peterson

Abstract: Aerosol-based detection methods for HPLC in which HPLC effluent is converted to an aerosol and detected optically have been employed in the past. This paper describes a new aerosol-based detection method for HPLC, which we name aerosol charge detection. Ibis detection method also involves generation of an aerosol but with aerosol detection by charging aerosol particles and measuring the current from the charged particle flux. A commercial electrical aerosol size analyzer was used for the aerosol detection. The constructed detector was tested using flow injection analysis with water as the mobile phase, and the signal response was found to be linear for sodium sulfate over the concentration ranges of 0.2-100 µg mL-1 using one of the nebulizers. Minimum mass and concentration detection limits using the more efficient nebulizer were estimated to be 0.2 ng and 10 ng mL-1, respectively. Behavior for most of the other compounds tested was similar with some differences in sensitivity. Testing the detector using reversed phase HPLC for glucose gave a range of linear response and detection Emits that were similar to the flow injection analysis studies. Under most HPLC conditions, the noise will primarily be a function of solvent impurities; however, the electrical aerosol size analyzer allows the removal of small charged particles to improve the signal-to-noise ratio.

"Electrochemical Sensors"
Anal. Chem. 2002 Volume 74, Issue 12 Pages 2781-2800
Eric Bakker and Martin Telting-Diaz

Abstract: The last fundamental review on this general topic was entitled Dynamic Electrochemistry and diverted significantly from the earlier series on chemical sensors by Janata in scope and content. The last chemical sensors review, which appeared in 1998, had made it already clear that modern electronic searching and abstracting tools were becoming so powerful and widespread that it no longer made sense to write comprehensive reviews on a large topic. Indeed, the electrochemical sensors section of that review alone covered already some 1000 references, which made it nearly impossible for the authors to write a truly critical review. It was therefore decided that future fundamental reviews in this series should be drastically more selective. This is a difficult task, as any authors of this series will invariably report on papers that caught their eye and that no longer represent a near-complete coverage of the literature.

"Application Of Isotopically Labeled Methylmercury For Isotope Dilution Analysis Of Biological Samples Using Gas Chromatography/ICPMS"
Anal. Chem. 2002 Volume 74, Issue 11 Pages 2505-2512
R. C. Rodr&iacute;guez Mart&iacute;n-Doimeadios, E. Krupp, D. Amouroux, and O. F. X. Donard

Abstract: An isotope dilution (ID) procedure for the determination of methylmercury (MMHg) in biological samples using an inductively coupled plasma mass spectrometer as detector after the capillary gas chromatographic separation (CGC/ICPMS) has been developed. For the first time, open-focused-microwave pretreatment has been used in conjunction with ID. Optimum conditions for the measurement of isotope ratios on the fast transient chromatographic peaks have been established. Mass bias was found to be about 1.5%/mass unit and was corrected by using the simultaneously measured thallium signals at Tl-203 and Tl-205. After mass-bias correction, deviation of the theoretical mercury ratio values was found to be as low as 0.2%. Isotope ratio precisions based on the peak areas measurements were 0.3% RSD for 20 pg injected (as Hg absolute). The absolute detection limits were in the range of 20-30 fg for Hg-202 and Hg-201. Methylmercury enriched in Hg-201 has been synthesized by direct reaction with methylcobalamine. The concentration of the MMHg spike has been measured by reverse isotope dilution with a natural MMHg standard. The capabilities of CGC/ICPMS to measure isotope ratios were used to optimize sample derivatization by aqueous ethylation with NaBEt4 with respect to MMHg degradation pathways and quantitative recovery. The accuracy of the method developed has been validated with biological certified reference materials (CRM-463, DORM-1).

"Flow Injection On-line Sorption Preconcentration Coupled With Hydride Generation Atomic Fluorescence Spectrometry For Determination Of (Ultra)trace Amounts Of Arsenic(III) And Arsenic(V) In Natural Water Samples"
Anal. Chem. 2002 Volume 74, Issue 9 Pages 2162-2166
Xiu-Ping Yan, Xue-Bo Yin, Xi-Wen He, and Yan Jiang

Abstract: A flow injection on-line sorption pre-concentration and separation in a knotted reactor (KR) was coupled to hydride generation atomic fluorescence spectrometry (HG-AFS) for speciation of inorganic arsenic in natural water samples. The method involved on-line formation of the As(III)-pyrrolidinedithiocarbamate (PDC) complex over a sample acidity of 0.001-0.1 mol L-1 HCl, its adsorption onto the inner walls of the K-R made from 150-cm long x 0.5-mm i.d. PTFE tubing, elution with 1 mol U L-1 HCl, and detection by HG-AFS. Total inorganic arsenic was determined after prereduction of As(V) to As(III) with 1% (m/v) L-cysteine. The concentration of AS(V) was calculated by the difference of the total inorganic arsenic and As(III). A 1 mol L-1 concentration of HCl was employed not only as the efficient eluent but also as the required medium for subsequent hydride generation. Potential factors that affect adsorption, rinsing, elution, and hydride generation were investigated in detail. The low cost, easy operation, and high sensitivity are the obvious advantages of the present system. The consumption of a 6 mL sample solution, an enhancement factor of 11 and a detection limit (3s) of 0.023 µg L-1 As(III) were obtained at a sample throughput of 32 h-1. The precision for 14 replicate measurements of 1 µg L-1 As(III) was 1.3% (RSD). The recoveries from natural water samples varied from 96.7 to 105% for 2 µg L-1 of As(III) spike and from 97.1 to 107% for 2 µg L-1 of As(V) spike. The analytical results obtained by the present method for total arsenic in the certified reference materials, SLRS-4 (river water) and NASS-5 (seawater), agreed well with the certified values. The developed method was also successfully applied to the speciation of inorganic arsenic in local natural water samples.
Knotted reactor

"Three-Layer Flow Membrane System On A Microchip For Investigation Of Molecular Transport"
Anal. Chem. 2002 Volume 74, Issue 9 Pages 2014-2020
Mariana Surmeian, Maxim N. Slyadnev, Hideaki Hisamoto, Akihide Hibara, Kenji Uchiyama and Takehiko Kitamori

Abstract: A stable three-layer flow system, water/organic solvent/water, has been successfully applied for the first time in a microchannel to get rapid transport through an organic liquid membrane. In the continuous laminar flow region, the analyte (methyl red) was rapidly extracted across the microchannel from the donor to the acceptor phase through the organic solvent phase (cyclohexane). Thermal lens microscopy was used to monitor the process. The thickness of the organic phase, sandwiched by the two aqueous phases, was ~64 µm, and it was considered as a thin liquid organic membrane. Permeability studies showed the effects of molecular diffusion, layer thickness, and organic solvent-water partition coefficient on the molecular transport. In the microchip, complete equilibration was achieved in several seconds, in contrast to a conventionally used apparatus, where it takes tens of minutes. The thickness of the organic and aqueous boundary layers was defined as equal to the microchannel dimensions, and the organic solvent-water partition coefficient was determined on a microchip using the liquid/liquid extraction system. Experimental data on molecular transport across the organic membrane were in agreement with the calculated permeability based on the three-compartment water/organic solvent/water model. This kind of experiment can be performed only in a microspace, and the system can be considered as a potential biological membrane for future in vitro study of drug transport.

"Optimal Environment For Glucose Oxidase In Perfluorosulfonated Ionomer Membranes: Improvement Of First-generation Biosensors"
Anal. Chem. 2002 Volume 74, Issue 7 Pages 1597-1603
Arkady A. Karyakin, Elena A. Kotel'nikova, Lilia V. Lukachova, Elena E. Karyakina, and Joseph Wang

Abstract: An optimal environment for glucose oxidase (GOx) in Naflon membranes is achieved using an advanced immobilization protocol based on a nonaqueous immobilization route. Exposure of glucose oxidase to water-organic mixtures with a high (85-95%) content of the organic solvent resulted in stabilization of the enzyme by a membrane-forming polyelectrolyte. Such an optimal environment leads to the highest enzyme specific activity in the resulting membrane, as desired for optimal use of the expensive oxidases. Casting solution containing glucose oxidase and Naflon is completely stable over 5 days in a refrigerator, providing almost absolute reproducibility of GOx-Nafion membranes. A glucose biosensor was prepared by casting the GOx-Nafion membranes over Prussian Blue-modified glassy carbon disk electrodes. The biosensor operated in the FIA mode allows the detection of glucose down to the 0.1 muM level, along with high sensitivity (0.05 A M-1 cm-2), which is only 10 times lower than the sensitivity of the hydrogen peroxide transducer used. A comparison with the recently reported enzyme electrodes based on similar H2O2 transducers (transition metal hexacyanoferrates) shows that the proposed approach displays a dramatic (100-fold) improvement in sensitivity of the resulting biosensor. Combined with the attractive performance of a Prussian Blue-based hydrogen peroxide transducer, the proposed immobilization protocol provides a superior performance for first-generation glucose biosensors in term of sensitivity and detection limits.
Glucose

"Electrogenerated Chemiluminescence Derivatization Reagents For Carboxylic Acids And Amines In High-performance Liquid Chromatography Using Tris(2,2 -bipyridine)ruthenium(II)"
Anal. Chem. 2002 Volume 74, Issue 7 Pages 1584-1589
Hirotoshi Morita and Masaharu Konishi

Abstract: 2-(2-Aminoethyl)-1-methylpyrrolidine and N-(3-aminopropyl)pyrrolidine (NAPP) were found to be selective and sensitive derivatization reagents for carboxylic acid by high-performance liquid chromatography (HPLC) with electrogenerated chemiluminescence detection using tris(2,2-bipyridine)ruthenium(II). Free fatty acids and ibuprofen were used as model compounds of carboxylic acids, and the derivatization conditions were optimized with myristic acid as a representative of free fatty acids. All the fatty acids tested were reacted with NAPP to produce highly sensitive derivatives under the mild reaction conditions of room temperature for 30 min in acetonitrile containing 2-bromo-1-ethylpyridinium tetrafluoroborate and 9-methyl-3,4-dihydro-2H-pyrido[1,2-a]-pyrimidin-2-one. The chemiluminescence intensities were similar for all fatty acids. The derivatives obtained from 10 free fatty acids were completely separated by reversed-phase chromatography under isocratic elution conditions. The on-column detection limits (signal-to-noise ratio of 3) with proposed HPLC separation and chemiluminescence detection were 70 and 45 fmol for myristic acid and ibuprofen, respectively. The free fatty acids in human plasma were successfully determined using the present method. Histamine, a model compound of primary amines, was also determined after precolumn derivatization with 3-(diethylamino)propionic acid at room temperature for 60 min in acetonitrile containing N,N-dicyclohexylcarbodiimide and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine with the detection limit of 70 fmol.

"Continuous-flow Chemical Processing On A Microchip By Combining Microunit Operations And A Multiphase Flow Network"
Anal. Chem. 2002 Volume 74, Issue 7 Pages 1565-1571
Manabu Tokeshi, Tomoko Minagawa, Kenji Uchiyama, Akihide Hibara, Kiichi Sato, Hideaki Hisamoto, and Takehiko Kitamori

Abstract: A new design and construction methodology for integration of complicated chemical processing on a microchip was proposed. This methodology, continuous-flow chemical processing (CFCP), is based on a combination of microunit operations (MUOs) and a multiphase flow network. Chemical operations in microchannels, such as mixing, reaction, and extraction, were classified into several MUOs. The complete procedure for Co(II) wet analysis, including a chelating reaction, solvent extraction, and purification was decomposed into MUOs and reconstructed as CFCP on a microchip. Chemical reaction and molecular transport were realized in and between continuous liquid flows in a multiphase flow network, such as aqueous/aqueous, aqueous/organic, and aqueous/organic/aqueous flows. When the determination of Co(II) in an admixture of Cu(II) was carried out using this methodology, the determination limit (2?) was obtained as 18 nM, and the absolute amount of Co chelates detected was 0.13 zmol, that is, 78 chelates. The sample analysis time was faster than that of a conventional processing system. Moreover, troublesome operations such as phase separation and acid and alkali washing, all necessary for the conventional system, were simplified. The CFCP methodology proposed here can be applied to various on-chip applications.

"Simultaneous Determination Of Trace Cadmium And Arsenic In Biological Samples By Hydride Generation-double Channel Atomic Fluorescence Spectrometry"
Anal. Chem. 2002 Volume 74, Issue 7 Pages 1525-1529
Yun-kai L&uuml;, Han-Wen Sun, Chun-Gang Yuan, and Xiu-Ping Yan

Abstract: Hydride generation atomic fluorescence spectrometry (HG-AFS) has been used for determination of hydride-forming elements because of its high sensitivity, simplicity, and low costs, but most of such work has been concentrated on single element analysis, and reports dealing with multielement determination by HG-nondispersive (ND)AFS are rare. In this work, a sensitive HG-NDAFS method was developed for simultaneous determination of trace cadmium and arsenic in biological materials. The conditions for the generation of volatile cadmium and arsenic species from the reaction with KBH4 in aqueous solution were investigated using a double-channel AFS integrated with an intermittent flow reactor. Like thiourea and Co(II), ascorbic acid was found to significantly enhance the generation efficiency of volatile Cd and As species. The interferences of coexisting ions were evaluated. Under optimal conditions, the detection limits for Cd and As were determined to be 10 and 150 ng L-1, respectively. The precision for 11 replicate determinations at the I µg L-1 Cd level and the 10 µg L-1 As level were 3.5 and 2.7% (RSD), respectively. The recoveries of spike analytes in the biological samples studied ranged from 94 to 109%. The proposed method was successfully applied to the simultaneous determination of Cd and As in a variety of biological samples.

"SERRS. In Situ Substrate Formation And Improved Detection Using Microfluidics"
Anal. Chem. 2002 Volume 74, Issue 7 Pages 1503-1508
Ruth Keir, Eishi Igata, Martin Arundell, W. Ewen Smith, Duncan Graham, Callum McHugh, and Jonathan M. Cooper

Abstract: Surface-enhanced resonance Raman scattering (SERRS) of a model derivative of TNT was detected using a microflow cell designed within the framework of the lab-on-a-chip concept, using only the analyte and readily available reagents. The SERRS substrate, silver colloid, was prepared in situ, on-chip, by borohydride reduction of silver nitrate. The silver colloid was imaged within the chip using a white light microscope in either transmission or, due to the high reflectivity of the colloid, reflection mode. A fine stream of colloid similar to30 mum in width was formed in a 250-mum-wide channel at the point where the colloid preparation reagents met. The chip was designed to produce a concentrated stream of colloid within a laminar regime, such that particles did not readily disperse into the fluid. One result of this was to reduce the effective volume of analysis. Attempts to deliberately disrupt this stream with microstructured pillars, fabricated in the fluidic channels, were unsuccessful. The chip was also designed to have the appropriate dimensions for detection using a modern Raman microscope system, which collects scattering from a very small volume. A dye derived from TNT was used as a model analyte. Quantitative behavior was obtained over 4 orders of magnitude with a detection limit of 10 fmol. This performance is between 1 and 2 orders of magnitude better than that achieved using a macroflow SERRS cell. The technique has the added advantage that both reagent consumption and effluent production are greatly reduced, leading to reduced operating costs and a decreased environmental impact.

"Determination Of Mercury In Coal By Isotope Dilution Cold-vapor Generation Inductively Coupled Plasma Mass Spectrometry"
Anal. Chem. 2002 Volume 74, Issue 7 Pages 1477-1483
Stephen E. Long and W. Robert Kelly

Abstract: A method based on isotope dilution cold-vapor inductively coupled plasma mass spectrometry (ID-CV-ICPMS) has been developed for high-accuracy determinations of mercury in bituminous and sub-bituminous coals. A closed-system digestion process employing a Carius tube is used to completely oxidize the coal matrix and chemically equilibrate the mercury in the sample with a Hg-201 isotopic spike. The digestates are diluted with high-purity quartz-distilled water, and the mercury is released as a vapor by reduction with tin(H) chloride. Measurements of Hg-201/Hg-202 isotope ratios are made using a quadrupole ICPMS system in time-resolved analysis mode. The new method has some significant advantages over e existing methods. The instrument detection limit is less than 1 pg/mL. The average blank (n = 17) is 30 pg, which is roughly 1 order of magnitude lower than the equivalent microwave digestion procedure. The detection limit in coal is blank limited and is similar to40 pg/g. Memory effects are very low. The relative reproducibility of the analytical measurements is similar to0.5% for mercury concentrations in the range 10^-150 ng/g. The method has been used to measure mercury concentrations in six coal reference materials, SRM 1632b (77.4 ng/g), SRM 1632 c (94.3 ng/g), BCR 40 (433.2 ng/g), BCR 180 (125.0 ng/g), BCR 181 (135.8 ng/g), and SARM 20 (252.6 ng/g), as well as a coal fly ash, SRM 1633b (143.1 ng/g). The method is equally applicable to other types of fossil fuels including both crude and refined oils.
Extraction

"Chemiluminometric Sensor For Simultaneous Determination Of L-glutamate And L-lysine With Immobilized Oxidases In A Flow Injection System"
Anal. Chem. 2002 Volume 74, Issue 6 Pages 1269-1274
Nobutoshi Kiba, Takao Miwa, Masaki Tachibana, Kazue Tani, and Hitoshi Koizumi

Abstract: A chemiluminometric flow-through sensor for simultaneous determination of L-glutamate (Glu) and L-lysine (Lys) in a single sample has been developed. Immobilized uricase, immobilized peroxidase, support material, coimmobilized glutamate oxidase/peroxidase, support material, and coimmobilized lysine oxidase/peroxidase were packed sequentially in a transparent PTFE tube, and the tube was placed in front of a photomultiplier tube as a flow cell. A three-peak recording was obtained by one injection of the sample solution. The peak height of the first peak was due to the concentrations of urate and other reductants in the sample; the immobilized uricase was used to decompose urate, and the hydrogen peroxide produced was decomposed with a luminol-hydrogen peroxide reaction by immobilized peroxidase. The peak heights of the second and third peaks were free from the interferences from the reductants and were dependent only on the concentrations of Glu and Lys, respectively. Calibration graphs for Glu and Lys were linear at 40-1,000 and 50-1,200 nM, respectively. The sampling rate was 11/h without carryover. The sensor was stable for two weeks. The sensor system was applied to the simultaneous determination of Glu and Lys in serum. [Journal Article; In English; United States]

"Selective Detection Of O-diphenols On Copper-plated Screen-printed Electrodes"
Anal. Chem. 2002 Volume 74, Issue 5 Pages 1202-1206
Jyh-Myng Zen, Hsieh-Hsun Chung, and Annamalai Senthil Kumar

Abstract: Selective detection of o-diphenols (e.g., catechol, dopamine, and pyrogallol) in the presence of simple phenols, m- and p- derivative diphenols, and ascorbic acid has been demonstrated on copper-plated screen-printed electrodes (CuSPEs) in pH 7.4 phosphate buffer solution. The CuSPE showed, an unusual catalytic response at -0.05 V versus Ag/AgCl selectively to o-diphenolic compounds. The o-diphenols can thus be determined amperometrically through direct electrochemical oxidation in low potentials (similar to0 V), where the CuSPE is much less subject to interfering reactions. Such a catalytic phenomenon cannot be observed on conventional Pt and glassy carbon electrodes. The selective mechanism is explained in terms of the formation of cyclic five-member complex intermediate (Cu(II)-o-quinolate). Most important of all, the common drawbacks of electrode fouling through polymerization were completely overcome in this system.

"Single-channel Microchip For Fast Screening And Detailed Identification Of Nitroaromatic Explosives Or Organophosphate Nerve Agents"
Anal. Chem. 2002 Volume 74, Issue 5 Pages 1187-1191
Joseph Wang, Martin Pumera, Madhu Prakash Chatrathi, Alberto Escarpa, Mustafa Musameh, Greg Collins, Ashok Mulchandani, Yuehe Lin, and Khris Olsen

Abstract: A single-channel chip-based analytical microsystem that allows rapid flow injection measurements of the total content of organic explosive or nerve agent compounds, as well as detailed micellar chromatographic identification of the individual ones, is described. The protocol involves repetitive rapid flow injection (screening) assays--to provide a timely warning and alarm--and switching to the separation (fingerprint identification) mode only when harmful compounds are detected. While micellar electrokinetic chromatography, in the presence of sodium dodecyl sulfate (SDS), is used for separating the neutral nitroaromatic explosive and nerve agent compounds, an operation without SDS leads to high-speed measurements of the total explosives or nerve agent content. Switching between the flow injection and separation modes is accomplished by rapidly exchanging the SDS-free and SDS-containing buffers in the separation channel. Amperometric detection was used for monitoring the separation. Key factors influencing the sample throughput, resolution, and sensitivity have been assessed and optimized. Assays rates of about 360 and 30/h can thus be realized for the total screening and individual measurements, respectively. Ultimately, such development will lead to the creation of a field-deployable microanalyzer and will enable transporting the forensic laboratory to the sample source. [Journal Article; In English; United States]

"A Flow Injection On-line Multiplexed Sorption Preconcentration Procedure Coupled With Flame Atomic Absorption Spectrometry For Determination Of Trace Lead In Water, Tea, And Herb Medicines"
Anal. Chem. 2002 Volume 74, Issue 5 Pages 1075-1080
Yan Li, Yan Jiang, Xiu-Ping Yan, Wen-Jie Peng, and Yue-Ying Wu

Abstract: One of the limitations in previous flow injection (FI) sorption pre-concentration procedures in a knotted reactor (KR), which have been carried out exclusively with a single continuous sample injection over a certain period, is the relatively low retention efficiency (typically 40-50%). Although the sensitivity of such systems could be improved by properly increasing sample pre-concentration time, sample loading flow rate., or both, further improvement of the sensitivity has been limited by the narrow linearity of the relationship between signal intensity and pre-concentration time or sample loading time. In this work, a novel on-line FI multiplexed sorption pre-concentration procedure with repetitive sample injections was developed to overcome the above problems in the previous systems. In contrast to previous FI pre-concentration systems, the proposed multiplexed pre-concentration procedure evenly divides a single longer sample injection step into several shorter substeps while the total pre-concentration time is still kept constant. To demonstrate its merits, the proposed FI on-line KR multiplexed sorption pre-concentration system was combined with flame atomic absorption spectrometry (FAAS) for determination of trace lead in water, tea, and herb medicines. The lead in the sample solution on-line reacted with ammonium pyrrolidine dithiocarbamate, and the resultant analyte complex was sorbed on the inner walls of the KR. The residual sample solution was then removed from the KR with an air flow. The above two steps were repeated eight times with a total pre-concentration time of 120 s. The sorbed analyte was eluted from the KR with 4.5 mol L-I HCl for on-line FAAS detection. The present;multiplexed pre-concentration procedure with eight repetitive sample injections for a total pre-concentration time of 120 s gave a retention efficiency of 92%, twice that obtained by one single sample injection pre-concentration (47%). In addition, the linear ranges of the diagrams of absorbance against sample loading flow rate and sample loading time were extended, offering more potential for achieving high sensitivity by increasing sample loading rates or sample loading time compared to the previous one single continuous sample injection pre-concentration procedure. At a sample loading flow rate of 3.6 mL min-1 for a total pre-concentration period of 120 s, an enhancement factor of 57 and a detection limit (3s) of 8 µg L-1 were obtained. The precision was 1.4% (RSD, n 11) at the 200 µg L-1 level. The developed method was successfully applied to the determination of trace lead in various water samples, herb medicines, and a certified tea reference material.
Knotted reactor

"A Method For Screening Total Mercury In Water Using A Flow Injection System With Piezoelectric Detection"
Anal. Chem. 2002 Volume 74, Issue 4 Pages 921-925
Lisbeth Manganiello, Angel R&iacute;os, and Miguel Valc&aacute;rcel

Abstract: An automatic microgravimetric screening system based on piezoelectric detection and the use of acidic stannous chloride as reductant was developed for the fast detection and determination of total mercury in water. Reduced mercury is detected as an amalgam by using a gold-coated piezoelectric crystal, the sensor subsequently being regenerated by passing it through a peroxydisulfate solution. The gold-coated piezoelectric crystal is a highly efficient retention unit for the main soluble mercury species (inorganic, complexed, and organometallic) previously reduced to elemental mercury and is free of interferences from other metal ions. This detector exhibits good sensitivity: it allows the determination of mercury at sub-parts-per-billion concentration levels (0.30-1.00 µg/L). The precision, expressed as relative standard deviation, was±2.7% (n = 11; P = 0.05) at 0.5 µg/L total mercury. The proposed method was successfully used as a rapid screening method for mercury monitoring in natural waters. [Journal Article; In English; United States]

"Flow-through Cell For On-line Amperometric Determination Of Ce(IV) During Polymerization Reactions"
Anal. Chem. 2002 Volume 74, Issue 4 Pages 915-920
P. Westbroek, J. De Strycker, P. Dubruel, E. Temmerman, and E. H. Schacht

Abstract: In this paper, a method is described for the chronoamperometric determination of Ce(IV) in acidic aqueous solutions in order to study the kinetics and the mechanism of polymerization reactions with Ce(IV) as the initiator. The method is based on the FIA principle, in which small samples are injected in a continuous 1.0 mol L-1 H2SO4 flow, and Ce(M is detected chronoamperometrically by reduction at the surface of a gold CDtrode. Such an electrode is made from a commercial CD that has a gold coating acting as a reflecting layer for the laser beam. It was found that a detection limit of 1.5 10^-7 Mol L-1 could be obtained with this method, that high concentrations (order of 10^-2 mol L-1) can be detected without IR drop and that the system can be used over several days without renewal or recalibration of the gold CDtrode.

"Ultra-Low-Volume, Real-Time Measurements Of Lactate From The Single Heart Cell Using Microsystems Technology"
Anal. Chem. 2002 Volume 74, Issue 4 Pages 908-914
Xinxia Cai, Norbert Klauke, Andrew Glidle, Peter Cobbold, Godfrey L. Smith and Jonathan M. Cooper

Abstract: The fabrication of microelectrodes integrated within ultra-low-volume microtiter chambers for the amperometric determination of metabolites continues to be of interest in the subject of single-cell and high-throughput screening. The microsystem described in this paper consists of a two-microelectrode sensor with a microfluidic dispensation technology, which is able to deliver both very low titers (6.5 pL) and single heart cells into a low-volume microphotoelectrochemical cell. Devices were fabricated using photolithography and liftoff giving reproducible sensors integrated within high aspect ratio titer chambers (with a volume of 360 pL), made of the photoepoxy SU8. In this paper, the determination of lactate was optimized using an enzyme-linked assay based upon lactate oxidase, involving the amperometric determination of hydrogen peroxide at +640 mV versus an internal Ag[verbar]AgCl pseudoreference. The microsystem (including the microfluidic dispensers and structures as well as the microsensor) was subsequently used to measure the lactate content of single heart cells. Dynamic electrochemical measurements of lactate during cell permeabilization are presented. We also show the use of respiratory uncouplers to simulate ischemia in the single myocyte and show that, as expected, the rate of lactate production from the hypoxic heart cell is greater than that within the normoxic healthy myocyte.

"Electrochemical Oxidation Of Chlorophenols At A Boron-doped Diamond Electrode And Their Determination By High-performance Liquid Chromatography With Amperometric Detection"
Anal. Chem. 2002 Volume 74, Issue 4 Pages 895-902
C. Terashima, Tata N. Rao, B. V. Sarada, D. A. Tryk, and A. Fujishima

Abstract: Anodically pretreated. diamond electrodes have been used for the detection of chlorophenols (CPs) in environmental water samples after high-performance liquid chromatographic (HPLC separation. The anodization of as-deposited boron-doped polycrystalline diamond thin-film electrodes has enabled the stable determination of phenols over a wide concentration range. Prior to the HPLC analysis, a comparative study with ordinary glassy carbon, as-deposited diamond, and anodized diamond was made to examine the oxidative behavior of phenols by cyclic voltammety and flow injection analysis with amperometric detection. At anodized diamond electrodes, reproducible, well-defined cyclic voltammograms were obtained even at high CP concentration (5 mM), due to a low proclivity for adsorption of the oxidation products on the surface. In addition, after prolonged use, the partially deactivated diamond could be reactivated on line by applying a highly anodic potential (2.64 Vvs SCE) for 4 min, which enabled the destruction of the electrodeposited polymer deposits. Hydroxyl radicals produced by the high applied potential, in which oxygen evolution occurs, are believed to be responsible for the oxidation of the passivating layer on the surface. When coupled with flow injection analysis (FIA), anodized diamond exhibited excellent stability, with a response variability of 2.3% (n = 100), for the oxidation of a high concentration (5 mM) of chlorophenol. In contrast, glassy carbon exhibited a response variability of 39.1%. After 100 injections, the relative peak intensity, for diamond decreased by 10%, while a drastic decrease of 70% was observed for glassy carbon. The detection limit obtained in the FIA mode for 2,4-dichlorophenol was found to be 20 nM (S/N = 3), with a linear dynamic range up to 100 muM. By coupling with the column-switching technique, which enabled on-line pre-concentration (50 times), the, detection limit was lowered to 0.4 nM (S/N = 3). By use of this technique, anodized diamond electrodes were demonstrated for the analysis of CPs in drainwater that was condensed from the flue gas of waste incinerators.

"Sequential Injection 90Sr Determination In Environmental Samples Using A Wetting-Film Extraction Method"
Anal. Chem. 2002 Volume 74, Issue 4 Pages 826-833
Manuel Mir&oacute;, Enrique G&oacute;mez, Jos&eacute; Manuel Estela, Montserrat Casas, and V&iacute;ctor Cerd&agrave;*

Abstract: A sequential injection procedure involving a flow-reversal wetting-film extraction method for the determination of the radionuclide 90Sr has been developed. The methodology is based on the coating of the inner walls of an open tubular reactor with a film prepared from a 0.14 M 4,4'(5')-bis(tert-butylcyclohexano)-18-crown-6 (BCHC) solution in 1-octanol, which allows the selective isolation of strontium from the sample matrix. Selection of the optimum extractant diluent attending its physical properties, investigation of the extraction kinetics features, and choice of the proper elution procedure are discussed in detail in this paper. The noteworthy aspects of using a wetting-film phase instead of a solid-phase material described to date in the literature are the reduction of crown ether consumption and the simplification of both the operational sequence and the automation of the extractant-phase renewal between consecutive samples, which is of interest to avoid analyte carryover and reduction of the resin capacity factor caused by irreversible interferences. The proposed method has been successfully applied to different spiked environmental samples (water, milk, and soil), with 90Sr total activities ranging between 0.07 and 0.30 Bq, measured using a low-background proportional counter. The standard deviation of the automated analytical separation procedure is lower than 3% (n = 10), and the 90Sr isolation process under the studied conditions may be carried out with a yield up to 80%.
Strontium-90 Environmental Environmental Cow Radiochemical Scintillation counter Sequential injection Extraction Interferences

"Easily Constructed Spectroelectrochemical Cell For Batch And Flow Injection Analyses"
Anal. Chem. 2002 Volume 74, Issue 3 Pages 720-723
Paul A. Flowers, Margaret A. Maynor, and Donald E. Owens

Abstract: The design and performance of an easily constructed spectroelectrochemical cell suitable for batch and flow injection measurements are described. The cell is fabricated from a commercially available 5-mm quartz cuvette and employs 60 ppi reticulated vitreous carbon as the working electrode, resulting in a reasonable compromise between optical sensitivity and thin-layer electrochemical behavior. The spectroelectrochemical traits of the cell in both batch and flow modes were evaluated using aqueous ferricyanide and compare favorably to those reported previously for similar cells. [Journal Article; In English; United States]

"Response Times Of Carbon Fiber Microelectrodes To Dynamic Changes In Catecholamine Concentration"
Anal. Chem. 2002 Volume 74, Issue 3 Pages 539-546
B. Jill Venton, Kevin P. Troyer, and R. Mark Wightman

Abstract: The electrode response time and the measured concentrations during dynamic catecholamine changes were compared using constant potential amperometry and fast-scan cyclic voltammetry. The amperometric response to a rectangular pulse of catecholamine is more rectangular than the cyclic voltammetric response; however, the response times are very similar when, during cyclic voltammetry, the temporal tag due to adsorption and desorption of catecholamine to the electrode is removed by deconvolution. Deconvolution of cyclic voltammetry data was applied to stimulated dopamine release in vivo, allowing for modeling of release and uptake kinetics and to measure catecholamine release from single cells, resulting in better resolution of peaks from single vesicles. In vitro postcalibrations were performed to calculate concentrations of catecholamine measured with cyclic voltammetry and amperometry. The addition of 600 muM ascorbic acid to the postcalibration buffer, allowing a catalytic reaction to regenerate dopamine, resulted in similar calculated concentrations for stimulated release of dopamine using amperometry and cyclic voltammetry. Using deconvoluted cyclic voltammetry to remove the response time lag and adding ascorbic acid to the calibration buffer, the shape and concentration of dynamic catecholamine changes are very similar when measured with constant potential amperometry and cyclic voltammetry.
Diffusion

"Toward A Fully Integrated Positive-pressure Driven Microfabricated Liquid Analyzer"
Anal. Chem. 2002 Volume 74, Issue 1 Pages 177-184
Paul G. Vahey, Sean A. Smith, Colin D. Costin, Younan Xia, Anatol Brodsky, Lloyd W. Burgess, and Robert E. Synovec

Abstract: A versatile integrated analyzer with a flow-programmed injection strategy and multiwavelength detection is described with applications toward sampling, flow injection analysis, and capillary separations. Continuous near-real-time sampling is a major benefit of the flow-programmed injection technique. Injection volumes ranging from 250 pL to several microliters were made without electrophoretic flow. Multiwavelength grating light reflection spectroscopy (GLRS) and transmission absorbance spectroscopy were performed simultaneously in a detection volume of 150 pL The utility of these detection methods for refractive index (RI) and absorbance detection in capillary channels is demonstrated through analysis of salt, indicator, and dyes. GLRS is a unique, selective, and path-length-independent technique for probing RI, absorbance, and other optical properties. A limit of detection (LOD) of 170 muM was achieved for GLRS interferometric detection of FD&C Red #3, which corresponded to 2.6 fmol of analyte in the 150 pL detection volume. A LOD of 2 mM for phosphate buffer, or 3 fmol in the 150 pL detection volume will also be demonstrated. A siloxane coating on the GLRS grating was employed as a sensing layer to probe interactions between the sample and stationary phase. The combined GLRS interferometric response provided insight into both optical and chromatographic properties of samples. Open tubular capillary liquid chromatography with multidimensional multiwavelength detection is demonstrated for the analysis of three food dyes. Separation efficiency, N, of 16 000 was achieved for an unretained dye peak eluting at 12 min. Integration of novel sampling and detection schemes makes this a broadly applicable liquid analyzer.

"Selective Continuous Monitoring And Analysis Of Mixtures Of Acesulfame-K, Cyclamate, And Saccharin In Artificial Sweetener Tablets, Diet Soft Drinks, Yogurts, And Wines Using Filter-supported Bilayer Lipid Membranes"
Anal. Chem. 2001 Volume 73, Issue 24 Pages 5945-5952
Dimitrios P. Nikolelis and Spyros Pantoulias

Abstract: This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring and analysis of mixtures of the artificial sweeteners acesulfame-K, cyclamate, and saccharin using stabilized systems of filter-supported bilayer lipid membranes (BLMs). Injections of artificial sweeteners were made into flowing streams of a carrier electrolyte solution, and a transient current signal with duration of seconds reproducibly appeared in less than < 1 min after exposure of the lipid membranes to the artificial sweeteners. The magnitude of this signal was linearly related to the concentration of artificial sweeteners, which could be determined at micromolar levels. Repetitive cycles of injection of artificial sweeteners have shown no signal degradation during each cycle (30 sequential injections). The time of appearance of the transient response was different for each artificial sweetener and increased in the order of cyclamic acid, acesulfame-K, and saccharin. The difference in time of response has allowed selective detection and analysis of these artificial sweeteners in mixtures. The effect of potent interferences, including a wide range of compounds usually found in foods, proteins, and lipids was investigated. The results showed no interferences from these constituents of real food samples. The major interference from proteins (most common in lipid-film-based biosensors) can be eliminated by modulation of the carrier solution that does not allow adsorption of these compounds in BLMs. The technique was applied in real food samples, that is, in artificial sweetener tablets, diet soft drinks, wines, and yogurts that contain mixtures of these artificial sweeteners with aspartame and other compounds. A comparison of results using the present method and that of an Official Method of Analysis showed good agreement between the two methods. [Journal Article; In English; United States]

"Water Soxhlet Extraction Assisted By Focused Microwaves: A Clean Approach"
Anal. Chem. 2001 Volume 73, Issue 24 Pages 5903-5908
J. L. Luque-Garc&iacute;a and M. D. Luque de Castro

Abstract: A prototype of a Soxhlet extractor that enables the use of water as extractant and application of focused microwaves on the cartridge zone has been designed and checked. The approach consists of a single unit in which the shortening of the distillation glassware allows reception of the water vapor in a refrigerant connected to the top of the sample cartridge vessel, its condensation, and dropping on the solid sample. When the water into the cartridge has reached a given level, irradiation with focused microwaves during an optimized time accelerates the leaching of the target analytes. Then, a valve is actuated and the extract is driven to the distillation flask where the leached species are concentrated as new vapor is formed and sent to the refrigerant and then to the cartridge. This new hybrid discontinuous-continuous approach has been used for leaching acid herbicides from different types of soil. The time required for total removal of the target compounds was 48 min. A flow injection manifold made posible the integration of the subsequent steps (pre-concentration, chromatographic separation, UV detection) for the determination of the analytes, the recoveries of which range from 105.24 to 96.44%. [Journal Article; In English; United States]

"Chemiluminescence Detection For Hybridization Assays On The Flow-Thru Chip, A Three-Dimensional Microchannel Biochip"
Anal. Chem. 2001 Volume 73, Issue 24 Pages 5777-5783
Brady J. Cheek, Adam B. Steel, Matthew P. Torres, Yong-Yi Yu and Hongjun Yang

Abstract: Chemiluminescence (CL) detection is seldom used in two-dimensional solid support microarray platforms because adequate sensitivity and spatial resolution is difficult to achieve. The three-dimensional ordered microchannels of the Flow-thru Chip increase both the sensitivity and spatial resolution required for quantitative CL measurements on microarrays. Enzyme-catalyzed CL reactions for the detection of hybridizations on microchannel glass were imaged using a CCD camera. Signal uniformity, sensitivity, and dynamic range of the detection method were determined. The relative standard deviation of signal intensities across an array of 64 spots was 8.1%. A detection limit of 250 amol of target with a linear dynamic range of 3 orders of magnitude was obtained for a 3-h assay. Similar to two-color fluorescence measurements, multiple enzyme labels were employed to demonstrate two-channel chemiluminescence. A unique method for measuring the relaxation time of a chemiluminescent species is also described.

"Reduction Of Signal Suppression Effects In ESI-MS Using A Nanosplitting Device"
Anal. Chem. 2001 Volume 73, Issue 23 Pages 5635-5644
Eric T. Gangl, Meg Annan, Neil Spooner, and Paul Vouros

Abstract: Electrospray ionization mass spectrometry is a valuable tool in the identification and quantification of drug metabolites in biological fluids. However, there are many instances where matrix components present in these fluids interfere with analyte detection and prevent the acquisition of accurate or complete results. In some instances, the matrix can suppress ionization to such an extent that analytes are completely undetectable by MS. In this work, we investigate how ionization and ion-transfer efficiencies are affected by drastically reducing the flow into the MS. A post-column concentric flow-splitting device was constructed to allow the measurement of analyte signal and ionization suppression across a range of flow rates (0.1-200 µL/min). Using this device, the effects of flow rate on signal intensity and ionization suppression were measured in analytical experiments that included flow injection analysis MS, post-column addition LC-MS, and on-line LC-MS analysis of metabolites generated from rat liver microsomes. The device used to deliver 0.1 µL/min flows is referred to as a nanosplitter because it achieved high split ratios (2000: 1), producing flow rates comparable to those observed in nanoelectrospray. The nanosplitter maintained chromatographic integrity with high fidelity and allowed the direct comparison of analyte signal across a range of flow rates (0.1-200 µL/min). A significant improvement in concentration and mass sensitivity as well as a reduction in signal suppression is observed when the performance at 200 versus 0.1 µL/min flow rate is compared. Using this specially designed concentric splitting device, the advantages of ultralow flow ESI were easily exploited for applications employing large bore chromatography.
Interface

"Carbon Electrodes Modified With Ruthenium Metallodendrimer Multilayers For The Mediated Oxidation Of Methionine And Insulin At Physiological PH"
Anal. Chem. 2001 Volume 73, Issue 22 Pages 5607-5610
Long Cheng, Gilbert E. Pacey, and James A. Cox

Abstract: A pentaerythritol-based metallodendrimer with ruthenium(II) terpyridine units, Ru(II)Den, catalyzed the oxidation of L-methionine and insulin at pH 7.0. The Ru(II)Den was immobilized on a carbon surface through layer-by-layer electrostatic deposition; the negatively charged polymer, poly(styrene sulfonate), was its counterpart. These bilayers were assembled on a glassy carbon electrode that was first modified by deposition of a layer of the conjugate base of sulfanilic acid and then with quaternized poly(4-vinylpyridine). Reversible voltammetry for the Ru(II/III) redox couple was observed, the current for which increased linearly with layer number, n, of Ru(II)Den up to n = 12. Cyclic voltammetry was used to demonstrate the mediation of L-methionine oxidation by a Ru(II)Den-containing multilayer assembly. Flow injection amperometric determination of insulin at pH 7.0 at this modified electrode yielded a calibration curve with the following characteristics: linear dynamic range, 6 nM - 0.4 µM; sensitivity, 225 nA µM-1; detection limit (k = 3 criterion), 2 nM. Of particular importance was that the sensitivity was proportional to the number of Ru(II)Den layers.

"Optical Determination Of Low-level Water Concentrations In Organic Solvents Using Fluorescent Acridinyl Dyes And Dye-immobilized Polymer Membranes"
Anal. Chem. 2001 Volume 73, Issue 21 Pages 5339-5345
Daniel Citterio, Katsuya Minamihashi, Yuka Kuniyoshi, Hideaki Hisamoto, Shin-ichi Sasaki, and Koji Suzuki

Abstract: The fluorescent acridinyl indicators 4-(9-acridinyl)-N-(5-hexenyl)-N-methylaniline (KD-F0011), 6-(9-acridinyl)-1,2,2,3-tetramethyl-2,3-dihydro-1H-perimidine (KD-F0021), and 6-(9-acridinyl)-2-(3-butenyl)-1,2,3-trimethyl-2,3-dihydro-1H-perimidine (KD-F0022) were designed, synthesized, and applied for highly sensitive optical determination of low-level water in organic solvents. All these dyes were found useful as fluorescence indicators for the detection of water below 1% (v/v) in different solvent media with a low detection limit of 0.002% (v/v) or 20 mg/L (22 ppm by weight) for KD-F0021 in THF solution. Sensing membranes made from poly(ethylene glycol) dimethacrylate by photocopolymerization with the indicator KD-F0011 were also prepared. Using the membrane sensor, the lowest detection limit of 0.001% (v/v) or 14 mg/L (20 ppm) water was achieved in diethyl ether samples. This system enables the continuous monitoring of the water content in a flow-through arrangement, where single-wavelength excitation (404 nm) and single-wavelength detection (532 nm) can be used for the fluorescence determination, allowing a simple measurement setup. In a continuous-flow experiment using THF samples, fully reversible and fast signal changes with t95% = 1-2 min for water concentrations up to 0.50% (v/v) were observed. A detection limit of 0.004% (v/v) or 40 mg/L (45 ppm) water in THF was achieved. These characteristics make this type of sensor a useful tool for the online continuous monitoring of water present as an impurity in organic media, which is difficult to achieve using a Karl Fischer instrument.

"Microfluidic Arrays Of Fluid-Fluid Diffusional Contacts As Detection Elements And Combinatorial Tools"
Anal. Chem. 2001 Volume 73, Issue 21 Pages 5207-5213
Rustem F. Ismagilov, Jessamine M. K. Ng, Paul J. A. Kenis and George M. Whitesides

Abstract: This paper describes microfluidic systems that can be used to investigate multiple chemical or biochemical interactions in a parallel format. These three-dimensional systems are generated by crossing two sets of microfluidic channels, fabricated in two different layers, at right angles. Solutions of the reagents are placed in the channels; in different modes of operation, these solutions can be either flowing or stationary-the latter is important when one set of channels is filled with viscous gels with immobilized reagents. At every crossing, the channels are separated either by a single membrane or by a composite separator comprising a membrane, a microwell, and a second membrane. These components allow diffusive mass transport and minimize convective transport through the crossing. Polycarbonate membranes with 0.1-1-µm vertical pores were used to fabricate the devices. Each crossing of parallel channels serves as an element in which chemical or biochemical interactions can take place; interactions can be detected by monitoring changes in fluorescence and absorbance. These all-organic systems are straightforward to fabricate and to operate and may find applications as portable microanalytical systems and as tools in combinatorial research.

"Determination Of Cabergoline By Electrospray Ionization Tandem Mass Spectrometry: Picogram Detection Via Column Focusing Sample Introduction"
Anal. Chem. 2001 Volume 73, Issue 20 Pages 4972-4976
Bruce A. Kimball, Thomas J. DeLiberto, and John J. Johnston

Abstract: An electrospray ionization tandem mass spectrometric method was developed for low-picogram detection of an ergot alkaloid, cabergoline, in coyote plasma extracts. Cabergoline is under investigation as an abortifacient in canid species. Central to the successful development of this method was the ability to introduce relatively large sample volumes into the mass spectrometer. This was achieved by focusing the analyte on a conventional high-performance liquid chromatography guard column prior to elution into the spectrometer. Volumes up to at least 900 µL could be injected onto the guard column using a 100% aqueous mobile phase. Cabergoline retained on the column was eluted as a discrete band into the mass spectrometer by the rapid addition of methanol (30%) to the mobile phase. As compared to flow injection sample introduction, the ability to inject larger sample volumes led to a greatly lowered detection limit. Using this technique and a modification of a previously reported extraction procedure, cabergoline could be determined in coyote plasma at concentrations as low as 9 pg of cabergoline/mL of plasma.

"Microfluidic Device For Airborne BTEX Detection"
Anal. Chem. 2001 Volume 73, Issue 19 Pages 4688-4693
Yuko Ueno, Tsutomu Horiuchi, Takashi Morimoto and Osamu Niwa

Abstract: We fabricated a microfluidic device for the optical detection of airborne benzene, toluene, ethylbenzene and xylenes (BTEX). The device consists of concentration and detection cells formed of 3 cm x 1 cm Pyrex plates. The concentration cell is composed of an adsorbent to concentrate the BTEX gases and a thin-film heater to desorb the concentrated gases from the adsorbent thermally. The collected gases are introduced into the detection cell, which is connected to optical fibers, to measure their absorption spectra. We optimized the device's operating conditions by studying the thermal characteristics of the concentration cell and the time profile of the gas concentration flowing in the detection cell. We used the device under optimized operating conditions to detect toluene gas as a typical example BTEX. The gas concentration amplification rate was ~2 orders of magnitude, and we successfully measured parts-per-million levels of toluene gas with this device.

"A Miniaturized Liquid Core Waveguide-capillary Electrophoresis System With Flow Injection Sample Introduction And Fluorometric Detection Using Light-emitting Diodes"
Anal. Chem. 2001 Volume 73, Issue 18 Pages 4545-4549
Shi-Li Wang, Xiao-Jing Huang, Zhao-Lun Fang, and Purnendu K. Dasgupta

Abstract: 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 Fl 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 µL samples containing a mixture of of fluorescein isothiocyanate (FITC)-labeled amino acids allowed a throughput rate up to 144 samples/ h, with similar to2% 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 mum 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-mum i.d.). Detection limits (S/N = 3) were 1.3 muM for arginine and 1.9 muM for phenylalanine and glycine.

"Nonaqueous Catalytic Fluorometric Trace Determination Of Vanadium Based On The Pyronine B-hydrogen Peroxide Reaction And Flow Injection After Cloud Point Extraction"
Anal. Chem. 2001 Volume 73, Issue 18 Pages 4428-4433
E. K. Paleologos, M. A. Koupparis, M. I. Karayannis, and P. G. Veltsistas

Abstract: The catalytic effect of vanadium on the pyronine B-H2O2 system is examined. Enhancement of the catalytic reaction rate along with the efficiency and selectivity against vanadium is achieved in a formic acid environment in the presence of a nonionic surfactant (Triton X-114). Elimination of drastic interference caused by inorganic acids and aqueous matrix along with a 50-fold pre-concentration of vanadium are facilitated through cloud point extraction of its neutral complex with 8-quinolinol in an acidic solution. Subsequent flow injection analysis (FIA) with fluorometric detection renders the proposed method ideal for selective and cost-effective determination of as little as 0.020 µg L-1 vanadium in environmental, biological, and food substrates. The pre-concentration step can be applied simultaneously to multiple samples, allowing for massive preparation prior to analysis, compensating, thus, for the time-consuming procedure.
Speciation

"Development Of A Flow Injection Capillary Chemiluminescent ELISA Using An Imprinted Polymer Instead Of The Antibody"
Anal. Chem. 2001 Volume 73, Issue 17 Pages 4388-4392
Ioana Surugiu, Juraj Svitel, Lei Ye, Karsten Haupt, and Bengt Danielsson

Abstract: A flow injection competitive assay analogous to enzyme immunoassays has been developed using a molecularly imprinted polymer instead of the antibody. A glass capillary was modified by covalently attaching an imprinted polymer to the inner capillary wall. The herbicide 2,4-dichlorophenoxyacetic acid was used as a model analyte. The analyte was labeled with tobacco peroxidase, and chemiluminescence was used for detection in combination with a photomultiplier tube or a CCD camera. In a competitive mode, the analyte-peroxidase conjugate was passed together with the free analyte through the polymer-coated capillary mounted in a flow system. After a washing step, the chemiluminescent substrate was injected and the bound fraction of the conjugate was quantified by measuring the intensity of the emitted light. Calibration curves corresponding to analyte concentrations ranging from 0.5 ng mL-1 to 50 µg mL-1 (2.25 nM - 225 µM) were obtained. A lowered detection limit by 2 orders of magnitude was obtained when detection was done in discontinuous mode and the chemiluminescence light was conducted inside the photomultiplier tube by an optical fiber bundle, thus yielding a dynamic range of 5 pg mL-1 - 100 ng mL-1 (22.5 pM - 450 nM).

"Optical Biosensors. Monitoring Studies Of Glycopeptide Antibiotic Fermentation Using White Light Interference"
Anal. Chem. 2001 Volume 73, Issue 17 Pages 4313-4318
Rolf T&uuml;nnemann, Martin Mehlmann, Roderich D. S&uuml;ssmuth, Bernd B&uuml;hler, Stefan Pelzer, Wolfgang Wohlleben, Hans-Peter Fiedler, Karl-Heinz Wiesm&uuml;ller, G&uuml;nter Gauglitz, and G&uuml;nther Jung

Abstract: This paper describes the design, characterization, and use of an optical biosensor suited for the process control of biotechnological processes. The detector principle is based on reflectometric interference spectroscopy (RIfS). RIfS enables a label-free, product-specific monitoring, with a future outline for on-line process control. The potential of the RUS biosensor is exemplified by the qualitative and quantitative monitoring of the microbial production of vancomycin-type glycopeptide antibiotics.

"Prediction Of The Chemiluminescent Behavior Of Pharmaceuticals And Pesticides"
Anal. Chem. 2001 Volume 73, Issue 17 Pages 4301-4306
L. Lahuerta Zamora, Y. Fuster Mestre, M. J. Duart, G. M. Ant&oacute;n Fos, R. Garc&iacute;a Dom&eacute;nech, J. G&aacute;lvez &Aacute;lvarez, and J. Mart&iacute;nez Calatayud

Abstract: The present paper deals with the first attempt to apply molecular connectivity calculations to predict a chemical property with analytical usefulness: the chemiluminescent behavior of substances when reacted with common oxidants in a liquid phase. Preliminary evidence when searching for new direct CL methods consisted of the examination of analyte reaction with a wide range of oxidants and media. This task, which results in time-consuming and trial-and-error expensive procedures, is necessary due to ensure empirical or theoretical rules for CL prediction are available. On the other hand, in quantitative structure-activity relationship studies, molecular connectivity is a topological method capable of describing the structure of a molecule by means of numbers named indices; subsequent regression in relation to the experimental values of the physical, chemical, or biological properties yields a series of functions called connectivity functions. Discriminant analysis was applied to 200 either chemiluminescent or nonchemiluminescent substances found either bibliographically or in an experimental screening. The method used for the selection of descriptors was a stepwise linear discriminant analysis from the Snedecor F-parameter. The classification criterion used was the minimum,value of Mahalanobis. The quality of the discriminant function was calculated through the Wilks U-statistical parameter. Finally, the function was applied to a database including of more than 50 000 structurally heterogeneous compounds. The theoretical predictions were faced with the empirical evidence obtained through a continuous-flow manifold.

"Continuous-flow Analysis Of Dissolved Inorganic Carbon Content In Seawater"
Anal. Chem. 2001 Volume 73, Issue 17 Pages 4111-4116
M. H. C. Stoll, K. Bakker, G. H. Nobbe, and R. R. Haese

Abstract: A rapid, continuous-flow determination of total inorganic carbon (TIC) in seawater samples is presented. The method runs on an autoanalyzer Traacs 800 spectrophotometric system and is calibrated versus certified reference materials readily available. Atypical analysis speed of 45 samples/h can be reached with an accuracy of 2-3 muM and a precision of similar to2.5 muM. The analysis requires only a small amount of sample and is thus ideally suited for pore water samples and samples taken from cultures where sample volume is at a premium. The speed of the analysis makes mapping of oceanic surface water characteristics possible. Potential interference of sulfide in anoxic (e.g., pore water) samples can be masked by the addition of a hydrogen peroxide step. Although the latter is a strong oxidative reagent, no significant effect on TIC concentration due to oxidation of (labile) organic matter could be found.
Calibration

"Photothermal Temperature Control Of A Chemical Reaction On A Microchip Using An Infrared Diode Laser"
Anal. Chem. 2001 Volume 73, Issue 16 Pages 4037-4044
Maxim N. Slyadnev, Yuki Tanaka, Manabu Tokeshi and Takehiko Kitamori

Abstract: We have demonstrated that a miniaturized device with IR laser heating of the solvent, based on a photothermal effect, is capable of fast and localized control of an enzymatic reaction on a microchip under flow conditions. Using noncontact spectroscopic temperature-sensing techniques, we measured temperature dynamics and spatial distribution and compared the measurements with results of numerical simulation analysis. The device was operated at ultrafast heating and cooling rates of 67 and 53°C/s, respectively, which is 30 times faster than conventional systems and 3-6 times faster than electrothermal miniaturized thermocyclers. The IR laser-mediated heater is characterized by a significantly reduced heated volume of only 5 nL, compared to existing chip-based systems with electrothermal heating. Direct heating of a sample with extremely small heat capacity led us to a fast heating rate, and efficient heat removal through heat transfer to the glass substrate resulted in a fast cooling rate. Reproducible temperature levels with dwell times shorter than 0.5 s were achieved. The enzyme reaction on a chip was successfully controlled with 0.6-s time resolution, using periodic photothermal heating by IR laser. The IR diode laser is compact and thus suits well the miniaturized system design. Our work gives the basis for integration in a chip format of a variety of chemical processes that require fast temperature control.

"Continuous-flow, On-line Monitoring Of Biospecific Interactions Using Electrospray Mass Spectrometry"
Anal. Chem. 2001 Volume 73, Issue 16 Pages 3816-3823
A. C. Hogenboom, A. R. de Boer, R. J. E. Derks, and H. Irth

Abstract: A continuous-flow analytical screening system is presented using electrospray mass spectrometry to measure the interaction of biologically active compounds with soluble affinity proteins. The biochemical detection system is based on a solution-phase, homogeneous assay. In a first step, compounds to be screened (e.g., biotinylated compounds, concentration range 10^-1,000 nmol/L) are injected into a continuous-flow reaction system and allowed to react with the affinity protein (e.g., streptavidin, concentration range 3-48 nmol/L). Subsequently, a reporter ligand (fluorescein-labeled biotin 96 nmol/L) is added to saturate the remaining free binding sites of the affinity protein and the concentration of unbound reporter ligand is measured using electrospray MS in the selected ion monitoring mode. The presence of active compounds in the sample results in an increase of the concentration of unbound reporter ligands. The feasibility of a homogeneous MS-based biochemical assay is demonstrated using streptavidin/biotin and anti-digoxigenin/digoxin as model systems. Compared to radioactive or fluorescence-based biochemical assays, the present assay format does not require the synthesis and purification of labels. Various analytical conditions were investigated to determine the ability of MS to measure the biochemical interactions. The availability of a single ligand that can be detected at 10^-50 nmol/L concentrations by electrospray MS is sufficient to set up the biochemical assay. For the biospecific interactions studies, detection limits of 10^-100 nmol/L were obtained.

"Ultrasonic Extraction Followed By Sonolysis-ozonolysis As A Sample Pretreatment Method For Determination Of Reactive Arsenic Toward Sodium Tetrahydroborate By Flow Injection-hydride Generation AAS"
Anal. Chem. 2001 Volume 73, Issue 15 Pages 3732-3736
J. L. Capelo, I. Lavilla, and C. Bendicho

Abstract: A new sample pretreatment method based on ultrasonic extraction in HCl medium and subsequent oxidation of the extracts by sonozone (i.e., sonolysis-ozonolysis) has been developed for determination of reactive arsenic toward sodium tetrahydroborate [mainly As(III) + As(V)] by flow injection-hydride generation atomic absorption spectrometry. This method avoids the use of intensive treatments with concentrated and corrosive acids, high pressures, and temperatures that are inherent with traditional wet or dry ashing procedures and entails reduced waste production and reagent consumption. A sonozone process at room temperature was optimized to break the bond of As to proteins and macromolecular constituents which was an essential requirement for effective reduction by L-cysteine prior to arsine generation. Spiking experiments showed that As(III), As(V), MMA, and DMA were fully recovered from several matrixes on applying the above treatment. On the other hand, a nonreducible As species such as arsenobetaine that is predominant in some biological samples remained unchanged. Application of the method to sediment, soil, fly ash, and plant CRMs demonstrated that, in general, a good agreement existed between certified and found As contents, thereby indicating the absence of nonreducible As forms. Low As recoveries were observed for fish CRMs, as a result of the nondegradability of arsenobetaine by sonozone. The detection limit of As in the samples investigated was in the range 0.19-2.8 µg g-1.
Speciation Optimization

"Online Incorporation Of Cloud Point Extraction To Flow Injection Analysis"
Anal. Chem. 2001 Volume 73, Issue 14 Pages 3502-3505
Qun Fang, Ming Du, and Carmen W. Huie

Abstract: The on-line incorporation of cloud point extraction (CPE) to flow injection analysis (FIA) is demonstrated for the first time, The technical difficulties of inducing the cloud point phenomenon, separating the surfactant-rich phase from the aqueous phase, and detecting trace amounts of analyte(s) in the presence of the highly scattering surfactant medium in an on-line FIA system were resolved by the following: (1) mixing the sample solution containing the analyte(s) and CPE surfactant with an appropriate salting-out agent, (2) using a collection column to entrap the analyte-containing surfactant aggregates, and ( 3) employing the peroxyoxalate chemiluminescence reaction for the sensitive and selective determination of the analyte(s) in the presence of surfactant micelles, The figures of merit for the determination of coproporphyrin in pretreated urine samples were as follows: precision, 1.1-2.2% (RSD); limit of detection, 2.0 µg/L; and the calibration curve was linear from 46 to 2319 (µ/L(r = 0.9996).
Preconcentration

"Application Of ABTS Radical Cation For Selective On-line Detection Of Radical Scavengers In HPLC Eluates"
Anal. Chem. 2001 Volume 73, Issue 14 Pages 3373-3381
Irina I. Koleva, Harm A. G. Niederl&auml;nder, and Teris A. van Beek

Abstract: The radical cation 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate), (ABTS*+) was utilized in an on-line HPLC method for the detection of radical scavengers in complex matrixes. The HPLC-separated analytes react post-column with the preformed ABTS*+, and the induced bleaching is detected as a negative peak by an absorbance detector at 734 nm. An optimized instrumental and experimental setup is presented. The method is suitable for both isocratic and gradient HPLC runs using mobile phases containing 100% organic solvent or its solution in water, weak acids, or buffers (pH 3-7.4). The method is sensitive, selective, relatively simple, applicable to compounds of different chemical natures; uses common instruments and inexpensive reagents; and has a time-saving, nonlaborious experimental protocol. It can also be used for quantitative analysis. The method was applied to several pure natural antioxidants and plant extracts. The minimum detectable concentration varied from 0.02 to 0.13 µg/mL, depending on the compound tested. The method can be applied to perform kinetic studies, which is illustrated by determination of Trolox equivalent antioxidant capacities (TEAC) of several known antioxidants in flow injection mode. [Journal Article; In English; United States]

"Sensitive And Selective Flow Injection Analysis Of Hydrogen Sulfite/sulfur Dioxide By Fluorescence Detection With And Without Membrane Separation By Gas Diffusion"
Anal. Chem. 2001 Volume 73, Issue 13 Pages 3187-3192
Hasson Mana and Uwe Spohn

Abstract: Highly sensitive and selective FIA flow injection analysis procedures for the determination of sulfite/hydrogen sultite/sulfur dioxide were developed on the basis of an in situ-generated o-phthalaldehyde (OPA)/ammonium reagent and fluorescence detection. The highest sensitivity was achieved at an excitation wavelength of 330 nm, an emission wavelength of 390 nm, and at pH 6.5. Sulfite concentrations between 2.5 nM and 5 muM can be determined with relative standard deviations between 10.5 and 1.0% (n = 5, confidence level α = 0.05) by utilization of a reagent that contains 0.2 mM OPA and 0.4 M NH4Cl in 50 mM potassium phosphate buffer. A concentration of 0.1 mM sulfite can be selectively detected in the presence of thiosulfate, thioglycolate, tetrathionate, cysteine, and ascorbate, The fluorometric sulfite detection was combined with a membrane gas diffusion step to improve the selectivity with respect to nonvolatile fluorescing substances. The total sulfite content can be quantitatively separated as sulfur dioxide into an acceptor solution before its now detection. Between 40 nM and 0.1 mM sulfite can be determined. After 1000-fold dilution, the total sulfite content can be determined in white and red wines.

"Identification Of Volatile Selenium Compounds Produced In The Hydride Generation System From Organoselenium Compounds"
Anal. Chem. 2001 Volume 73, Issue 13 Pages 3181-3186
Amit Chatterjee, Yasuyuki Shibata, Minoru Yoneda, Rupendranath Banerjee, Masao Uchida, Hiroyuki Kon, and Masatoshi Morita

Abstract: We report a novel aqueous derivatization of selenomethionine (Semet), selenoethionine (Seet) and trimethylselenonium ion (TmSe) by NaBH4 and HCl to volatile selenium species, namely, diethyldiselenide (DeDSe), dimethyldiselenide (DMDSe), dimethylselenide (DmSe) and ethylhydrogenselenide (ESeH), in the hydride generation (HG) system. The volatile selenium compounds produced in the HG system were on-line trapped and concentrated in a U-tube that was immersed in the liquid nitrogen trap. The trapped volatile Se compounds were volatilized at 80°C in a water bath, and 50-500 µL of volatile gas was injected into the GC/AED and GC/MS, respectively. It has been established that DmSe, DmDSe, and DeDSe are the predominant Se compounds that are produced in the HG system from TmSe, Semet, and Seet, respectively, followed by ESeH from Seet, Analytical methods previously employed have stated that these compounds are inactive in the HG system, Prior decomposition of Semet, Seet, and TmSe to selenous acid is essential before HG. To the best of our knowledge, current findings for the production and identification of volatile selenium compounds in the HG system are new and different from existing reports; hence, direct estimation of Semet, Seet, and TmSe is possible when coupling with a HG system using a suitable Se-specific detector.
Speciation

"Adaptation Of A Surface Plasmon Resonance Biosensor With Microfluidics For Use With Small Sample Volumes And Long Contact Times"
Anal. Chem. 2001 Volume 73, Issue 13 Pages 2828-2835
Miguel Abrantes, M. Teresa Magone, Lisa F. Boyd and Peter Schuck

Abstract: The efficient delivery of sample to surface-immobilized sites is a key element in biosensing. For a surface plasmon resonance (SPR) biosensor, this has been addressed by constant flow through a microfluidic system with a sample injection loop (Sjölander, S.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345). The present study describes an alternative mode of sample delivery without constant unidirectional flow. It was implemented on a commercial Biacore X SPR biosensor equipped with a microfluidic cartridge, but with the fluidic handling performed by an externally computer-controlled syringe pump. We demonstrate that sample volumes as low as 2 µL can be reproducibly positioned to cover the sensor surfaces, manipulated in a serial fashion, efficiently mixed by applying an oscillatory flow pattern, and fully recovered. Compared to the traditional continuous unidirectional flow configuration, we found very similar kinetic responses at high analyte concentrations and slightly slower responses at low concentrations, most likely due to depletion of analyte from the small sample volumes due to surface binding. With the antibody-antigen systems tested, binding parameters were obtained that are generally within 10% of those from conventional experiments. In the new configuration, biosensor experiments can be conducted without the usual constraints in the surface contact time that are correlated with sample volume and mass transport rate. This can translate to improved detection limits for slow reactions and can facilitate kinetic and thermodynamic binding studies.

"Pharmaceuticals And Related Drugs"
Anal. Chem. 2001 Volume 73, Issue 12 Pages 2805-2816
R. K. Gilpin and L. A. Pachla

Abstract: The current article represents an overview of the pharmaceutical methods that have appeared in either Analytical Abstracts or Chemical Abstracts during the past two years. As has been the case in past reviews, it does not cover biochemical or clinical aspects of the topic but concentrates on procedures that are concerned with unformulated and formulated products. However, because of space and citation (i.e., no more than a maximum of 300) limitations from Analytical Chemistry, several major changes in organization of the review and the topics to be covered were necessary. Instead of 11 major sections as has been the case in past reviews, there are now six topical areas: General Information; Alkaloids; Antibiotics; Nitrogen-Oxygen-Containing Compounds; Peptides, Proteins, and Related Compounds; and Techniques. Major sections eliminated are Inorganics, Steroids, Sulfur-Containing Compounds, Vitamins, and Miscellaneous. Compounds that have formally appeared in these latter categories are not considered or in some cases they appear in a related section. In addition, routine procedures, less often used techniques, and more common approaches typically are not included and a citation generally appears only in a single section. Clearly, the articles selected represent only a very small fraction (i.e., probably <5%) of the total number of papers published.

"Determination Of Total Sulfur At Microgram Per Gram Levels In Geological Materials By Oxidation Of Sulfur Into Sulfate With In Situ Generation Of Bromine Using Isotope Dilution High-resolution ICPMS"
Anal. Chem. 2001 Volume 73, Issue 11 Pages 2547-2553
Akio Makishima and Eizo Nakamura

Abstract: We have developed a new, simple, and accurate method for the determination of total sulfur at microgram per gram levels in milligram-sized silicate materials with isotope dilution high-resolution inductively coupled plasma mass spectrometry equipped with a flow injection system. In this method, sulfur can be quantitatively oxidized by bromine into sulfate with achievement of isotope equilibrium between the sample and spike. Detection limits for 32S+ and 34S+ in the ideal solution and silicate samples were 1 and 6 ng mL-1 and 0.07 and 0.3 µg g-1, respectively. The total blank was 46 ng, so that a 40 mg silicate sample containing 10 µg g-1 sulfur can be measured with a blank correction of < 10%. This total blank can be lowered to 8 ng if a low-blank air system is used for evaporations. To evaluate the applicability of this method, we analyzed not only silicate reference materials with sulfur content of 5.25-489 µg g-1 and sample sizes of 13-40 mg but also the Allende meteorite with a sulfur content of 2%. The reproducibility for various rock types was <9%, even though blank corrections in some samples of low sulfur content were up to 24%. This method is suitable for analyzing geological samples as well environmental samples such as soils, sediments, and water samples.

"Determination Of Picomolar Levels Of Iron In Seawater Using Catalytic Cathodic Stripping Voltammetry"
Anal. Chem. 2001 Volume 73, Issue 11 Pages 2522-2528
Hajime Obata and Constant M. G. van den Berg

Abstract: A new procedure for the direct determination of picomolar levels of iron in seawater is presented. Cathodic stripping voltammetry (CSV) is preceded by adsorptive accumulation of the iron(III) -2,3-dihydroxynaphthalene (DHN) complex from seawater, containing 20 muM DHN at pH 8.0, onto a static mercury drop electrode, followed by reduction of the adsorbed species. The reduction current is catalytically enhanced by the presence of 20 mM bromate. Optimized conditions include a 60-s adsorption period at -0.1 V and a voltammetric scan using sampled de modulation at 10 Hz, In these conditions, a detection limit of 13 pM iron in seawater was achieved which can be lowered further by extending the adsorption time to 300 s. The new catalytic CSV method is similar to5 times more sensitive than existing CSV methods and was tested on samples from the Atlantic Ocean.
Preconcentration

"Development Of Isotope Dilution Cold Vapor Inductively Coupled Plasma Mass Spectrometry And Its Application To The Certification Of Mercury In NIST Standard Reference Materials"
Anal. Chem. 2001 Volume 73, Issue 10 Pages 2190-2199
S. J. Christopher, S. E. Long, M. S. Rearick, and J. D. Fassett

Abstract: An isotope dilution cold vapor inductively coupled plasma mass spectrometry (ID-CV-ICPMS) method featuring gaseous introduction of mercury via tin chloride reduction has been developed and applied to the quantification and certification of mercury in various NIST standard reference materials: SRM 966 Toxic Metals in Bovine Blood (30 ng mL-1); SRM 1641d Mercury in Water (1.6 µg mL-1); and SRM 1946 Lake Superior Fish Tissue (436 ng g-1). Complementary mercury data were generated for SRMs and NIST quality control standards using cold vapor atomic absorption spectroscopy (CVAAS). Certification results for the determination of mercury in SRM 1641d using two independent methods (ID-CV-ICPMS and CVAAS) showed a degree of agreement of 0.3% between the methods. Gaseous introduction of mercury into the ICPMS resulted in a single isotope sensitivity of 2 x 10(6) counts s-1 ng-1 g for Hg-201 and significantly reduced the memory and washout effects traditionally encountered in solution nebulization ICPMS. Figures of merit for isotope ratio accuracy and precision were evaluated at dwell times of 10, 20, 40, 80, and 160 ms using SRM 3133 Mercury Spectrometric Solution. The optimum dwell time of 80 ms yielded a measured Hg-201/Hg-202 isotope ratio within 0.13% of the theoretical natural value and a measurement precision of 0.34%, on the basis of three replicate injections of SRM 3133.
Mercury NIST 3133 NIST 1641d NIST 966 NIST 1946 Mass spectrometry Mass spectrometry Spectrophotometry Amalgamation Volatile generation Reference material Method comparison

"Simultaneous Screen For Microsomal Stability And Metabolite Profile By Direct Injection Turbulent-laminar Flow LC-LC And Automated Tandem Mass Spectrometry"
Anal. Chem. 2001 Volume 73, Issue 9 Pages 2140-2146
H. K. Lim, K. W. Chan, S. Sisenwine, and J. A. Scatina

Abstract: A LC-LC/MS/MS method has been developed that significantly increases the throughput in metabolism screening of drug candidates during lead optimization in discovery. This was accomplished by the reduction of sample preparation time through an on-line extraction of a drug and its metabolites from microsomal proteins using turbulent flow chromatography. Following its injection onto a column at turbulent flow, the drug and its metabolites are backwashed onto a reverse-phase column sia online column switching and resolved chromatographically at a laminar noir: of 2 mL/min. This tandem turbulent-laminar flow chromatographic system in a total cycle time of 8 min can achieve adequate separation of isomeric metabolites of venlafaxine, haloperidol, or adatanserin, Further improvement in throughput can be achieved by multiplexing both microsomal stability assessment and metabolite profiling into a single analysis. This is made possible by the ability of the ion-trap mass spectrometer to perform simultaneously multiple-reaction monitoring for microsomal stability and data-dependent multiple-stage mass spectrometric analysis for metabolite profiling within a single LC analysis. Such a LC-LC/MS/MS approach call dramatically shorten the time for providing metabolism feedback to the drug discovery process.

"Integrated Plastic Microfluidic Devices With ESI-MS For Drug Screening And Residue Analysis"
Anal. Chem. 2001 Volume 73, Issue 9 Pages 2048-2053
Yun Jiang, Pen-Cheng Wang, Laurie E. Locascio and Cheng S. Lee

Abstract: For this work, two different plastic microfluidic devices are designed and fabricated for applications in high-throughput residue analysis of food contaminants and drug screening of small-molecule libraries. Microfluidic networks on copolyester and poly(dimethylsiloxane) substrates are fabricated by silicon template imprinting and capillary molding techniques. The first device is developed to perform affinity capture, concentration, and direct identification of targeted compounds using electrospray ionization mass spectrometry. Poly(vinylidene fluoride) membranes sandwiched between the imprinted copolyester microchannels in an integrated platform provide continuous affinity dialysis and concentration of a reaction mixture containing aflatoxin B1 antibody and aflatoxins. The second microfluidic device is composed of microchannels on the poly(dimethylsiloxane) substrates. The device is designed to perform miniaturized ultrafiltration of affinity complexes of phenobarbital antibody and barbiturates, including the sequential loading, washing, and dissociation steps. These microfabricated devices not only significantly reduce dead volume and sample consumption but also increase the detection sensitivity by at least 1-2 orders of magnitude over those reported previously. Improvements in detection sensitivity are attributed to analyte pre-concentration during the affinity purification step, limited analyte dilution in the microdialysis junction, minimal sample loss, and the amenability of ESI-MS to nanoscale sample flow rates.

"Chromatographic Immunoassays"
Anal. Chem. 2001 Volume 73, Issue 7 Pages 198A-205A
David S. Hage and Mary Anne Nelson

Abstract: When a simple and selective method is required, there are few analytical techniques that compare with immunoassays. This group of methods uses antibodies or antibody-related molecules as reagents for chemical analysis (1, 2). Immunoassays are among the most specific of the analytical techniques, provide low detection limits, and can be used for a wide range of substances. These features, plus the fact that many samples need little or no pretreatment, have made immunoassays common in clinical testing, pharmaceutical analysis, environmental monitoring, and food safety testing. There are many ways in which immunoassays can be performed, but a recent approach based on LC, known as a chromatographic or flow immunoassay, has received particular attention (3 10). We examine this method s basic principles, discuss various formats in which it can be performed, and describe the potential advantages, limitations, and applications.
Preconcentration

"In Line Valve Injection For Capillary Electrophoresis"
Anal. Chem. 2001 Volume 73, Issue 9 Pages 1974-1978
Lisa M. Ponton and Christine E. Evans

Abstract: Direct in-line injection is successfully demonstrated for capillary electrophoresis using a commercially available injection valve designed for liquid chromatographic applications. The internal, fluid-contacting materials in this valve injector are composed of ceramics and PEEK (polyetheretherketone). In studies up to 20 kV, this materials design provides a sufficient dielectric interface to insulate the high-voltage buffer from the metal valve body. Partial-loop injections from 6 to > 60 nL are shown to be highly reproducible and generally consistent with direct electrokinetic injections under the same experimental conditions. The small extracolumn variance contributed hy the valve injection system is symmetrical, and the measured theoretical plates for 75-mum- and 100-mum-i.d., separation capillaries are 1.6 x 10(5) and 2.5 x 10(5), respectively, As a result, the separation performance is quite good, demonstrating the viability of in-line valve injection for capillary electrophoresis. This development in capillary electrophoretic instrumentation has important implications for the advancement of electrophoretic applications as well as for the design of completely integrated analysis systems.

"A Picoliter-Volume Mixer For Microfluidic Analytical Systems"
Anal. Chem. 2001 Volume 73, Issue 9 Pages 1942-1947
Bing He, Brian J. Burke, Xiang Zhang, Roujian Zhang and Fred E. Regnier

Abstract: Mixing confluent liquid streams is an important, but difficult operation in microfluidic systems. This paper reports the construction and characterization of a 100 pL mixer for liquids transported by electroosmotic flow. Mixing was achieved in a microfabricated device with multiple intersecting channels of varying lengths and a bimodal width distribution. All channels running parallel to the direction of flow were 5 µm in width whereas larger 27-µm-width channels ran back and forth through the parallel channel network at a 45°C angle. The channel network composing the mixer was ~10 µm deep. It was observed that little mixing of the confluent solvent streams occurred in the 100-µm-wide, 300-µm-long mixer inlet channel where mixing would be achieved almost exclusively by diffusion. In contrast, after passage through the channel network in the ~200-µm-length static mixer bed, mixing was complete as determined by confocal microscopy and CCD detection. Theoretical simulations were also performed in an attempt to describe the extent of mixing in microfabricated systems.

"On-Chip Thermopneumatic Pressure For Discrete Drop Pumping"
Anal. Chem. 2001 Volume 73, Issue 8 Pages 1831-1838
K. Handique, D. T. Burke, C. H. Mastrangelo and M. A. Burns

Abstract: A class of 'lab-on-a-chip' devices use external air pressure for pumping discrete drops in a microchannel network. External air connectors can be cumbersome and are real-estate intensive. We have developed an on-chip technique to generate pressures required for metering and pumping of nanoliter-volume discrete drops. This is achieved by heating of trapped air in a pressure-generating chamber. The pressure-generating chamber is connected to the point of pressure application in the liquid-conveying microchannel through an air-delivery channel. The trapped air volume on the order of 100 nL is heated by resistive metal heaters by tens of °C to generate air pressures on the order of 7.5 kN/m2. The rate of discrete drop pumping is electronically controlled in the microchannel device by controlling the rate of air heating. Flow rates on the order of 20 nL/s are obtained in the microchannel (300 µm x 30 µm) by heating the air chamber at the rate of ~6°C/s. In this paper, we describe the design, fabrication, and operation of this new technique of generating on-chip air pressure, used for metering and pumping nanoliter discrete drops in microchannels.

"A Polyaniline-modified Electrode-based FIA System For Sub-ppb-level Chromium(VI) Analysis"
Anal. Chem. 2001 Volume 73, Issue 6 Pages 1377-1381
Yun-Jung Yang and Hsuan-Jung Huang

Abstract: By taking advantage of the electrocatalytic effect of polyaniline, a polyaniline/polystyrene composite electrode was prepared and used as a sensitive FIA detector for the analysis of Cr(VI). Optimal conditions, such as applied potential, pH, and now rate of carrier for flow injection analysis, were studied. A linear dynamic range from 0.08 to 32.00 ppb with a linear correlation coefficient of 0.9999 and a relative standard deviation (n = 17) of 1.67% for successive injection of 2.0 ppb Cr(VI) were obtained. By incorporating a Dowex 1 x 4 anion-exchange column into the FIA system, interference to the Cr(VI) analysis can be eliminated and the detection limit for Cr(VI) analysis can be lowered to 0.004 ppb. The contents of Cr(VI) in seawater samples were analyzed to demonstrate the capability of the developed system.
Polymer Preconcentration

"Plant Tissue-based Chemiluminescence Flow Biosensor For Glycolic Acid"
Anal. Chem. 2001 Volume 73, Issue 6 Pages 1203-1206
Baoxin Li, Zhujun Zhang, and Yan Jin

Abstract: A novel plant tissue-based chemiluminescence (CL) biosensor for glycolic acid combined with flow injection analysis is proposed in this paper. The spinach tissue acts as the molecular recognition element. Glycolic acid is oxidized by oxygen under the catalysis of glycolate oxidase in the tissue column to produce hydrogen peroxide, which can react with luminol in the presence of peroxidase of spinach tissue to generate a CL signal. The CL emission intensity was linear with glycolic acid concentration in the range of 4 x 10^-3-4 x 10^-6 mol/L and the detection limit was 1.3 x 10^-6 mol/L. The biosensor was stable for about 3 weeks. A complete analysis, including sampling and washing, could be performed in 1.5 min with a relative standard deviation of 1.7%. [Journal Article; In English; United States]

"Electrocatalytic Oxidation And Sensitive Detection Of Cysteine On A Lead Ruthenate Pyrochlore Modified Electrode"
Anal. Chem. 2001 Volume 73, Issue 6 Pages 1169-1175
Jyh-Myng Zen, Annamalai Senthil Kumar, and Jyh-Cheng Chen

Abstract: Electrocatalytic oxidation of cysteine (CySH) at Nafion/ lead ruthenate pyrochlore (Py) chemically modified electrodes was thoroughly studied. Electrochemical ac impedance spectroscopy analysis indicated the formation of Py microparticles in the interfacial galleries of Nafion. Experiments with benchmark systems of Fe(CN)(6)(3-/4-)and RU(bpy)(2+/3+) reveal the suppression of Nafions anionic character after the in situ precipitation of Py. Michaleis-Menten-type kinetics with the rate determination step of CSS-Py-Ru-VI --> Py-Ru-IV + CyS-SCy was proposed for this catalytic oxidation. The electrocatalytic behavior is further developed as a sensitive detection scheme for CySH by square-wave voltammetry (SWV) and now injection analysis (FIA). Under the optimized conditions, the calibration curve is linear up to 560 muM with a detection limit (signal/noise 3) of 1.91 muM in SWV. The detection limit can be improved to 1.70 nM (i.e., 24.22 ng in a 20 µL sample loop) in EIA. This is the lowest value ever reported for direct CySH determination without preliminary accumulation.

"Fluorescent Liposome Flow Markers For Microscale Particle-Image Velocimetry"
Anal. Chem. 2001 Volume 73, Issue 5 Pages 1057-1061
Anup K. Singh, Eric B. Cummings and Daniel J. Throckmorton

Abstract: Unilamellar liposomes carrying both encapsulated and surface-immobilized fluorophores have been synthesized as novel fluorescent markers to image flow profiles in microfabricated structures. The unilamellar liposomes were made with phospholipids and cholesterol by extrusion through a polycarbonate membrane. They contained carboxyfluorescein in the aqueous core and fluorescein-labeled lipids in the bilayer to render them both a surface and volume fluorescer, maximizing their fluorescence intensity. The lipid composition was chosen to impart a net negative charge to liposomes to minimize self-aggregation as well as interaction with negatively charged glass surfaces of the channels. These liposomes were monodisperse (mean diameter 283 nm), neutrally buoyant, and hydrophilic and exhibited no adsorption on glass surfaces. Unlike polystyrene spheres, they were readily broken up by surfactants, thereby allowing for easy and complete removal from microfluidic channels. The fluorescent liposomes were used to investigate pressure-driven flow in an offset cross intersection in a microfluidic chip and provided images with excellent signal-to-noise ratio. A novel computational scheme that is particularly suitable for analyzing particle-image velocimetry data in micrometer-scale flow channels was employed to analyze the images. These liposomes are easily synthesized and can be custom-made for various applications to offer a broad range of surface and volume characteristics such as charge, size, and surface chemistry.

"Self Referencing Ceramic Based Multisite Microelectrodes For The Detection And Elimination Of Interferences From The Measurement Of L-glutamate And Other Analytes"
Anal. Chem. 2001 Volume 73, Issue 5 Pages 1037-1042
Jason J. Burmeister and Greg A. Gerhardt

Abstract: A self-referencing technique utilizing two microelectrodes on a ceramic-based multisite array is employed for confirmation and elimination of interferences detected by enzyme-based microelectrodes. The measurement of L-glutamate using glutamate oxidase was the test system; however, other oxidase enzymes such as glucose oxidase can be employed. One recording site was coated with Nafion with L-glutamate oxidase and bovine serum albumin (BSA) cross-linked with glutaraldehyde while the other had Nafion with BSA cross-linked with glutaraldehyde. Differences in the chemistry of the two recording sites allowed for identification and elimination of interfering signals to be removed from the analyte response. The electrode showed low detection limits (LOD = 0.98±0.09 muM, signal-to-noise ratio of 3), fast response tithes (T-90 similar to1 s), and excellent linearity (R-2 = 0.999±0.000) over the concentration range of 0-200 muM for calibrations of L-glutamate in vitro. The selectivity and dimensions of the multisite electrode allow in vivo glutamate measurements. This electrode has been applied to in vivo measurements of the clearance of locally applied glutamate and release of glutamate in the prefrontal cortex of anesthetized rats. In addition, a similar approach has been applied to the development of a microelectrode for measures of glucose.
Enzyme

"Diamond Optically Transparent Electrodes: Demonstration Of Concept With Ferri-ferrocyanide And Methyl Viologen"
Anal. Chem. 2001 Volume 73, Issue 5 Pages 908-914
Jerzy K. Zak, James E. Butler, and Greg M. Swain

Abstract: A new type of optically transparent electrode is reported on-an electrically conductive diamond thin film. The electrode was free-standing (0.38 mm thick and 8 mm in diameter), mechanically polished to a 7 nm rms roughness over a 10 m linear distance, boron-doped (0.05% B/C in the reactant gas mixture), and mounted in a thin-layer transmission cell. The electrode has a short-wavelength cutoff of ~225 nm, which is the indirect band gap of the material, and transmits light out to at least 1000 nm. In theory, the electrode has an optical window from 225 nm well out into the far-infrared, except for the boron acceptor band and the intrinsic multiphonon absorptions. The electrode was used to electrooxidize ferrocyanide to ferricyanide, and the absorbance change associated with the formation of the oxidized product (max = 420 nm) was spectroscopically monitored. The electrode was also used to electroreduce methyl viologen (MV2+) to the cation radical (MV+) and the neutral (MV0). The depletion of MV2+ (max = 257 nm) and formation of MV+ (max = 398 and 605 nm) were spectroscopically monitored.
Selectivity

"Needle-type Dual Microsensor For The Simultaneous Monitoring Of Glucose And Insulin"
Anal. Chem. 2001 Volume 73, Issue 4 Pages 844-847
Joseph Wang and Xueji Zhang

Abstract: A miniature needle-type sensor suitable for the simultaneous amperometric monitoring of glucose and insulin is described. The integrated microsensor consists of dual (biologically and chemically) modified carbon-paste working electrodes inserted into a 14-guage needle. The glucose probe is based on the biocatalytic action of glucose oxidase, and the insulin one relies on the electrocatalytic activity of ruthenium oxide. The analytical performance of the dual sensor is assessed under flow injection conditions, The needle dual detector exhibits a very rapid response to dynamic changes in the concentrations of glucose and insulin. No apparent cross reactivity is observed in mixtures containing millimolar glucose levels and nanomolar insulin concentrations, The response is highly linear (to at least 1000 nM insulin and 14 mM glucose) and reproducible (RSD = 2.6-4.1%). The combination microsensor holds great promise for real-time measurements of the insulin/glucose ratio and for improved management of diabetes.

"Electroosmotic Flow In Composite Microchannels And Implications In Microcapillary Electrophoresis Systems"
Anal. Chem. 2001 Volume 73, Issue 4 Pages 829-836
F. Bianchi, F. Wagner, P. Hoffmann, and H. H. Girault

Abstract: The electroosmotic flow in laminated excimer laser-ablated microchannels has been studied as a function of the depth of the rectangular channels, and particular emphasis has been given to the difference in the g-potentials between the lamination layer and the ablated substrate. Experimental electroosmotic flow follows the tendency predicted by a recently published model. The g-potentials of lamination and ablated surfaces were determined for poly(ethylene terephthalate) and poly(carbonate) substrates by fitting the experimental data with a numerical implementation of this model. In the experimentally investigated range of channel cross sections, a linear fit to the data gives a good approximation of the zeta -potentials for both materials. Moreover, a flow injection analysis of fluorescein dye has been performed to show the severe loss in numbers of theoretical plates, caused by Taylor dispersion, when such microchannels, dedicated to microcapillary electrophoresis, are used.

"Microflow-based Automated Chemistries: Application To Protein Sequencing"
Anal. Chem. 2001 Volume 73, Issue 4 Pages 776-786
Michael Powell and Paul Tempst

Abstract: We describe a microflow-based instrument, consisting of multiple rotary valves, capillary tubing, and miniaturized reaction vessels, for the purpose of performing automated chemical and biochemical reactions on a very small scale (i.e. submicroliter volumes). The novelty is that close to 100% of the reaction end products are available in a minimal volume (?5 µL) inside a pressurized microvial for subsequent analysis. This makes the system compatible with capillary HPLC and, in principle, with continuous-flow nanoelectrospray mass spectrometry. Total control of flow path combinations and directions, temperatures and gas pressures enables precise execution of complex biochemical laboratory procedures. Instrument performance was convincingly demonstrated by partially sequencing 100 fmol of an intact protein using classical Edman chemistry in combination of capillary-bore liquid chomatography. To our knowledge, this is the smallest amount of protein ever reported to be successfully analyzed in this way. Additional application that merge enzymatic and chemical procedures with high-sensitivity analytical devices can be envisioned in the future.

"Electrochemiluminescence Determination Of 2 ,6 -difluorophenyl 10-methylacridan-9-carboxylate"
Anal. Chem. 2001 Volume 73, Issue 4 Pages 763-767
Robert Wilson, Hashem Akhavan-Tafti, Renuka DeSilva, and A. Paul Schaap

Abstract: Electrochemical oxidation of the acridan 2,6-difluorophenyl-10-methylacridan-9-carboxylate produces the corresponding acridinium ester, which reacts with hydrogen peroxide forming a dioxetanone intermediate. Decomposition of the dioxetanone generates light at 430 nm when it relaxes to the ground state. The effect of pH and hydrogen peroxide concentration on this ECL reaction and on the stability of the acridan were investigated. At pH 8.0 and a hydrogen peroxide concentration of 10 mM, light emission from the ECL reaction was used to determine the acridan concentration with a detection limit of 54 pmol L-1. Results suggest that acridan esters could be used as labels in ECL immunoassays and nucleotide assays.

"Multispectral Imaging Microscope With Millisecond Time Resolution"
Anal. Chem. 2001 Volume 73, Issue 4 Pages 732-739
Oleg Khait, Sergey Smirnov, and Chieu D. Tran

Abstract: A new multispectral imaging microscope with micrometer spatial resolution and millisecond temporal resolution has been developed. The imaging microscope is based on the use of an acousto-optic tunable filter (AOTF) for spectral tuning and a progressive scan camera capable of snapshot operation for recording, It can operate in two modes: images are recorded as a function of time or wavelength. When operated as a function of time, the microscope is configured so that as many images as possible are recorded, grabbed, and stored per one wavelength. Upon completion, the AOTF is scanned to a new wavelength, and a new set of images are recorded, Up to 33 images/second (i.e., 30 ms/image) can be recorded in this mode. In the other configuration, the recording wavelength is rapidly scanned (by means of the AOTF) and only one image is rapidly recorded, grabbed, and stored for each wavelength. Because additional time is needed to scan the AOTF, the maximum number of images can be grabbed in this case is 16 frames/s, Preliminary applications of the imaging microscope include measurements of photoinduced changes of a single unit cell in temperature-sensitive cholesteric liquid crystals as a function of time and wavelength. The changes were found to be varied with time and wavelength. Interestingly, the photoinduced changes of unit cells in the liquid crystal are not the same but different from cell to cell. This imaging microscope is particularly useful for measurements of small-size samples that undergo rapid chemical or biochemical reactions, e.g,, activities of a single biological cell.

"Electrokinetic Injection For Stacking Neutral Analytes In Capillary And Microchip Electrophoresis"
Anal. Chem. 2001 Volume 73, Issue 4 Pages 725-731
James Palmer, Dean S. Burgi, Nicole J. Munro, and James P. Landers

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

"A Compactly Integrated Flow Cell With A Chemiluminescent FIA System For Determining Lactate Concentration In Serum"
Anal. Chem. 2001 Volume 73, Issue 2 Pages 373-378
Hideaki Nakamura, Yuji Murakami, Kenji Yokoyama, Eiichi Tamiya, Isao Karube, Masayuki Suda, and Shunichi Uchiyama

Abstract: We have fabricated an integrated flow cell as a total microanalysis system (µTAS). This flow cell (size, 15 x 20 mm; total inner volume, 12.2 µL) was designed for a rational analyzing system of lactate determination for serum. This cell was made by micromachining techniques and consisted of two hollows of a lactate oxidase (LOD) reactor and a mixing cell, a spiral groove, and three penetrated holes. To form the reactor and capillary, these patterns, etched on a silicon wafer, were attached to a glass plate by the anodic bonding method. A photodiode was put under part of the spiral capillary. The compactly accumulated devices were integrated into a flow injection analysis (FIA) system. In the flow cell, lactate was catalyzed to pyruvate and hydrogen peroxide at the LOD reactor; subsequently, hydrogen peroxide reacted with the luminol-ferricyanic reagent at the mixing cell. The resulting chemiluminescent light was detected by the photodiode. Using the miniaturized flow cell, the sample volume for one measurement was greatly reduced to 0.2 µL. The response to lactate was obtained within 30 s and was linear between 0.5 and 5.0 mM (4.5 and 45 mg/dL) lactate with excellent correlative variances of 3.2% (average of three measurements at 5.0 mM). For practical application, the lactate concentration in control human serum was determined using this system. The results showed a good correlation coefficient (r = 0.979) with the results obtained by the spectrophotometric reference method. No difference in sera (normal or pathological) was found. Consequently, this integrated flow cell shows potential as a clinical device for lactate determination in serum. In this article, the effect of the design on the chemiluminescent FIA system is also described.

"Techniques For Increasing The Throughput Of Flow Injection Mass Spectrometry"
Anal. Chem. 2001 Volume 73, Issue 2 Pages 247-252
Kenneth L. Morand, Thomas M. Burt, Brian T. Regg, and Thomas L. Chester

Abstract: Improvements to the design and operation of a Gilson 215 multiprobe liquid-handling system have resulted in a significant increase in the throughput for flow injection molecular weight characterization of combinatorial chemistry libraries. The rapid injection sequence, and subsequent increased sample throughput, is effected by directing the entire mobile-phase flow through each of the injection loops sequentially while isolating or dead-ending the remaining nonactive loops. This mode of operation was accomplished by incorporating column-switching valves prior to and following the set of eight parallel injectors. Analysis rates are achieved without sacrificing the integrity of the flow injection peak profile as baseline resolution is maintained for all samples. Using this system, the total analysis time for a 96-well microtiter plate has been reduced to approximately 5 min. [Journal Article; In English; United States]

"Determination Of Mycophenolic Acid And Mycophenolate Mofetil By High-performance Liquid Chromatography Using Postcolumn Derivatization"
Anal. Chem. 2001 Volume 73, Issue 1 Pages 41-46
Ulf D. Renner, Christian Thiede, Martin Bornh&auml;user, Gerhard Ehninger, and Hans-Michael Thiede

Abstract: An efficient method to lower the optical detection limit is described using file displacement of an absorption and emission band of an analyte after a polarity change in different solvents. This solvatochromic effect was used in a RP-HPLC assay for the fluorescence detection of mycophenolic acid (6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl)-4-methyl-4-hexenoic acid, MPA) and the prodrug mycophenolate mofetil (MMF), the N-(2-hydroxyethyl)morpholino ester of MPA. The rational to use fluorescence detection is based on the behavior of MMF and MPA, which fluoresce in a basic medium (pH >9.5). Following a simple protein precipitation, the anatytes were separated in an isocratic RP-HPLC system. The post-column generation of the phenolate anions of MPA and MMF was achieved by addition of an aqueous sodium hydroxide solution regulated by a newly developed continuous-flow liquid control system. MPAG, not directly accessible for fluorescence detection, was analyzed after enzymatic deglucuronidation to MLPA. Compared to published quantification limits for MPA and MMF by UV detection, this method is more than 100-fold more sensitive, with a lower limit of quantification of 45 fmol for both MPA and MMF.

"Subsecond Adsorption And Desorption Of Dopamine At Carbon-fiber Microelectrodes"
Anal. Chem. 2000 Volume 72, Issue 24 Pages 5994-6002
Bradley D. Bath, Darren J. Michael, B. Jill Trafton, Joshua D. Joseph, Petrise L. Runnels, and R. Mark Wightman

Abstract: High-repetition fast-scan cyclic voltammetry and chronoamperometry were used to quantify and characterize the kinetics of dopamine and dopamine-o-quinone adsorption and desorption at carbon-fiber microelectrodes. A flow injection analysis system was used for the precise introduction and removal of a bolus of electroactive substance on a sub-second time scale to the disk-shaped surface of a microelectrode that was fabricated from a single carbon fiber (Thornel type T650 or P55). Pretreatment of the electrode surfaces consisted of soaking them in purified isopropyl alcohol for a minimum of 10 min, which resulted in S/N increasing by 200-400% for dopamine above that for those that were soaked in reagent grade solvent. Because of adsorption, high scan rates (2000 V/s) are shown to exhibit equivalent S/N ratios as compared to slower, more traditional scan rates. In addition, the steady-state response to a concentration bolus is shown to occur more rapidly when cyclic voltammetric scans are repeated at short intervals (4 ms). The new methodologies allow for more accurate determinations of the kinetics of neurotransmitter release events (10-500 ms) in biological systems. Brain slice and in vivo experiments using T650 cylinder microelectrodes show that voltammetrically measured uptake kinetics in the caudate are faster using 2000 V/s and 240 Hz measurements, as compared to 300 V/s and 10 Hz.

"On-line Separation And Determination Of Bromate In Drinking Waters Using Flow Injection ICP Mass Spectrometry"
Anal. Chem. 2000 Volume 72, Issue 22 Pages 5725-5730
A. R. Elwaer, C. W. McLeod, and K. C. Thompson

Abstract: A flow injection (FI) system with a microcolumn of anion exchanger has been used to effect rapid on-line separation of bromate and bromide prior to quantitation by ICP mass spectrometry. Basic performance studies are described including the effect of key Fl parameters, i.e., sample injection volume, carrier stream now rate, and eluent concentration on system response. The new approach permitted ultratrace determinations of bromate in drinking waters, the main benefits being low limit of detection (0.13 µg/L based on a 500 µL sample injection), rapid analysis time (10 min/sample), and good precision (2.8% at the 5 µg/L level). Accuracy was checked via an EC-sponsored interlaboratory trial.

"Electrochemiluminescent Metallopolymer Coatings: Combined Light And Current Detection In Flow Injection Analysis"
Anal. Chem. 2000 Volume 72, Issue 22 Pages 5576-5582
Robert J. Forster and Conor F. Hogan

Abstract: The application of thin films of the metallopolymer [Ru(bpy)(2)PVP10](2+) for the electrochemiluminescent (ECL) detection of oxalate in a flow injection analysis system is reported, where bpy is 2,2-bipyridyl and PVP is poly(4-vinylpyridine). Immobilization of the ECL reagent means that it Can Be regenerated in situ, eliminating the need to constantly: deliver it to the reaction zone. Electrochemically:generated Ru3+ reacts with the analyte to form the excited-state [Ru2+]*, which luminesces at 610 nm. The reaction is optimal at low pH, where the layer is swollen and homogeneous charge transport through the layer is more facile.: Unlike traditional approaches, we simultaneously monitor both the amperometric and luminescent response of the modified electrode. The precision of both signals is:similar at similar to2% (n = 10). However, the ECL response has a larger dynamic range extending from the low-micromolar to higmillimolar range and a lower limit of detection, similar to0.2 muM or 4 pmol of oxalate injected. The ECL approach displays excellent selectivity for oxalate over:a wide range of potential interferences including oxygen, amines, iron sulfate, ammonium nitrate, urea, and glucose. Ascorbic acid represents the most significant ECL interference. However, the signal observed for a 1 mM solution of ascorbic acid is still. only 2.6% of the response Observed for the injection of a similar concentration of oxalate.

"Pulse Amperometric Detection Of Salt Concentrations By Flow Injection Analysis Using Ionodes"
Anal. Chem. 2000 Volume 72, Issue 22 Pages 5562-5566
Hye Jin Lee, Carlos M. Pereira, Ant&oacute;nio F. Silva, and Hubert H. Girault

Abstract: A sensitive novel approach of using an amperometric ion detector for the now injection analysis of salts has been developed. The detection methodology is based on measuring the-current associated with the transfer of ions across polarized microinterfaces between the aqueous sample solution and a 2-nitrophenyloctyl ether-poly(vinyl chloride) gel phase, referred to as ionodes, Different sodium salts of fluoride, chloride, bromide, nitrate, and sulfate were investigated. It was found that by employing an amperometric pulse detection mode and pure water as eluent; the detection limit of the ionode detector could be lowered to ppt level of salt concentrations under flowing conditions.

"Detection Of Native Amino Acids And Peptides Utilizing Sinusoidal Voltammetry"
Anal. Chem. 2000 Volume 72, Issue 22 Pages 5542-5548
Sara A. Brazill, Pankaj Singhal, and Werner G. Kuhr

Abstract: Native amino acids and peptides were detected at a copper microelectrode using sinusoidal voltammetry (SV). Traditionally, these molecules can only be measured after derivatization with either a fluorescent or electroactive tag. In this work, an electrocatalytic oxidation reaction at copper is used to detect underivatized peptides and amino acids. The oxidation reaction is somewhat independent of peptide structure (i.e., it is not limited to the detection of aromatic amino acids) and is therefore able to produce nanomolar detection limits for all amino acids and peptides tested. A scanning technique, sinusoidal voltammetry, is used to provide the sensitivity of constant-potential techniques but also provide selectivity gained through utilization of the frequency domain. The frequency spectrum due to the oxidation of each molecule has a unique 'fingerprint' response resulting from the kinetics of oxidation at the electrode surface. Through examination of the frequency spectra, even structurally similar molecules can be easily distinguished from one another. Flow injection analysis is used to demonstrate the sensitive and selective detection of a variety of amino acids and peptides. This technique can also be easily coupled to a separation step, i.e., high-performance liquid chromatography or capillary electrophoresis without electrode fouling from the adsorption of the analytes.

"Measurement Of Atmospheric Hydrogen Peroxide And Hydroxymethyl Hydroperoxide With A Diffusion Scrubber And Light Emitting Diode-liquid Core Waveguide-based Fluorometry"
Anal. Chem. 2000 Volume 72, Issue 21 Pages 5338-5347
Jianzhong Li and Purnendu K. Dasgupta

Abstract: We describe a new automated instrument for measuring gas- and aqueous-phase H2O2 The chemistry relies on the hematin-catalyzed oxidation of nonfluorescent thiamine to fluorescent thiochrome by H2O2; this reaction is 35-fold more selective for hydrogen peroxide than its nearest alkyl hydroperoxide congener, CK3HO2. The optical characteristics of the fluorescent product are such that it is ideally excited by newly available GaN-based light emitting diodes emitting in the near-UV. A stable long-life miniature flow-through fluorescence detector based on a transversely illuminated liquid core waveguide is thus used for this purpose. The limit of detection (LOD, S/N = 3) for liquid-phase H2O2 is 11 nM. A temperature-controlled high-efficiency Nafion membrane diffusion scrubber is used to collect gaseous hydrogen peroxide with near-quantitative efficiency with an S/N = 3 LOD of 13.5 pptv. The system responds to hydrogen peroxide and hydroxymethyl hydroperoxide but not to methyl hydroperoxide. The use of very inexpensive and stable reagents, highly sensitive detection, benign chemistry, and a fluorescence detector using a solid-state illumination source results in a particularly affordable automated instrument. Design and performance details and illustrative results from a 1999 field campaign (Atlanta Supersite Study) are presented.

"Voltammetry On Microfluidic Chip Platforms"
Anal. Chem. 2000 Volume 72, Issue 21 Pages 5285-5289
Joseph Wang, Ronen Polsky, Baomin Tian, and Madhu Prakash Chatrathi

Abstract: Microfluidic chip devices are shown to be attractive platforms for performing microscale voltammetric analysis and for integrating voltammetric procedures with on-chip chemical reactions and fluid manipulations. Linear-sweep, square-wave, and adsorptive-stripping voltammograms are recorded while electrokinetically pumping the sample through the microchannels, The adaptation of voltammetric techniques to microfluidic chip operation requires an assessment of the effect of relevant experimental variables, particularly the high voltage used for driving the electroosmotic now, upon the background current, potential window, and size or potential of the voltammetric signal. The exact potential window of the chip detector is dependent upon the driving voltage. Manipulation of the electroosmotic now opens the door to hydrodynamic modulation (stopped-flow) and reversed-flow operations. The modulated analyte velocity permits compensation of the microchip voltammetric background. Reversal of the driving voltage polarity offers extended residence times in the detector compartment, Rapid square-wave voltammetry/flow injection operation allows a detection limit of 2 x 10^-12 mol (i.e., 2 pmol) of 2,4,6-trinitrotoluene (TNT) in connection with 47 nL of injected sample. The ability of integrating chemical reactions with voltammetric detection is demonstrated for adsorptive stripping measurements of trace nickel using the nickel-dimethylglyoxime model system. The voltammetric response is characterized using catechol, hydrazine, TNT, and nickel as test species. The ability to perform on-chip voltammertic protocols is advantageous over nanovial voltammetric operations that lack a liquid-handling capability. Coupling the versatility of microfluidic chips with the rich information content of voltammetry thus opens an array of future opportunities.

"Immunologic Trapping In Supported Liquid Membrane Extraction"
Anal. Chem. 2000 Volume 72, Issue 21 Pages 5280-5284
Eddie Thordarson, Jan &Aring;ke J&ouml;nsson, and Jenny Emn&eacute;us

Abstract: To obtain a high degree of selectivity in sample preparation, supported liquid membrane (SLM) extraction was combined with immunologic recognition, The SLM employs a hydrophobic polymer for supporting the immobilization of an organic solvent, thus forming a nonporous membrane. Said membrane separates the aqueous sample on one side (donor) from a receiving aqueous phase on the other (acceptor), The extraction involves the partitioning of neutral compounds between the sample solution, continuously pumped alongside the membrane, and the membrane. From the membrane, re-extraction takes place into a second aqueous phase containing antibodies specific for the target compound(s), Hence, there is a formation of an antibody-antigen complex at the heart of the sample preparation (ImmunoSLM). When the immunocomplex: forms, the antigen can no longer redissolve in the organic membrane, thus being trapped in the acceptor. Consequently, the concentration gradient of free antigen over the membrane is ideally unaffected, this being the driving force for the process. With a surplus of antibody, the concentration of analyte in the receiving phase will easily exceed the initial sample concentration. In this work, the so formed immunocomplex was quantified on-line, using a fluorescein flow immunoassay in a sequential injection analysis (SIA) setup, The outlined ImmunoSLM-SIA scheme was successfully applied for the extraction of 4-nitrophenol from spiked water solutions as well as from a spiked wastewater sample, indicating that the immunoextraction can be suitable when dealing with difficult matrixes.

"Speciation Of Key Arsenic Metabolic Intermediates In Human Urine"
Anal. Chem. 2000 Volume 72, Issue 21 Pages 5172-5177
X. Chris Le, Xiufen Lu, Mingsheng Ma, William R. Cullen, H. Vasken Aposhian, and Baoshan Zheng

Abstract: Biomethylation is the major human metabolic pathway for inorganic arsenic, and the speciation of arsenic metabolites is essential to a better understanding of arsenic metabolism and health effects. Here we describe a technique for the speciation of arsenic in human urine and demonstrate its application to the discovery of key arsenic metabolic intermediates, monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), in human urine. The study provides a direct evidence in support of the proposed arsenic methylation pathway in the human. The finding of MMA(III) and DMA(III) in human urine, along with recent studies showing the high toxicity of these arsenicals, suggests that the usual belief of arsenic detoxification by methylation needs to be reconsidered, The arsenic speciation technique is based on ion pair chromatographic separation of arsenic species on a 3-mum particle size column at 50°C followed by hydride generation atomic fluorescence detection. Speciation of MMA(III), DMA(III), arsenite (As-III), arsenate (As-V), monomethylarsonic acid(MMA(V)), and dimethylarsinic acid (DMA(V)) in urine samples is complete in 6 min with detection limits of 0.5-2 µg/L. There is no need for any sample pretreatment. The capability of rapid analysis of trace levels of arsenic species, which resulted in the findings of the key metabolic intermediates, makes the technique useful for routine arsenic speciation analysis required for toxicological and epidemiological studies.
Temperature

"Room Temperature Sonolysis-based Advanced Oxidation Process For Degradation Of Organomercurials: Application To Determination Of Inorganic And Total Mercury In Waters By Flow Injection-cold Vapor Atomic Absorption Spectrometry"
Anal. Chem. 2000 Volume 72, Issue 20 Pages 4979-4984
J. L. Capelo, I. Lavilla, and C. Bendicho

Abstract: A new oxidation method based on room-temperature ultrasonic irradiation (sonolysis) is proposed for Conversion of organomercurials into inorganic mercury and subsequent determination by now injection-cold vapor atomic absorption spectrometry. This advanced oxidation process eliminates the need for chemical oxidants, high temperature, and pressure for degradation of organomercurials so that total mercury can be determined with sodium tetrahydroborate(III) or tin(II) chloride as reducing agents. Complete oxidations can be accomplished within 3 min, using a 40% sonication amplitude (100 W nominal power) provided by a probe ultrasonic device (20 kHz frequency) and a 1. mol L-1 HCl liquid medium. The presence of HCl was seen to be necessary for fast oxidation of organomercurials, in contrast to other chemical oxidants such as H2O2 or HNO3 which yielded incomplete oxidation. Further advantages of the proposed method over existing methods which are currently employed for oxidation prior to total Hg determination are the removal of hazardous wastes and the decreased risk of Hg losses by volatilization. Oxidation kinetics indicated a pseudo first-order reaction with apparent rate constants (k) of 3.2 x 10^-2 and 1.6 x 10^-2 s-1 for methylmercury and phenylmercury, respectively. Oxidation experiments in the presence of foreign substances acting as OH radical scavengers showed a tolerance at least up to a concentration of 1000 mg L-1. likewise, model wastewaters with chemical oxygen demand of up to 1000 mg L-1 could be processed without diminishing the oxidation efficiency. The method was applied to determination of inorganic and total mercury in simulated wastewaters and spiked environmental waters in combination with selective reduction.

"Ferricyanide Reduction By Escherichia Coli: Kinetics, Mechanism, And Application To The Optimization Of Recombinant Fermentations"
Anal. Chem. 2000 Volume 72, Issue 20 Pages 4949-4956
Peter Ertl, Birgit Unterladstaetter, Karl Bayer, and Susan R. Mikkelsen

Abstract: Ferricyanide reduction was studied by flow injection analysis (FIA) and chronoamperometry (CA) using two host strains and one recombinant strain of E. coli. Samples taken from batch cultures off, coli JM105 and HB101 showed maximal specific ferricyanide reduction rates in the late exponential phase of growth, with values (µmol/min.g) of 24 (FIA) and 17 (CA) for JM105, and 36 (FIA) for HB101, when shake-flask cultures were sampled, and 70 for HB101, when a chemostat was used to control pH and dissolved oxygen concentration throughout the cultivation. Remarkably higher ferricyanide reduction rates were obtained with HB101 cells; cultivated continuously at very slow growth rate, when chilled, resuspended cell samples were incubated for 5 min in solutions containing 10 mM succinate or formate. These compounds are substrates for primary, membrane-bound dehydrogenases that transfer electrons via ubiquinone to the cytochrome oxidase complexes. Apparent Michaelis-Menten kinetics were observed with respect to ferricyanide concentration when 10 mM succinate was included in the assay buffer; apparent K-m values of 10.1±0.6 mRI and 14.4±1.2 mM ferricyanide were obtained for exponential- and stationary-phase E. coli JM105, respectively. Cyanide inhibition studies show that ferricyanide is reduced mainly by cytochrome o oxidase in exponentially growing cells. The large difference in ferricyanide reduction rates observed in the absence and presence of succinate and formate were used to signal stationary-phase entry 5 h after induction of recombinant human Cu/Zn superoxide dismutase expression in a batch fermentation off. coli HMS174(DE3)(pET3ahSOD). This new method can be used as an adjunct to the quantitation of medium components for the optimization of recombinant fermentations.

"Continuous-flow Microextraction Exceeding1000-fold Concentration Of Dilute Analytes"
Anal. Chem. 2000 Volume 72, Issue 18 Pages 4462-4467
Wuping Liu and Hian Kee Lee

Abstract: A novel liquid-liquid microextraction method, that we have termed continuous-flow microextraction (CFME), is described. In a 0.5 mL glass chamber, an organic drop (1-5 µL) is held at the outlet tip of a polyetheretherketone (PEEK) connecting tubing which is immersed in a continuously flowing sample solution and acts as the fluid delivery duct and as a soh ent holder. Extraction takes place between the organic drop and the flowing sample solution that is continuously ejected out of the PEEK tubing, Concentration factors of between 260- to 1600-fold are achieved within 10 min of extraction, Aspects relevant to CFME were studied. In combination with gas chromatography-electron capture detection, CFME allows analytes to be detected at femtogram-per-milliliter levels. The performance of this technique was evaluated on the basis of the analysis of trace nitroaromatic compounds and chlorobenzenes in environmental samples.
Extraction

"Reduction Of Mass Bias And Matrix Effects In Inductively Coupled Plasma Mass"
Anal. Chem. 2000 Volume 72, Issue 18 Pages 4435-4440
Narong Praphairaksit and R. S. Houk

Abstract: Electrons from a heated tungsten filament are created inside the extraction lens and driven out toward the skimmer. These electrons move through the ion path and reduce space charge effects between positive ions in the beam. The ion transmission efficiency is improved by factors of two (for Pb+) to 27 (for Li+). The greater sensitivity improvement for low-mass ions leads to a substantial reduction in mass bias. With the additional electrons, MO+/M+ and M2+/M+ abundance ratios increase but can be minimized with a small reduction in aerosol gas flow rate. No new background ions are observed with this technique. Matrix effects can be significantly diminished when the electron source is operated under the high electron current mode. The mass dependence of matrix-induced suppression of analyte signals is essentially eliminated. Using flow injection analysis to minimize solid deposition, the technique can tolerate Na matrix up to 10 000 ppm (1%) with only ?15% loss of analyte sensitivity.

"Nanoparticle Characterization In Nanoliter Volumes By Grating Light Reflection Spectroscopy"
Anal. Chem. 2000 Volume 72, Issue 18 Pages 4428-4434
Sean A. Smith, Anatol M. Brodsky, Paul G. Vahey and Lloyd W. Burgess

Abstract: We present both theoretical and experimental results demonstrating that grating light reflection spectroscopy (GLRS) can provide information about the concentration and average size of particles of nanometer dimensions distributed in liquid-phase media. To demonstrate this, we have performed experiments on various concentrations of dendrimeric oligomers in water. Our results show that, with GLRS, we can determine the mean radius of particles with sizes on the order of molecular dimensions. The measurements were carried out in a continuous-flow format using a microchannel flow system and in a detection volume of less than 200 nL.

"Determination Of Arsenic Compounds By High-performance Liquid Chromatography-ultrasonic Nebulizer-high Power Nitrogen-microwave-induced Plasma Mass Spectrometry: An Accepted Coupling"
Anal. Chem. 2000 Volume 72, Issue 18 Pages 4402-4412
Amit Chatterjee, Yasuyuki Shibata, Jun Yoshinaga, and Masatoshi Morita

Abstract: To establish a sensitive, accurate, and precise determination of arsenic compounds, a high power nitrogen microwave-induced plasma (1.3 kW) mass spectrometer (N-2-MIP-MS) has been successfully coupled with an ultrasonic nebulizer (HPLC-USN) that is attached to a high-performance liquid chromatograph. It is examined as an element-specific detector for its applicability to the optimization and determination of seven arsenic compounds [arsenic acid, methylarsonic acid(MA), dimethylarsinic acid (DMA), arsenobetaine (AB), arsenocholine (AC), trimethylarsine oxide (TMAO), and tetramethylarsonium ion (TMI)]. This HPLC-USN-MIP-MS coupling is an encouraging combination as an alternative method for mass spectroscopy for elemental speciation analysis. Interchanging of the MIP-MS fabricated nebulizer (concentric) with an ultrasonic nebulizer, increases 3-6 times the ion signals for the anionic and 6-12 times those for the cationic arsenic compounds as compared to traditional methods. The HPLC-USN-MIP-MS combination used is excellent, amplifying the ion signals about 1.5-2 times for cationic and 1.3-3.8 times for the anionic arsenic compounds as compared to the HPLC-ICPMS coupling, The detection limits for As(V), MA, DMA, AB, TMAO, AC, and TMI (in Milli-Q-water) obtained with the optimized HPLC-USN-N-2-MIP-MS system are 0.46, 0.36, 0.73, 0.21, 3.64, 0.39, and 0.32 µg L-1, respectively, about 13-50 times lower than the HPLC-MIP-MS and about 3-11 times lower than the HPLC-ICPMS. The detection limits of As(V), MA, DMA, AB, TMAO, AC, and TMI, which spike in the urine, are deteriorated by 1.7-4.2 times compared with the detection limits of the seven different As compounds, which are prepared in the Milli-Q-water, The repeatability (RSD for three successive analyzes) and reproducibility (RSD for three successive analyzes performed on three different days), considering peak area and peak height, achieved for seven different arsenic compounds are 0.5-7 and 0.7-8%, comparable with the HPLC-ICPMS (0.3-8.5%; 4-12%) and HPLC-MIP-MS (0.4-9%; 5-12%) systems. The combined HPLC-USN-N-2-MIP-MS has been adequately applied to the determination of AB in NIES Candidate Human Urine CRM. The results agree with the HPLC-ICPMS values. Chloride interference as (ArCl+)-Ar-40-Cl-35 is not found in the urine and with the high chloride matrix (10 000 mg L-1).

"Multidimensional Analysis Of Poly(ethylene Glycols) By Size Exclusion Chromatography And Dynamic Surface Tension Detection"
Anal. Chem. 2000 Volume 72, Issue 18 Pages 4372-4380
Keith E. Miller, Emilia Bramanti, Bryan J. Prazen, Marina Prezhdo, Kristen J. Skogerboe, and Robert E. Synovec

Abstract: Substantial improvements in a multidimensional dynamic surface tension detector (DSTD) are presented. Rapid, online calibration and measurement of the dynamic surface tension for high-performance liquid chromatography separations is achieved. Dynamic surface tension is determined by measuring the differential. pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 2 s) is achieved, for each eluting drop, providing insight into the kinetic behavior of molecular orientation processes at the liquid-air interface. Three-dimensional data are obtained, with surface tension first concerted to surface pressure, which is plotted as a function of elution time axis versus drop time axis. Two key innovations will be reported. First, a novel calibration procedure is described and implemented. Differential pressure signals from three drops (mobile phase, standard in mobile phase, and analyte in mobile phase) are utilized to make the dynamic surface tension measurement, thereby eliminating the need for optical imaging, and viscosity and hydrostatic pressure corrections, as required by other methods. Only pressure signals from one mobile-phase drop and one standard drop pressure signal are required, while the analyte drop pressure signal is measured along the chromatographic time axis. Second, corrections for drop elongation are not required, because the drops are precisely detached bq an air burst actuation method in a regime were the surface tension forces significantly dominate gravitational forces. Drops that would fall with a volume of similar to 10 µL due to gravity are precisely and repeatedly detached earlier at a volume of 2 µL. The sensitivity and unique selectivity of the DSTD opens up new possibilities in the analysis of small molecular weight polymers of varying degrees of surface activity, as illustrated for the size-exclusion chromatography analyzes of complex poly(ethylene glycol) (PEG) samples. Using partial least squares for data analysis, polydispersity of complex PEG samples is determined at a relative precision of similar to 1%.
Interface

"The Capillary Cold Trap As A Suitable Instrument For Mercury Speciation By Volatilization, Cryogenic Trapping, And Gas Chromatography Coupled With Atomic Absorption Spectrometry"
Anal. Chem. 2000 Volume 72, Issue 17 Pages 4178-4184
C. Dietz, Y. Madrid, C. C&aacute;mara, and P. Quevauviller

Abstract: An innovative accessory for speciation analysis has been developed, The system is based on the combination of cryogenic trapping and gas chromatographic separation, carried out within the same capillary. The instrument, hyphenating derivatization, gas-phase extraction, pre-concentration, and analyte separation, is semiautomated, and all operational parameters are adjustable via an in-house-developed control unit, which regulates the selected parameters throughout the analysis process. Species detection nas carried out by atomic absorption spectrometry, The detection limits achieved were 33, 39, and 71 ng L-1 for dimethylmercury, methylmercury, and inorganic mercury, respectively. A complete chromatogram could be obtained within three minutes, resulting in the duration of one whole analysis cycle of about 15 min. The proposed method was applied to mercury speciation in freeze-dried tuna fish powder after microwave-assisted extraction, finding that mercury is present at 80% as methylmercury and about 20% as inorganic mercury, in this kind of biological material.
Preconcentration

"Rotating Rod Renewable Microcolumns For Automated, Solid-phase DNA Hybridization Studies"
Anal. Chem. 2000 Volume 72, Issue 17 Pages 4135-4141
Cynthia J. Bruckner-Lea, Mark S. Stottlemyre, David A. Holman, Jay W. Grate, Fred J. Brockman, and Darrell P. Chandler

Abstract: The development of a new temperature-controlled renewable microcolumn now cell, for solid-phase nucleic acid hybridization in an automated sequential injection system is described. The now cell included a stepper motor-driven rotating rod with the working end cut to a 45 degrees angle. In one position, the end of the rod prevented passage of microbeads while allowing fluid now; rotation of the rod by 180 degrees releases the beads. This system was used to rapidly test many hybridization and elution protocols to examine the temperature and solution conditions required for sequence-specific nucleic acid hybridization. Target nucleic acids labeled with a near-infrared fluorescent dye were detected immediately post-column during all column perfusion and elution steps using a now-through fluorescence detector. Temperature control of the column and the presence of Triton X-100 surfactant were critical for specific hybridization. Perfusion of the column with complementary oligonucleotide (200 µL, 10 nM) resulted in hybridization with 8% of the DNA binding sites on the microbeads with a solution residence time of less than 1 s and a total sample perfusion time of 40 s. The use of the renewable column system for detection of an unlabeled PCR product in a sandwich assay was also demonstrated.
Sequential injection

"Model For Nonequilibrium Binding And Affinity Chromatography: Characterization Of 8-hydroxyquinoline Immobilized On Controlled Pore Glass Using A Flow Injection System With A Packed Microcolumn"
Anal. Chem. 2000 Volume 72, Issue 16 Pages 3927-3933
Maury E. Howard and James A. Holcombe

Abstract: This paper discusses the use of pulse techniques for analysis of zonal elution data for the determination of mass-transfer and axial dispersion constants for porous support materials with adsorption to the surface or to a surface-bonded phase. As an example, this paper considers the case of controlled pore glass (CPG) with a bonded phase that is used with microcolumns and a now injection analysis system, For the CPG, axial dispersion in the form of eddy mixing can be described by l = 0.203, and the overall mass-transfer term, K-OL = 3.9 x 10^-6 cm/s. Additionally an affinity chromatography model was adapted to effectively describe systems employing CPG as the support material through modification of equations describing typical affinity chromatography systems and by inclusion of an axial dispersion term in the calculation of N. This model was used to predict breakthrough curves for cadmium adsorption by 8-hydroxyquinolinol immobilized on CPG packed in microcolumns. In general, the information from the model can be used to extract equilibrium-based constants (binding strengths and site capacities) from a nonequilibrium flow system. The data and model can also be employed in determining the performance for scaled-up extraction systems. The modified model is available in EXCEL spreadsheet format as Supporting Information.

"Approach For Independent-matrix Removal Of Polycyclic Aromatic Hydrocarbons From Solid Samples Based On Microwave-assisted Soxhlet Extraction With On-line Fluorescence Monitoring"
Anal. Chem. 2000 Volume 72, Issue 15 Pages 3627-3634
L. E. Garc&iacute;a-Ayuso, J. L. Luque-Garc&iacute;a, and M. D. Luque de Castro

Abstract: A flow injection interface for fluorometric monitoring of focused microwave-assisted Soxhlet extraction (FMASE) is presented. This assembly allows real-time on-line monitoring of the PAHs extracted from solid samples in each Soxhlet cycle and provides qualitative and semiquantitative information from natural and spiked samples. Hence, the extraction kinetics can be followed and the end of the leaching step determined independently of the sample matrix, thus avoiding excessive extraction times, The research also involves the study of the multivariate effects of the FMASE parameters by a central composite design. The method was applied to a certified reference material (CRM 524, BCR, industrial soil/organics) for quality assurance/validation. The total content of each analyte was determined by HPLC-fluorometric detection, and the results obtained demonstrated that FMASE is a technique as efficient as conventional Soxhlet to extract PAHs from soils but with a drastic reduction of both extraction time and organic solvent disposal.

"Chemical Vapor Generation: Atomic Absorption By Ag, Au, Cu, And Zn Following Reduction Of Aquo Ions With Sodium Tetrahydroborate(III)"
Anal. Chem. 2000 Volume 72, Issue 15 Pages 3523-3531
Aderval S. Luna, Ralph E. Sturgeon, and Reinaldo C. de Campos

Abstract: Volatile species of Ag, Au, Cu, Zn, Cd, and As were generated at room temperature by the addition of sodium tetrahydroborate(III) to an acidified solution of the analytes, The vapor-phase species were rapidly transported to a heated quartz tube atomizer (QTA) for detection by atomic absorption spectrometry. A univariate approach was used to achieve optimized conditions and derive analytical figures of merit. The analytes are released from solution as molecular species (likely as metal hydrides) and are (partially) atomized in the QTA under nonoptimized conditions. Detection limits range from 1.8 (Zn) to 420 ng (Au). The efficiency of the generation process is estimated to be 92±4% for An. Loss of analyte during transport to the QTA was minimized through use of the minimum length of narrow-bore Teflon transfer line possible.

"Thermometric Sensing Of Peroxide In Organic Media. Application To Monitor The Stability Of RBP-retinol-HRP Complex"
Anal. Chem. 2000 Volume 72, Issue 15 Pages 3443-3448
Kumaran Ramanathan, Birgitta R. J&ouml;nsson, and Bengt Danielsson

Abstract: The stability of horseradish peroxidase (HRP) in aqueous and organic solvents is applied to develop a simple thermometric procedure to detect the binding of retinoic acid-HRP conjugate to retinol binding protein (RBP). Butanone peroxide (BP) in organic phase and hydrogen peroxide in aqueous phase is detected thermometrically on a HRP column, immobilized by cross-linking with glutaraldehyde on controlled pore glass (CPG). Acetone, acetonitrile, methanol, and 2-butanol are used for detection of BP, in the flow injection analysis (FIA) mode. A linear range between 1 and 50 mM BP is obtained in all the organic solvents with a precision of 5-7% (CV%). The magnitude and nature of the thermometric response is significantly different in each organic solvent. The stability of HRP in the organic phase is used to study the stability of a retinoic acid-HRP conjugate bound to immobilized REP. The response of HRP (to 20 mM BP) in the retinoic acid- HRP conjugate is used as an indicator of the stability of the RBP-retinoic acid-HRP complex, after challenges with various organic/aqueous solvents. Both immobilized HRP and REP are stable at least for 6 months. The effect of o-phenylene diamine on the thermometric response of HRP is also investigated. A scheme for the design of a thermometric retinol (vitamin A) biosensor is proposed.

"Laser-induced Fluorescence Detection By Liquid Core Waveguiding Applied To DNA"
Anal. Chem. 2000 Volume 72, Issue 15 Pages 3423-3430
Anders Hanning, Peter Lindberg, Joakim Westberg, and Johan Roeraade

Abstract: A new laser-induced fluorescence detector for capillary electrophoresis (CE) is described. The detector is based on transverse illumination and collection of the emitted fluorescent light via total internal reflection along the separation capillary. The capillary is coated with a low refractive index fluoropolymer and serves as a liquid core waveguide (LCW). The emitted light is detected end-on with a CCD camera at the capillary exit. The observed detection limit for fluorescein is 2.7 pM (550 ymol) in the continuous-flow mode and 62 fM in the CE mode. The detector is applied to DNA sequencing. One-color G sequencing is performed with single-base resolution and signal-to-noise ratio ?250 for peaks around 500 bases. The signal-to-noise ratio is ~50 for peaks around 950 bases. Full four-color DNA sequencing is also demonstrated. The high sensitivity of the detector is suggested to partly be due to the efficient rejection of scattered laser light in the LCW. The concept should be highly suitable for capillary array detection.

"Photocatalytic Sensor For Chemical Oxygen Demand Determination Based On Oxygen Electrode"
Anal. Chem. 2000 Volume 72, Issue 14 Pages 3379-3382
Yoon-Chang Kim, Kyong-Hoon Lee, Satoshi Sasaki, Kazuhito Hashimoto, Kazunori Ikebukuro, and Isao Karube

Abstract: The construction and performance evaluation of a novel Chemical Oxygen Demand (COD) sensor is described. The sensor measures, using an oxygen electrode, a decrease of dissolved oxygen of a given sample resulting from photocatalytic oxidation of the organic compounds therein. As the photocatalyst, titanium dioxide (TiO2) fine particles adsorbed on a translucent poly(tetrafluoroethylene) (PTFE) membrane was used. The oxygen electrode with the membrane attached on its tip was used as the sensor probe. The operation characteristics of the sensor are demonstrated using an artificial wastewater and real water samples from lakes in Japan. This method is considered to be reliable, in that the observed parameter is close to the theoretical definition of chemical oxygen demand (COD), the amount of oxygen consumed for oxidation of organic compounds.

"Segregation Of Micrometer-Dimension Biosensor Elements On A Variety Of Substrate Surfaces"
Anal. Chem. 2000 Volume 72, Issue 14 Pages 3253-3259
Sunday A. Brooks, Narasaiah Dontha, C. Brandon Davis, Joan K. Stuart, Geoff O'Neill and Werner G. Kuhr

Abstract: With the rapid development of µtotal analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules to small areas of surfaces other than silicon. To this end, photolithographic techniques were used to derivatize micrometer-sized, spatially segregated biosensing elements on several different substrate surfaces. Both an interference pattern and a dynamic confocal patterning apparatus were used to control the dimensions and positions of immobilized regions. In both of these methods, a UV laser was used to initiate attachment of a photoactive biotin molecule to the substrate surfaces. Once biotin was attached to a substrate, biotin/avidin/biotin chemistry was used to attach fluorescently labeled or nonlabeled avidin and biotinylated sensing elements such as biotinylated antibodies. Dimensions of 2-10 µm were achievable with these methods. A wide variety of materials, including glassy carbon, quartz, acrylic, polystyrene, acetonitrile-butadiene-styrene, polycarbonate, and poly(dimethylsiloxane), were used as substrates. Nitrene- and carbene-generating photolinkers were investigated to achieve the most homogeneous films. These techniques were applied to create a prototype microfluidic sensor device that was used to separate fluorescently labeled secondary antibodies.

"A Continuous Film-recirculable Drop Gas-liquid Equilibration Device. Measurement Of Trace Gaseous Ammonia"
Anal. Chem. 2000 Volume 72, Issue 14 Pages 3165-3170
Zhang Genfa and Purnendu K. Dasgupta

Abstract: A miniature gas-liquid equilibrator or a gas collector, intended as a low-volume interface between a soluble gaseous sample and a liquid phase analyzer or between a liquid phase sample and a detector designed for use with gas samples, is described. This paper addresses the application of the device for the measurement of trace atmospheric ammonia. Gas collection occurs solely by diffusive sampling such that aerosol particles are not collected. The device essentially consists of a tube surrounded externally by a jacket. Gas flows through the jacket and contacts a liquid film flowing on the surface of the tube. The flowing film forms a drop at the tube terminus and is aspirated off through the inner bore of the tube. The collected analyte can be (a) directly sent to an analysis system or (b) pre-concentrated on a suitable stationary phase; the pre-concentrator effluent can be recycled, if desired. With a fluorometric flow injection analysis system harnessed to measure ammonia with such a collector, the limit of detection (LOD, S/N = 3) for a sample drawn for 18 min at 200 mL/min was 4.5 parts per trillion by volume, with the linear range extending up to 30 parts per billion.

"Equilibrium And Kinetic Measurements Of Muscarinic Receptor Antagonism On Living Cells Using Bead Injection Spectroscopy"
Anal. Chem. 2000 Volume 72, Issue 14 Pages 3109-3115
Peter S. Hodder, Craig Beeson, and Jaromir Ruzicka

Abstract: Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay. Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands. A now injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber. The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BTS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response. Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.

"Sample Preparation Based On Dynamic Ion-exchange Solid Phase Extraction For GC/MS Analysis Of Acidic Herbicides In Environmental Waters"
Anal. Chem. 2000 Volume 72, Issue 14 Pages 3077-3084
Nanqin Li and Hian Kee Lee

Abstract: The newly established enrichment technique, dynamic ion-exchange solid-phase extraction (DIE-SPE), was studied for sample preparation for GC/MS analysis of 16 acidic herbicides in environmental waters. C-18 bonded silica was the solid-phase material used, The optimal sample DR was weakly acidic to neutral. However, for common tap water and surface water, which run pH 6-9, all the acidic herbicides except for Chloramben could be effectively extracted from a sample of 1000 mL volume without pH adjustment. The humic acid could be concurrently extracted from water, but most of it was separated from the sample by using 3 mt of 10% methanol in acetone as the eluent, which would completely elute the analytes and leave a large part of the humic acid on the cartridge. The selective elution reduced the interference of humic acid and made the DIE-SPE an effective approach for the analysis of the acidic herbicides in surface water. Comparing DIE-SPE with conventional reversed phase SPE (RP-SPE), the former gave higher recoveries for the acidic herbicides and was less affected by sample matrixes. A tandem-cartridge system combining RP- and DIE-SPE in sequence was set up for the simultaneous isolation of the acidic herbicides and removal of the interfering substances. Despite some minimal retention on the upper RP-SPE cartridge, most of the acidic herbicides could be extracted on the lower DIE-SPE cartridge with recovery over 80% except for Chloramben (50%), fenoprop (73%), MCPB (67%), and 2,4-DB (70%) when a 500 mL aqueous sample of pH. 9.5 was percolated through the tandem-cartridge system, The effectiveness of the system in removing the long carbon chain fatty acids as well as the basic and neutral organic interfering substances from the sample was also demonstrated.
Preconcentration

"Analysis Of Ternary Mixtures With A Single Dynamic Microbial Sensor And Chemometrics Using A Nonlinear Multivariate Calibration"
Anal. Chem. 2000 Volume 72, Issue 13 Pages 2937-2942
Volker Plegge, Michael Slama, Benno S&uuml;selbeck, Dietrich Wienke, Friedrich Spener, Meinhard Knoll, and Christiane Zaborosch

Abstract: An amperometric biosensor based on immobilized bacterial cells of Alcaligenes eutrophus KT02 and an oxygen electrode was integrated in a now-through system. Because microorganisms metabolize various organic analytes in a specific manner, the sensor shows for different pure analytes distinct time-dependent oxygen consumption rates that can be treated as characteristic patterns. This behavior is conserved also when the biosensor is exposed to a mixture of these organic analytes; the sensor with a particular type of microorganisms responds with a total signal. The respiration curves as time-dependent amplitudes were subdivided into several time channels. This procedure creates an additional data dimension and makes the single sensor dynamic. Using multivariate calibration models with only one single biosensor, simultaneous quantitative analysis of ternary mixtures of acetate, L-lactate, and succinate was realized. A nonlinear algorithm that compensated for conceivable interactions of the analytes was superior to a partial least-squares algorithm. Each analyte was predicted more precisely by the nonlinear approach resulting in root-mean-square errors of prediction of 0.20 mg/L for acetate, 0.43 mg/L for L-lactate, and 0.73 mg/L for succinate.

"Amperometric Immunosensors Based On Protein A Coupled Polyaniline-perfluorosulfonated Ionomer Composite Electrodes"
Anal. Chem. 2000 Volume 72, Issue 13 Pages 2925-2929
Chih-Hung Liu, Kuo-Tang Liao, and Hsuan-Jung Huang

Abstract: A very sensitive immunosensor based on polyaniline/Nation/protein A (PA/NF/PrA) composite electrodes has been developed for the amperometric immunoanalysis with urease-labeled immunoreagents. The use of urease conjugated goat anti-RIgG (GaRIgG-Ur) as the labeled antibody and urea as the substrate with an amperometric detection at -200 mV (vs Ag/AgCl) resulted in a dynamic range of 50-2000 ng mL-1 and a low detection limit of 10 ng/mL (64 pM) for the immunoanalysis of rabbit immunoglobulin G (RIgG). Because of the special affinity between protein A and RIgG, the PA/NF/PrA electrode can be regenerated repetitively by changing the pH of the buffer solutions. Characteristics of the PA/NF/PrA/RIgG immunosensor and optimal conditions for the competitive immunoanalysis of RIgG with FIA were studied.

"Flow Injection Analysis: From Beaker To Microfluidics"
Anal. Chem. 2000 Volume 72, Issue 5 Pages 212A-217A
Jaromir Ruzicka and Elo Harald Hansen

Abstract: Conceived in Denmark, tested in Brazil, and developed by researchers worldwide; those who have believed in FIA have worked hard to gain acceptance of the method. Jaromir Ruzicka of the University of Washington and Elo Harald Hansen of the Technical University of Denmark recount the history of FIA and its related techniques.

"Room-Temperature Imprinting Method For Plastic Microchannel Fabrication"
Anal. Chem. 2000 Volume 72, Issue 8 Pages 1930-1933
Jingdong Xu, Laurie Locascio, Michael Gaitan and Cheng S. Lee

Abstract: A new plastic imprinting method using a silicon template is demonstrated. This new approach obviates the necessity of heating the plastic substrate during the stamping process, thus improving the device yield from ~10 devices to above 100 devices per template. The dimensions of the imprinted microchannels were found to be very reproducible, with variations of less than 2%. The channel depths were dependent on the pressures applied and the materials used. Rather than bonding the open channels with another piece of plastic, a flexible and adhesive poly(dimethylsiloxane) film is used to seal the microchannels, which offers many advantages. As an application, isoelectric focusing of green fluorescence protein on these plastic microfluidic devices is illustrated.

"Speciation Of Dissolved Iron(III) And Iron(II) In Water By On-line Coupling Of Flow Injection Separation And Preconcentration With Inductively Coupled Plasma Mass Spectrometry"
Anal. Chem. 2000 Volume 72, Issue 8 Pages 1879-1884
Xiu-Ping Yan, M. Jim Hendry, and Robert Kerrich

Abstract: A method has been developed for the speciation of trace dissolved Fe(III) and Fe(II) in water by on-line coupling of now injection separation and pre-concentration with inductively coupled plasma mass spectrometry (ICPMS), Selective determination of Fe(III) in the presence of Fe(II) was made possible by on-line formation and sorption of the Fe(III)-pyrrolidinecarbodithioate (PDC) complex in a PTFE knotted reactor over a sample acidity range of 0.07-0.4 mol L-1 HCl, eiution with 1 mol L-1 HNO3, and detection by ICPMS, Over a sample acidity range of 0.001-0.004 mol L-1 HCl, the sum of Fe(III) and Fe(II), i.e., Fe(III + II), could be determined without the need for preoxidation of Fe(II) to Fe(III), The concentration of Fe(II) was obtained as the difference between those of Fe(III + II) and Fe(III), With a sample flow rate of 5 mL min-1 and a 30-s pre-concentration time, an enhancement factor of 12, a retention efficiency of 80%, and a detection limit (3s) of 0.08 µg L-1 were obtained at a sampling frequency of 21 samples h-1. The relative standard deviation (n = 11) was 2.9% at;the 10 µg L-1 Fe(III) level. Recoveries of spiked Fe(III) and Fe(II) in local tap water, river water, and groundwater samples ranged from 95% to 103%. The concentrations of Fe(III) and Fe(II) in synthetic aqueous mixtures obtained by the proposed method were in good agreement with the spiked values. The result for total iron concentration in the river water reference material SLRS-3 was in good agreement with the certified value. The method was successfully applied to the determination of trace dissolved Fe(III) and Fe(II) in local tap water, river water, and groundwater samples.
Knotted reactor Extraction

"On-column Surface-enhanced Raman Spectroscopy Detection In Capillary Electrophoresis Using Running Buffers Containing Silver Colloidal Solutions"
Anal. Chem. 2000 Volume 72, Issue 8 Pages 1866-1871
William F. Nirode, Gerald L. Devault, Michael J. Sepaniak, and Roderic O. Cole

Abstract: Direct on-column surface-enhanced Raman spectroscopy (SERS) detection is demonstrated in capillary electrophoresis (CE). Distinctive SERS spectra of two test compounds, riboflavin and Rhodamine 6G, are obtained in 100 µm i.d. fused-silica capillaries under CE conditions using running buffers that contain silver colloidal solutions. Detection is performed using an unmodified commercial Raman spectrometer in a confocal microscope mode of operation. The effects of laser power, wavelength, spectra acquisition time, silver colloidal concentration, and applied voltage (i.e., now rate) on the quality of SERS spectra are evaluated. Using laser powers of 17 mW (at the sample) at 515 nm and employing 1 s spectral acquisition times, spectra with bands exhibiting signal-to-noise ratios greater than 10 could be obtained for 1.0 x 10^-6 M riboflavin and very low nanomolar concentrations of Rhodamine 6G. This was accomplished without optimization of silver colloidal solution compositions and by using a low-throughput spectrometer. Incorporation of the colloidal solutions into running buffers is shown to have little effect on the separation of the test compounds as monitored using a laser-induced fluorescence instrumental scheme. However, SERS spectra degrade if the capillary is not rinsed between experiments. Riboflavin and Rhodamine 6G spectra are obtained on-the-fly for actual CE separations. In the case of the latter solute, the injected quantity was approximately 90 amol.

"Amperometric Biosensor For Glutamate Using Prussian Blue-based Artificial Peroxldase As A Transducer For Hydrogen Peroxide"
Anal. Chem. 2000 Volume 72, Issue 7 Pages 1720-1723
Arkady A. Karyakin, Elena E. Karyakina, and Lo Gorton

Abstract: The specially deposited Prussian Blue denoted as artificial peroxidase was used as a transducer for hydrogen peroxide, The electrocatalyst as stable, highly active, and selective to hydrogen peroxide reduction in the presence of oxygen, which allowed sensing of H2O2 around 0.0 V (Ag/AgCl). Glutamate oxidase was immobilized on the surface of the Prussian Blue-modified electrode in a Nafion layer using a nonaqueous enzymology approach. The calibration range for glutamate in flow injection system was 1 x 10^-7-1 x 10^-4 M. The lowest concentration of glutamate detected (1 x 10^-7 M) and the highest sensitivity in the linear range of 0.21 A M-1 cm-2 were achieved. The influence of reductants was practically avoided using the low potential of an indicator electrode (0.0 V Ag/AgCl). The attractive performance characteristics of the glutamate biosensor illustrate the advantages of Prussian Blue-based artificial peroxidase as transducer for hydrogen peroxide detection.

"Solvent Extraction Coupled On-line To A Reversed Micellar Mediated Chemiluminescence Detection System For Trace-level Determination Of Atropine"
Anal. Chem. 2000 Volume 72, Issue 7 Pages 1715-1719
Terufumi Fujiwara, Imdad U. Mohammadzai, Katsumi Murayama, and Takahiro Kumamaru

Abstract: A fast and sensitive method for the determination of atropine, an alkaloid closely related to cocaine, is proposed. The principles of on-line ion-pair formation of alkaloid-metal complexes and liquid-liquid extraction are applied to the chemiluminescence determination of atropine. On mixing with a reversed micellar medium of cetyltrimethylammonium chloride in dichloromethane-cyclohexane(1:1 v/v)-water (0.3 M Na2CO3) containing luminol, the ion-pair complex of tetrachloroaurate(III) with atropinium produced an analytical chemiluminescence signal when it entered the reversed micellar water pool. Using the reverse-flow injection and chemical conditions optimized for atropine in aqueous samples, a detection limit of 1 ng/mL was achieved and a linear calibration graph was obtained with a wide dynamic range from 10 ng/mL to 100 µg/mL. The proposed method is simple and provides a good precision with a relative standard deviation (n = 6) of ~3% at the atropine concentration of 100 ng/mL. After a preliminary study involving the potential interference from species of organic, inorganic, and metallic nature, the method was applied to the determination of atropine in artificial urine samples and of atropine and scopolamine in pharmaceutical formulations.

"Mid-infrared Quantum Cascade Lasers For Flow Injection Analysis"
Anal. Chem. 2000 Volume 72, Issue 7 Pages 1645-1648
B. Lendl, J. Frank, R. Schindler, A. M&uuml;ller, M. Beck, and J. Faist

Abstract: A Fabry-Perot quantum cascade laser (QCL) was used as a powerful light source for mid infrared (MIR) detection in flow injection analysis. The QCL lased at several wavelengths close to each other within a few wavenumbers (990-1010 cm-1), hence fitting well to the broad absorption bands of molecules in liquid phase. As compared with that of a state-of-the-art Fourier transform spectrometer, the signal-to-noise ratio could be improved by a factor of 50. Additionally, by using a QCL as the light source, optical path lengths of more than 100 m could be used even in aqueous matrixes which reduced the danger of cell clogging. In the example shown here phosphate was determined in Diet Coke samples. The flow injection system used allowed the measurement of the sample at two pH values (5 and 13) at which the analyte was present as H2PO4- and PO43-, respectively. As the analytical readout the difference in IR absorption of H2PO4- and PO43- at the laser wavelengths was taken. The FIA-QCL measurements were corroborated by ion chromatography which was used as a reference method.

"Electrochemical Oxidation Of Histamine And Serotonin At Highly Boron Doped Diamond Electrodes"
Anal. Chem. 2000 Volume 72, Issue 7 Pages 1632-1638
B. V. Sarada, Tata N. Rao, D. A. Tryk, and A. Fujishima

Abstract: The electrochemistry of histamine and serotonin in neutral aqueous media (pH 7.2) was investigated using polycrystalline, boron-doped diamond thin-film electrodes. Cyclic voltammetry, hydrodynamic voltammetry, and flow injection analysis (FIA) with amperometric detection were used to study the oxidation reactions. Comparison experiments were carried out using polished glassy carbon (GC) electrodes. At diamond electrodes, highly reproducible and well-defined cyclic voltammograms were obtained for histamine with a peak potential at 1.40 V vs SCE. The voltammetric signal-to-background ratios obtained at diamond were 1 order of magnitude higher than those obtained for GC electrodes at and above 100 M analyte concentrations. A linear dynamic range of 3-4 orders of magnitude and a detection limit of 1 M were observed in the voltammetric measurements. Well-defined sweep rate-dependent voltammograms were also obtained for 5-hydroxytryptamine (5-HT). The characteristics of the voltammogram indicated lack of adsorption of its oxidation products on the surface. No fouling or deactivation of the electrode was observed within the experimental time of several hours. A detection limit of 0.5 M (signal-to-noise ratio 13.8) for histamine was obtained by use of the FIA technique with a diamond electrode. A remarkably low detection limit (10 nM) was obtained for 5-HT on diamond by the same method. Diamond electrodes exhibited a linear dynamic range from 10 nM to 100 M for 5-HT determination and a range of 0.5-100 M for histamine determination. The FIA response was very reproducible from film to film, and the response variability was below 7% at the actual detection limits.

"Uranyl-selective Electrode Based On A New Bifunctional Derivative Combining The Synergistic Properties Of Phosphine Oxide And Ester Of Phosphoric Acid"
Anal. Chem. 2000 Volume 72, Issue 7 Pages 1604-1610
Antonio Florido, Ignasi Casas, Josep Garc&iacute;a-Raurich, Rina Arad-Yellin, and Abraham Warshawsky

Abstract: Ion-selective electrodes based on the bifunctional chelating agent O-methyldihexylphosphine oxide O-hexyl-2-ethylphosphoric acid (HL) incorporated into a poly(vinyl chloride) membrane were developed. This new derivative is proposed as a single molecular unit combining the overall properties of the synergistic single components, di-2-ethylhexylphosphoric acid and trioctylphosphine oxide. Two different ionophores, HL and its uranyl complex (UO2L2), were studied. The response of the electrodes to uranyl ion was Nernstian for UO2L2 and super-Nernstian for HL ionophores, with detection limits of 3.0 x 10^-6 and 3.0 x 10^-5 M, respectively. Results indicate a more effective interaction with the analyte in the case of having a unique molecule incorporating the two functional groups immobilized into a polymeric membrane, rather than the separated two synergistic ligands. Flow-through tubular electrodes based on both ionophores were also used as potentiometric detectors in flow injection techniques.

"Redox Hydrogel-based Amperometric Bienzyme Electrodes For Fish Freshness Monitoring"
Anal. Chem. 2000 Volume 72, Issue 7 Pages 1591-1597
Mihaela Niculescu, Catalin Nistor, Ivo Fr&eacute;bort, Pavel Pe, Bo Mattiasson, and Elisabeth Cs&ouml;regi

Abstract: This work presents the design and optimization of amperometric biosensors for the determination of biogenic amines (e.g., histamine, putrescine, cadaverine, tyramine, cystamine, agmatine, spermidine), commonly present in food products, and their application for monitoring of freshness in fish samples. The biosensors were used as the working electrodes of a three-electrode electrochemical cell of wall-jet type, operated at -50 mV vs Ag/AgCl, in a flow injection system. Two different bienzyme electrode designs were considered, one based on the two enzymes [a newly isolated and purified amine oxidase (AO) and horseradish peroxidase (HRP)] simply adsorbed onto graphite electrodes, and one when they were cross-linked to an Os-based redox polymer. The redox hydrogel-based biosensors showed better biosensors characteristics, i.e., sensitivity of 0.194 A M-1 cm-2 for putrescine and 0.073 A M-1 cm-2 for histamine, and detection limits (calculated as three times the signal-to-noise ratio) of 0.17 ?M for putrescine and 0.33 ?M for histamine. The optimized redox hydrogel-based biosensors were evaluated in terms of stability and selectivity, and were used for the determination of total amine content in fish samples kept for 10 days in different conditions.

"Triangle-Programmed Coulometric Nanotitrations Completed By Continuous Flow With Potentiometric Detection"
Anal. Chem. 2000 Volume 72, Issue 7 Pages 1585-1590
Olivier T. Guenat, Bart H. van der Schoot, Werner E. Morf and Nicolaas F. de Rooij

Abstract: Coulometric nanotitrations were realized in a microchannel system using a continuous-flow titration technique with a triangle current-time profile. Redox and acid-base titrations were carried out on Fe(II) and nitric acid samples, respectively, with the same nanotitrator device. A linear relation between the concentration and the coulometric current transferred to the solution was found. The advantages of this universally applicable nanotitrator are fast response, low sample volume, high sensitivity, and high reproducibility as well as the convenience of handling an automated analyzer of the flow-through type.

"New Nucleophilic Catalysts For Bright And Fast Peroxyoxalate Chemiluminescence"
Anal. Chem. 2000 Volume 72, Issue 7 Pages 1373-1380
Tobias Jonsson and Knut Irgum

Abstract: Miniaturized detection applications based on chemiluminescence require fast reaction kinetics for optimum performance. in this work, high-intensity light from the analytically useful peroxyoxalate chemiluminescence reaction has been generated at high rates by employing both single-component and dual-component nucleophilic catalysis. 4-(Dimethylamino)pyridine and its derivatives were superior to all other bases in terms of reaction speed and intensity of the generated light and outshone imidazole, which hitherto has been considered as the best catalyst, The light intensity was related to the difference in pK(a) between the 4-aminopyridine catalyst and the leaving group of the reagent, and the optimum Delta pK(a) was found to be close to 0. Similarly, high light intensities were obtained when mixtures of the imidazole analogue 1,2,4-triazole and the strong, nonnucleophilic base 1,2,2,6,6-pentamethylpiperidine acted as catalysts, The mechanism behind this was concluded to be a base-induced nucleophilic catalysis, where the ancillary strong base assisted the production of the highly nucleophilic 1,2,4-triazolate anion, which as the actual catalyst then participated in the formation of a more reactive transient reagent. All the investigated catalysts reduced the light yield of the reaction due to base-catalyzed breakdown reactions of the reagents and/or intermediates. The intensity peak maximums of these bright and fast reactions typically appeared after less than 10 ms, whereafter the light decayed to darkness within a few seconds, These reaction characteristics are especially advantageous for sensitive detection applications where the observation volumes and times are limited, e.g., peaks emerging from a capillary-based separation process.

"Chemiluminescent Reaction Of Fluorescent Organic Compounds With KHSO5 Using Cobalt(II) As Catalyst And Its First Application To Molecular Imprinting"
Anal. Chem. 2000 Volume 72, Issue 6 Pages 1148-1155
Jin-Ming Lin and Masaaki Yamada

Abstract: The decomposition of peroxomonosulfate (HSO5-) has been investigated by chemiluminescence (CL). A weak CL was observed during mixing the HSO5- solution with the Co2+ solution in unbuffered conditions. An appropriate amount of fluorescent organic compounds (FOCs), such as dansyl amino acids and pyrene, was added to the KHSO5/Co2+ solution, a strong CL was recorded. A possible CL mechanism, based on studies of the fluorescence, CL, and UV-visible spectra and comparison of Co3+ oxidation ability with the SO4.- radical ion, was discussed. The CL from HSO5-/Co2+ is the emission of singlet oxygen produced from the catalytic decomposition of HSO5-. It was suggested that the decomposition of HSO5- in aqueous solution with Co2+ proceeds via one-electron transfer to yield SO4.- radical ion. The FOC was attacked by SO4.- radical ion and oxidized to decompose into small molecules, During this proceeding, CL emission was given out. The present CL system has been developed as a now injection analysis for FOCs. The detection limits (S/N = 3) were in the concentration range 10^-9 - 10^-7 M for FOCs. Oxidation decomposition and CL emission of the analytes have been used in the molecular imprinting recognition. As an initial attempt, dansyl-L-phenylalanine was used as a template molecule and methacrylic acid and 2-vinylpyridine were used as functional monomers. The network copolymer imprinted with dansyl-L-phenylalanine exhibits an affinity for the template molecule. When the flowing streams of HSO5- and Co2+ solutions mixing through the molecularly imprinted polymer particles filled the how cell, the template molecule, dansyl-L-phenylalanine reacted with the HSO5-/Co2+ solution and CL was emitted, The dansyl-L-phenylalanine was decomposed during the CL process, and the cavities of a defined shape and an arrangement of functional groups complementary to the template in the polymer were left for the next,sample analysis.

"Flame Hydrolysis Deposition Of Glass On Silicon For The Integration Of Optical And Microfluidic Devices"
Anal. Chem. 2000 Volume 72, Issue 5 Pages 1093-1097
Jesus M. Ruano, Vincent Benoit, J. Stewart Aitchison and Jonathan M. Cooper

Abstract: Flame hydrolysis deposition (FHD) of glasses has previously found applications in the telecommunications industry. This paper shows how the technology can be used to deposit silica with different refractive indices and thereby produce low-loss planar waveguides for use in analytical applications. We also show that the glasses can be patterned using a new reactive ion etch and sealed using a modification of anodic bonding, such that the resulting microstructures can be readily incorporated within a lithographically defined 'chip', integrating both optical and fluidic circuitry on the same device. In the example described in this paper, waveguides, analytical microtiter chambers and fluidic capillary channels, with the necessary high aspect ratio features (and with depths up to 40 µm) were all produced in glass, using the appropriate deposition and etching technologies. The performance of the chip was assessed in the framework of a low-volume fluorescence assay, using waveguides to address miniaturized microtiter chambers with volumes of 230 and 570 pL. Devices featuring different optical detection configurations, including both in-line and orthogonal waveguide geometries, were fabricated. In the optimal configuration, the experimental detection limit was determined as ~20 pM (equivalent to 10 zmol) of a cyanine fluorophore, Cy5. The applicability of the device as a biochip platform was further illustrated by analytical measurements on fluorescently labeled oligodeoxynucleotides.

"Voltammetric And Reference Microelectrodes With Integrated Microchannels For Flow Through Microvoltammetry. 2. Coupling The Microcell To A Supported Liquid Membrane Preconcentration Technique"
Anal. Chem. 2000 Volume 72, Issue 5 Pages 943-948
O. C. Keller and J. Buffle

Abstract: The paper describes the assembly and functioning of the microvoltammetric cell described in Part 1, with a hollow Fiber Supported Liquid Membrane (HFSLM), for trace metal analysis. Membrane stability, working-electrode behavior, mercury-film lifetime inside the HFSLM, hydrodynamic conditions, as well as transport kinetics of the metal through the SLM have been studied in detail. System calibrations have been performed in the range 5-120 nM Pb(II). The reproducibility and sensitivity of the whole microsystem is discussed as well as limitations and possible improvements.

"Voltammetric And Reference Microelectrodes With Integrated Microchannels For Flow Through Microvoltammetry. 1. The Microcell"
Anal. Chem. 2000 Volume 72, Issue 5 Pages 936-942
O. C. Keller and J. Buffle

Abstract: This paper describes the construction of 2 microsensor units for on-line voltammetric detection inside a cylindrical microcell (a working microsensor unit and a reference and auxiliary microsensor unit), for application to heavy-metal analysis in complex media such as natural waters. Both microsensor units include a channel for the solution renewal in the microcell after analysis. The working microsensor, a Hg-plated Ir microelectrode, is protected against fouling with an agarose gel including a hydrophobic chromatographic phase (C18). The fabrication steps and the quality tests related to long-term use and reliability, as well as to precision, are described. The application of the protective gel layer against fouling by hydrophobic or surface active small molecules is of general application, reliable, and very efficient. The reference and auxiliary unit is composed by an iridium oxide based mini reference electrode, an auxiliary Pt electrode, and a circulation channel. It is built to enable its use inside a 700-µm-diameter tubing connected to a hollow fiber supported liquid membrane lumen (volume, 5-10 µL) for heavy-metal analysis. However, it can be used in any other microanalytical system. The reference electrode is sufficiently stable for voltammetric applications (1-2 mV drift /day), and its lifetime is more than one year. The Ti/IrO2 core is immersed in a pH-buffered agarose gel, to guarantee potential stability even when the electrode is immersed in variable pH solutions.

"Fiber Optic Chemical Sensors And Biosensors"
Anal. Chem. 2000 Volume 72, Issue 12 Pages 81R-89R
Otto S. Wolfbeis

Abstract: A review with many references. This review is divided into sections on books and reviews (A), on specific sensors for gases and vapors (B), ions (C), and organic species (D), respectively, followed by sections on biosensors (E) and by chapters on application-oriented sensor types (F), sensing schemes (G), and sensor materials (H), respectively
Sol-gel Review

"Kinetic Determinations And Some Kinetic Aspects Of Analytical Chemistry"
Anal. Chem. 2000 Volume 72, Issue 12 Pages 53R-70R
Stanley R. Crouch, Alexander Scheeline, and Ewa S. Kirkor

Abstract: In this review, we have attempted to retain the overall organizational structure of the previous review (Crouch, S. R.; Cullen, T. F.; Scheeline, A.; Kirkor, E. S. Anal. Chem. 1998, 70, 53R-106R). The majority of the papers selected from Chemical Abstracts appeared there during the October 1997-October 1999 period. References to the patent literature, to conference proceedings, and to dissertations are not included. The papers considered were published in English unless accompanied by a Chemical Abstracts reference number. The scope of this review differs from those in the past. Because of the rapidly increasing growth in the number of articles considered, the editors have asked us to write a more critical review and to limit the number of references to approximately 200. Over 10 000 articles were generated from our search; hence, limiting the number of references has been a formidable task. Our final database included over 2000 references. The articles discussed here were deemed the most significant and useful by the authors. We have again this year chosen to include enzyme-catalyzed reactions, but to exclude immunoassay techniques including those involving enzymes. Because some readers desire a more comprehensive bibliography, we have included additional references classified according to our organizational scheme in the Supporting Information for the journal. We have chosen to restrict our review to those references citing specifically a dynamic or kinetic aspect and unlikely to be cited in other reviews that may overlap this review (e.g., those dealing with luminescence spectrometry, electrochemistry, chemometrics, thermal analysis, and various separation methods). The authors take complete responsibility for their omissions and any errors in classifying articles. The Sixth International Symposium on Kinetics in Analytical Chemistry took place in Kassandra, Chalkidiki, Greece, from September 16 to September 19, 1998. There were nearly 100 presentations (oral and poster) in the areas of catalytic methods (including enzyme-catalyzed reactions), multicomponent (differential) kinetic methods, continuous-flow processing, instrumentation for kinetic methods and studies, nonlinear dynamics, electrochemistry, and general applications of kinetic methods. Professors M. I. Karayannis (University of Ioannina) and C. Papadopoulos (Aristotelian University of Thessaloniki) served as Chairpersons of the Symposium. One interesting trend that was noted during this period is the tremendous increase in the number of biological applications involving kinetic methods or the measurement of some type of transient phenomena. A large fraction of the papers reviewed deal with the development or application of biosensors, often involving immobilized enzymes or immunoreagents. We expect to see continued emphasis on biological systems.

"A Rotating Ball Inlet For On-line MALDI Mass Spectrometry"
Anal. Chem. 2000 Volume 72, Issue 1 Pages 251-254
Henrik &Oslash;rsnes, Thomas Graf, Hans Degn, and Kermit K. Murray

Abstract: The rotating ball inlet (ROBIN) is presented in a new design for on-line matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). This method uses a capillary to deliver a matrix and analyte solution to the surface of a rotating ball upon which MALDI is carried out. The ball is in contact with a polymer gasket surrounding the capillary. Sample adhering to the surface of the ball is dragged past the gasket into the vacuum of the mass spectrometer where it is irradiated by a pulsed UV laser, and the resulting ions are mass-separated in a linear time-of-flight mass spectrometer. The mechanical sample introduction prevents clogging of the vacuum interface by matrix crystals or frozen solvent. Preliminary results from flow injection analysis (FIA) suggest that the new interface does not introduce a significant peak-tailing or memory effect. The system is capable of 20-30 h of continuous operation with a flow rate of 2 L/min before cleaning of the ball is needed. With the prototype inlet, concentration detection limits are at the low micromolar level.

"A Microwave-induced Plasma Based On Microstrip Technology And Its Use For The Atomic Emission Spectrometric Determination Of Mercury With The Aid Of The Cold-vapor Technique"
Anal. Chem. 2000 Volume 72, Issue 1 Pages 193-197
Ulrich Engel, Attila M. Bilgi&ccedil;, Oliver Haase, Edgar Voges, and Jos&eacute; A. C. Broekaert

Abstract: A new low-power, small-scale 2.45 GHz microwave plasma source at atmospheric pressure for atomic emission spectrometry based on microstrip technology is described. The MicroStrip Plasma (MSP) source was produced in microstrip technology on a fused-silica wafer and designed as an element-selective detector for miniaturized analytical applications. The electrodeless microwave-induced plasma (MIP) operates at microwave input power of 10^-40 W and gas flows of 50-1000 mL min-1 of Ar. Rotational (OH) and excitation (Fe) temperatures were found to be 650 and 8000 K, respectively. Spatially resolved measurements of the Hg I 253.7 nm atomic emission line with an electronic slitless spectrograph (ESS) showed that a cylindrically symmetric plasma with a diameter of about 1 mm is obtained. With the MSP, Hg could be determined by applying the now injection cold vapor (FI-CV) technique with a detection limit of 50 pg mL-1. In terms of the relative standard deviation, a time stability of <1.4% for 45 replicates within 80 min can be realized at a concentration level of 10 ng mL-1 of Hg. Hg could be determined in the leachate of a certified standard reference soil (STSD-4) obtained by treatment with aqua regia at the 930±76 ng g-1 level. Results obtained by calibration with aqueous solutions of Hg and with standard addition were found to be in good agreement with those of cold-vapor atomic absorption spectrometry.

"Flow Injection Monitoring And Analysis Of Mixtures Of Hydrazine Compounds Using Filter-supported Bilayer Lipid Membranes With Incorporated DNA"
Anal. Chem. 2000 Volume 72, Issue 1 Pages 180-186
Christina G. Siontorou, Dimitrios P. Nikolelis, and Ulrich J. Krull

Abstract: This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring and analysis of mixtures of hydrazine compounds using stabilized systems of filter-supported bilayer lipid membranes (BLMs) composed of egg phosphatidylcholine (egg PC) with incorporated DNA. Injections of hydrazines were made into flowing streams of a carrier electrolyte solution, and a transient current signal with a duration of seconds reproducibly appeared in less than one min after exposure of the DNA-modified lipid membranes to the hydrazines. The magnitude of this signal was linearly related to the concentration of hydrazines, which could be determined at sub-micromolar levels. Repetitive cycles of injection of hydrazines have shown no signal degradation during each cycle (30 sequential injections), The time of appearance of the transient response was different for each hydrazine and increased in the order of hydrazine, methylhydrazine or dimethylhydrazine, and phenylhydrazine. The difference in time of response has snowed selective detection and analysis of these hydrazines in mixtures.

"Producing Self-plasticizing Ion-selective Membranes"
Anal. Chem. 2000 Volume 72, Issue 1 Pages 42-51
Lee Yook Heng and Elizabeth A. H. Hall

Abstract: Polymer membranes have been explored for the analysis of ions that do not require plasticizers and with photo-curable properties, This work was focused on investigating the viability of the methacrylic-acrylic copolymers as new self-plasticizing membrane matrixes for ion-selective electrodes or other ion-sensor applications, Copolymers with glass transition temperatures ranging from -20 to -44°C could be prepared without added plasticizer and were found to be functional as ion-selective membranes, Both free-radical solution polymerization and photopolymerization could be used, and self-plasticizing behavior of copolymers was observed with a high alkylacrylate (R = C-4) content. This was found to be compatible with most commercially available ionophores, and sensors for potassium, sodium, calcium and pH were fabricated entirely by photocure procedures; single-step procedures for the immobilization of benzo-15-crown-5 ionophore on these self-plasticizing copolymer matrixes were also developed. Even though the ionophore was immobilized, potentiometric studies revealed that the ionophore remained functional, and thus, these copolymers have the advantage of suffering neither leaching of ionophore nor plasticizers. All these sensors exhibited a Nernstian or near Nernstian response with selectivity comparable to plasticized PVG membranes or other plasticized and photocurable polymer membranes. The long-term response of the potassium sensor with immobilized ionophore and the sodium sensor showed little deterioration for as long as one month and three months, respectively, under continuous use.

"Determination Of Perchlorate At Trace Levels In Drinking Water By Ion-pair Extraction With Electrospray Ionization Mass Spectrometry"
Anal. Chem. 2000 Volume 72, Issue 1 Pages 25-29
Matthew L. Magnuson, Edward T. Urbansky, and Catherine A. Kelty

Abstract: Perchlorate has been added to the U.S. Environmental Protection Agency's Drinking Water Contaminant Candidate List (CCL). The present work describes the analysis of perchlorate in water by liquid-liquid extraction followed by flow injection electrospray mass spectrometry (ESI/MS). Cationic surfactants, mostly alkyltrimethylammonium salts, are used to ion-pair aqueous perchlorate, forming extractable ion pairs. The cationic surfactant associates with the perchlorate ion to form a complex detectable by ESI/MS. The selectivity of the extraction and the mass spectrometric detection increases confidence in the identification of perchlorate. The method detection limit for perchlorate based on 3.14?(n-1) of seven replicate injections was 100 ng L-1 (parts per trillion). Standard addition was used to quantitate perchlorate in a drinking water sample from a contaminated source, and the concentration determined agreed within experimental error with the concentration determined by ion chromatography.

"Discovery Of Quasi-molecular Ions In Electrospray Spectra By Automated Searching For Simultaneous Adduct Mass Differences"
Anal. Chem. 1999 Volume 71, Issue 24 Pages 5557-5562
Ekkehard G&ouml;rlach and Ramsay Richmond

Abstract: Our high-throughput flow injection analysis mass spectrometry (FIA-MS) system using electrospray ionization steers the majority of ion current into quasi molecular ion and adduct m/z positions. One advantage of this dawned on us, i.e., that adducted electrospray spectra are amenable to automated quasi molecular ion discovery by searching for the simultaneous occurrence of adduct mass differences. Therefore, an adduct scanning algorithm was developed, given a graphical user interface, and then incorporated into a Visual Basic application originally developed for reporting of high-throughput FIA-MS results. Copyright 1999, American Chemical Society.

"Radionuclide Sensors Based On Chemically Selective Scintillating Microspheres: Renewable Column Sensor For Analysis Of Tc-99 In Water"
Anal. Chem. 1999 Volume 71, Issue 23 Pages 5420-5429
Oleg B. Egorov, Sandra K. Fiskum, Matthew J. O'Hara, and Jay W. Grate

Abstract: A method for chemically selective radiometric sensing of non-γ-emitting radionuclides in solution is described. Using scintillating microspheres with selective radionuclide uptake properties, radiochemical separation and radiometric detection steps are integrated within a sensor device. These microspheres are loaded into a renewable minicolumn that serves to capture, pre-concentrate, and separate radionuclides. The pre-concentrating minicolumn also localizes and retains radionuclides within a detector of well-defined geometry and emits a photometric signal. The sensor material in the column can either be regenerated with eluent chemistries or be renewed by fluidic replacement of the beads. The latter method allows the use of materials that bind analytes irreversibly or are unstable under regeneration conditions. Radionuclide-selective scintillating microspheres were prepared by coimmobilization of scintillating fluors and selective organic extractants within the pores of an inert polymeric support. Preparation and characterization of microspheres, and their use for selective quantitative sensing of Tc-99(VII), is described in detail. A sensor-based procedure for Tc-99(VII) analysis was developed and successfully applied toward the determination of Tc-99(VII) in groundwater samples from the Hanford site, using standard addition techniques for quantification, Using a 50 mL sample volume and signal accumulation time of 30 min, the detection limit for Tc-99(VII) was 0.37 dpm/mL (9.8 pg/mL).
Sequential injection Preconcentration

"Quantitative Analysis Of Molecular Interaction In A Microfluidic Channel: The T-Sensor"
Anal. Chem. 1999 Volume 71, Issue 23 Pages 5340-5347
Andrew Evan Kamholz, Bernhard H. Weigl, Bruce A. Finlayson and Paul Yager

Abstract: The T-sensor is a recently developed microfluidic chemical measurement device that exploits the low Reynolds number flow conditions in microfabricated channels. The interdiffusion and resulting chemical interaction of components from two or more input fluid streams can be monitored optically, allowing measurement of analyte concentrations on a continuous basis. In a simple form of T-sensor, the concentration of a target analyte is determined by measuring fluorescence intensity in a region where the analyte and a fluorescent indicator have interdiffused. An analytical model has been developed that predicts device behavior from the diffusion coefficients of the analyte, indicator, and analyte-indicator complex and from the kinetics of the complex formation. Diffusion coefficients depend on the local viscosity which, in turn, depends on local concentrations of all analytes. These relationships, as well as reaction equilibria, are often unknown. A rapid method for determining these unknown parameters by interpreting T-sensor experiments through the model is presented.

"Detection Of Oxygen Consumption Of Cultured Adherent Cells By Bead Injection Spectroscopy"
Anal. Chem. 1999 Volume 71, Issue 22 Pages 5248-5252
Ilkka L&auml;hdesm&auml;ki, Louis D. Scampavia, Craig Beeson, and Jaromir Ruzicka

Abstract: This paper describes a method for detecting oxygen consumption of adherent cell cultures. The sensing is based on oxygen-dependent quenching of the phosphorescence of a Pt-porphyrin complex immobilized on microcarrier beads, which are used as the cell culture substrate. Bead injection, a recent variant of the flow injection technique, is used to pack an aliquot of the beads into a small sensing layer that can be easily and rapidly renewed. The technique is tested on a model system of Chinese Hamster Ovary M1 cells grown on Cytodex-3 microcarrier beads. Cellular respiration is monitored through O2 consumption measured across a period of 3 min. The method is validated by detecting the impairment of aerobic metab. caused by 1.5 mM amobarbital. Further, it is shown to have enough precision to distinguish even more subtle changes, such as the increase in oxygen consumption caused by stimulation of the muscarinic m1 receptor with 100 µM carbachol.

"Microfluidic Assays Of Acetylcholinesterase Inhibitors"
Anal. Chem. 1999 Volume 71, Issue 22 Pages 5206-5212
Andrew G. Hadd, Stephen C. Jacobson, and J. Michael Ramsey

Abstract: A microfabricated device for flow injection analysis and electrophoretic separation of acetylcholinesterase (AChE) inhibitors is described. Solutions of inhibitor, enzyme, substrate, and derivatizing agent were mixed within the channels of the microchip using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was measured in an onchip reaction of thiocholine with coumarinylphenylmaleimide, and the resulting thioether was detected by laser-induced fluorescence. Inhibitors reduced the fluorescence signal and produced a negative peak diagnostic for the type of inhibition. A Gaussian peak was observed for competitive inhibitors, whereas a broad negative peak was observed for irreversible inhibitors. From a microchip assay for tacrine, an inhibition constant, K-i, of 1.5±0.2 nM was derived, which compared well with a standard cuvette assay, A now injection assay of two irreversible inhibitors, carbofuran and eserine, was performed. With a Ei-min stopped-flow reaction time, a detection limit of 10 nM carbofuran was obtained. As a potential multiplex screening device, a mixture of four cationic inhibitors, tacrine, edrophonium, and tetramethyl-and tetraethylammonium chloride, was separated and detected within 70 s.

"A Signal Amplification Scheme For Ultrasensitive Amperometric Detection In Flowing Streams"
Anal. Chem. 1999 Volume 71, Issue 22 Pages 5101-5108
Philip T. Radford, Marla French, and Stephen E. Creager

Abstract: A new signal amplification scheme for ultrasensitive amperometric electrochemical detection of redox-active molecules in quiescent solution and in flowing streams is described. The method is based upon a continuous regeneration of electrochemically oxidized analytes by reaction with a sacrificial electron donor in solution. It utilizes a selective coating on the electrode that is chosen to have properties which allow for relatively facile electrooxidation of analyte but which also inhibit electrooxidation of the sacrificial electron donor. Ultrasensitive detection of hydroxymethylferrocene (HMFc) as a model analyte using ferrocyanide as the sacrificial electron donor is demonstrated at a dodecanethiol-coated gold electrode. Signal amplification factors of several hundred to several thousand are obtained in flow injection mode for analyte injections in a concentration range between 10^-4 and 10^-7 M where peaks can be discerned both with and without amplification. Even higher amplification factors are estimated for analyte concentrations below approximately 10^-8 M, for which peaks without amplification are undetectable. Amperometric detection of 60 million injected HMFc analyte molecules (corresponding to either a 10 µL injection at 10^-11 M or a 1.0 mL injection at 10^-13 M) is demonstrated using the new method in flow injection mode.

"Influence Of Mixed Diffusional, Migrational, And Convective Mass Transport On The Response Of A Wall-tube Microelectrode In A Flow Injection System"
Anal. Chem. 1999 Volume 71, Issue 21 Pages 4926-4931
Fredrik Bj&ouml;refors, Joanna Gadomska, Miko&#322;aj Donten, Leif Nyholm, and Zbigniew Stojek

Abstract: The current response of a 10 µm wall-tube microelectrode in a flow injection system under the conditions of low ionic strength was examined experimental for several redox systems such as ferrocene in methanol, undiluted methanol, and water in acetone. The examination involved the dependence of the current on the positioning of the microelectrode relative to a 760 µm internal diameter capillary outlet, flow rate, potential pulse time, and ratio between the concentrations of the supporting electrolyte and electroactive species (Cel/Credox). For Cel/Credox ratios smaller than ~0.001 and a flow rate of 200 µL/min, the dependencies of the current on the flow rate and the positioning of the microelectrode vs. the capillary tip were reversed compared to the presence of excess supporting electrolyte. The current thus decreases with increasing flow rate while a local current max. could be seen when the microelectrode was center-aligned with the capillary tip. The changes in the current are the results of local differences in convective transport. These differences alter the rate of migrational accumulation of counterions at the electrode surface. Results similar to those obtained for the excess supporting electrolyte case can be obtained for a low value of Cel/Credox and a given flow rate if the electrode potential and time scale of the experiments are chosen appropriately.

"A Technique For The Determination Of Trimethylamine-N-oxide In Natural Waters And Biological Media"
Anal. Chem. 1999 Volume 71, Issue 21 Pages 4886-4891
Angela D. Hatton and Stuart W. Gibb

Abstract: Trimethylamine-N-oxide (TMAO) is a nitrogenous osmolyte widely distributed in marine organisms. The reduction of TMAO to TMA has long been implicated as characteristic reaction associated with fish and seafood spoilage. However, it is now apparent that, in the marine environment, TMAO can act as precursor to a range of reduced nitrogenous biogases that can play a significant role in the biogeochemical cycle of nitrogen and in the regulation of atmospheric pH, Although methods exist for the analysis of TMAO in some biological samples, they lack the sensitivity required for measurement of TMAO in natural waters. Here we present a new, safe and sensitive method for the determination of TMAO in aqueous and biological media, where TMAO is enzymatically reduced to TMA and subsequently quantified using Flow Injection Gas Diffusion-Ion Chromatography (Gibb et al, J, Autom. Chem. 1995, 17 (6), 205-212), The limit of detection was calculated to be 1.35 nmol L-1 TMAO, and the response was linear for both fresh and seawater (R-2 = 0.996 and 0.993, respectively). Precision (RSD) for standards in the range 40-600 nmol L-1 was within 3%, The specificity and competitive inhibition of the enzyme are addressed and the applicability of the technique demonstrated through analysis of a number of natural water and biological samples.

"Handling Of Picoliter Liquid Samples In A Poly(dimethylsiloxane)-based Microfluidic Device"
Anal. Chem. 1999 Volume 71, Issue 20 Pages 4781-4785
Kazuo Hosokawa, Teruo Fujii, and Isao Endo

Abstract: Transportation, metering, and mixing of picoliter-sized liquid samples were realized in a microfluidic device with a main working area of one square millimeter. The device was constructed by sealing microfabricated grooves on a chip made of poly(dimethylsiloxane) (PDMS). Two different samples were segmented into 600 pL droplets in a microchannel with a cross section of W (100 µm) x H (25 µm), and the droplets were merged together. For acceleration of the mixing, the merged droplet was shuttled back and forth. Recirculation in a moving droplet was proven to be effective for high-speed mixing in this diffusion-dominated scale. All the handling operations were carried out using air pressure transferred through microfabricated vent valves which have been newly developed. The demonstrated strategy, including fabrication, leads to high-performance and low-cost µtotal analysis systems (µTAS).
Segmented flow Dispersion

"Characterization And Mathematical Modeling Of A Bienzyme Electrode For L-malate With Cofactor Recycling"
Anal. Chem. 1999 Volume 71, Issue 20 Pages 4657-4662
Nenad Gajovic, Axel Warsinke, Tina Huang, Thomas Schulmeister, and Frieder W. Scheller

Abstract: The coimmobilization of a NADP(+)-dependent dehydrogenase with p-hydroxybenzoate hydroxylase (PHBH, EC 1.14. 13.2) in front of a Clark electrode yields a flexible design for highly selective, dehydrogenase-based biosensors, The use of L-malate dehydrogenase (decarboxylating, EC 1.1.1.40) as a model enzyme resulted in a novel L-malate sensor. It had improved characteristics compared with those of earlier sensor approaches: a strongly reduced NADP(+) requirement (0.01 mmol L-1), an extended linear range from 0.005 to 1.1 mmol L-1 L-malate, and a working stability of more than 30 days. Only inexpensive chemicals (p-hydroxybenzoate, MgCl2) were needed in millimolar amounts. A linear mathematical model for the steady state helped to elucidate the sensor operation. Both experimental and simulation results indicated that the bienzyme sensor behaved like a quasi monoenzyme electrode with a hypothetical L-malate hydroxylase: The response was determined by the substrate concentration and diffusivity only, indicating the perfect coupling of both enzyme reactions by the intermediate NADPH. The presented scheme based on PHBH and the Clark electrode is a promising and reliable approach for other NADP(+)-dependent dehydrogenases.
Diffusion

"Voltammetric And Amperometric Investigations Of Azide Oxidation At The Basal Plane Of Highly Oriented Pyrolytic Graphite"
Anal. Chem. 1999 Volume 71, Issue 20 Pages 4603-4608
Jishou Xu and Greg M. Swain

Abstract: The electrochemical oxidation of dissolved azide anion was studied at the basal plane of highly oriented pyrolytic graphite. Cyclic and linear sweep voltammetry and differential pulse voltammetry were employed to study the oxidation reaction mechanism in neutral pH as a function of the potential sweep rate, analyte concentration, and electrolyte composition and in the presence of adsorbed anthraquinone-2,6-disulfonate (2,6-AQDS). The linear dynamic range in the differential pulse voltammetric measurements was 4 orders of magnitude and the estimated limit of detection (SNR = 3) was ~2.3 x 10^-7 M (9.7 ppb). The electron-transfer kinetics for azide oxidation appear rapid at this surface, and the voltammetric features are independent of the fraction of exposed edge plane, the presence of surface oxides, the electrolyte composition, and the adsorption of 2,6-AQDS. The reaction proceeds by an EC(dim) mechanism. Amperometric detection results for flow injection analysis (FIA) and ion chromatography are also presented. A linear dynamic range of nearly 5 orders of magnitude, an estimated detection limit (SNR = 3) of 3.7 nM (0.16 ppb or 74 fmol injected), and a response variability of 2% or less were observed in the FIA measurements.

"Species-Selective Analysis By Microcolumn Multicapillary Gas Chromatography With Inductively Coupled Plasma Mass Spectrometric Detection"
Anal. Chem. 1999 Volume 71, Issue 20 Pages 4534-4543
Isaac Rodrigue&zdot;, Sandra Mounicou, Ryszard &#321;obi&#324;ski, Vladimir Sidelnikov, Youri Patrushev and Michiko Yamanaka

Abstract: A glass rod (5-20 cm long, 2 mm o.d.) containing more than 1200 parallel microchannels (<40 µm i.d.) was converted into a high-resolution (>100 theoretical plates cm-1) GC column by coating the inside of each channel in a way that compensated for the dispersion of the channel inner diameter. The columns were evaluated for the separation of mixtures of several organometallic (Hg, Sn, Pb) compounds prior to on-line sensitive metal-selective detection by ICPMS. Chromatographic separation conditions were optimized to enable a rapid (within a maximum 30 s) multielemental speciation analysis. Absolute detection limits were 0.1 pg for Hg, 0.05 pg for Sn, and 0.03 pg for Pb using the carrier gas flows of ~200 mL min-1. The microcolumn multicapillary GC/ICPMS developed was applied to the analysis of a number of environmental samples. The results were validated with certified reference materials for tin (BCR477, PACS-2) and mercury (DORM-1, TORT-1).

"Enhancement Of Extraction Efficiency And Reduction Of Boundary Layer Effects In Pulse Introduction Membrane Extraction"
Anal. Chem. 1999 Volume 71, Issue 19 Pages 4407-4412
Xuemei Guo and Somenath Mitra

Abstract: Membrane separation has emerged as an attractive alternative for interfacing an extraction step directly to a gas chromatograph or to a mass spectrometer. In pulse introduction (or flow injection type) membrane extraction, a sample pulse is introduced onto an eluent stream that transports it onto the membrane. Since a fixed sample volume is injected, the detector response is directly proportional to the extraction efficiency. This in turn depends on membrane module design, flow conditions, etc. Also, when water contacts a membrane, a static boundary layer is formed at the membrane surface that serves as an additional diffusional barrier to the permeation process, and consequently, permeation slows down which lowers the speed of anal. In this paper, methods of increasing the extraction efficiency and decreasing boundary layer effects are presented. The goal is to have higher sensitivity at a shorter anal. time. A stream of nitrogen is introduced into the membrane after sample elution to eliminate the aqueous boundary layer. This technique is found to be effective not only for faster anal., but also for increasing extraction efficiency.

"Online Coupling Of Flow Injection Microcolumn Separation And Preconcentration To Electrothermal Atomic Absorption Spectrometry For Determination Of (ultra)trace Selenite And Selenate In Water"
Anal. Chem. 1999 Volume 71, Issue 19 Pages 4353-4360
Xiu-Ping Yan, Michael Sperling, and Bernhard Welz

Abstract: A flow injection manifold with an air-segmented and air-transported operational sequence for online coupling of micro-column separation and pre-concentration. to electrothermal atomic absorption spectrometry (ETAAS) was developed to determine ultra-trace selenite and selenate in water. Selenite was determined by selective reaction with pyrrolidine dithiocarbamate (PDC), sorption of the resultant Se-PDC compound onto a conical micro-column (10.2 µL) packed with RP C18 sorbent, elution with ethanol, and ETAAS detection. Selenate selenate was determined as the difference between selenite concentrations. after and before pre-redn. of selenate to selenite. With the developed manifold and operation sequence, the dispersion during elution and eluate transport and the eluent volume required to completely elute the sorbed analyte were minimized. As a result, the sorbed analyte was quant. eluted from the column with only 26 µL of ethanol; all the eluate was automatically introduced into the graphite tube by an air flow without pre-heating the graphite tube or precise timing. Pretreating the graphite tube with Ir as a long-term permanent modifier effectively prevented analyte loss arising from the high volatility of the Se-PDC compound and greatly improved precision, sensitivity, and detection limit. One thermal pretreatment of the graphite tube with injection of 150 µg Ir made possible at least 200 repetitive atomization cycles. With a pre-concentration. time of 180 s and a sample flow rate of 1.4 mL/min, an enhancement factor of 112 was achieved vs. direct injection of 30 µL of aqueous solution The detection limit (3s) was 4.5 ng/L Se. The relative standard deviation (n = 7) was 3.8% at 20 ng/L Se. Selenite and selenate concentrations. determined in synthetic aqueous mixtures were in good agreement with expected values. Selenite recovery from spiked seawater samples was 98-102%. Selenite concentrations. in several seawater reference materials obtained with simple aqueous standard solutions for calibration agreed well with certified and information values, respectively. In addition, this method was successfully used to certify selenite and selenate in water.
Knotted reactor

"Single-Microparticle Measurements: Laser Trapping-Absorption Microspectroscopy Under Solution-Flow Conditions"
Anal. Chem. 1999 Volume 71, Issue 19 Pages 4338-4343
Haeng-Boo Kim, Osamu Kogi and Noboru Kitamura

Abstract: A laser trapping-microspectroscopy system combined with a fluid manifold was developed to manipulate and analyze 'single' microparticles. A sample solution containing microparticles was introduced to a flow cell set on a microscope stage, and a single particle was trapped by a 1064-nm laser beam. With the particle being trapped, the other particles were pumped out by flowing water to hold the unique microparticle in the flow cell. Under solution-flow conditions, a single microparticle was laser trapped in balance with the gradient (Fg) and Stokes forces (Fs) experienced by the particle, and thus, the trapped position was shifted to the downstream side of the 1064-nm laser beam focus. Flow rate and particle size dependencies of this particular positional displacement of the particle were discussed in terms of Fg and Fs. On the basis of these studies, optical requirements to conduct absorption microspectroscopy of a laser-trapped particle were optimized, and the technique was applied to study a time course of dye adsorption processes in single microparticles. The adsorption rate of Rhodamine B was determined for individual microparticles for the first time.

"Packed Column Supercritical Fluid Chromatography/mass Spectrometry For High-throughput Analysis. Part 2"
Anal. Chem. 1999 Volume 71, Issue 19 Pages 4223-4231
Manuel C. Ventura, William P. Farrell, Christine M. Aurigemma, and Michael J. Greig

Abstract: A supercritical fluid chromatograph was previously interfaced to a mass spectrometer (SFC/MS) and the system evaluated for applications requiring high sample throughput using negative-mode atmospheric-pressure chemical ionization (APCI) (Ventura et al. Anal. Chem. 1999, 71, 2410-2416). This report extends the previous work demonstrating the effectiveness of SFC/MS, using positive ion APCI for the analysis of compounds with a wide range of polarities. Substituting SFC/MS for LC/MS results in substantial time saving, increased chromatography efficiency, and more precise quantitation of sample mixtures Flow injection analysis (FIA) also benefits from our SFC/MS system. A broader range of solvents is compatible with the SFC mobile phase compared with LC/MS, and solutes elute more rapidly from the SFC/MS system, reducing sample carryover and cycle time. Our instrumental setup also allows for facile conversion between LC/MS and SFC/MS modes of operation.
Interface Optimization

"Application Of A Macrocycle Immobilized Silica Gel Sorbent To Flow Injection Online Microcolumn Preconcentration And Separation Coupled With Flame Atomic Absorption Spectrometry For Interference-free Determination Of Trace Lead In Biological And Environmental Samples"
Anal. Chem. 1999 Volume 71, Issue 19 Pages 4216-4222
Xiu-Ping Yan, Michael Sperling, and Bernhard Welz

Abstract: A simple and highly selective flow injection online pre-concentration and separation-flame atomic absorption spectrometric method was developed for routine anal. of trace amounts of lead in biological and environmental samples. The selective pre-concentration of lead was achieved in a wide range of sample acidity (0.075 to ≥3 mol L-1 HNO3) on a microcolumn (145 µL) packed with a macrocycle immobilized on silica gel. The lead retained on the column was effectively eluted with an EDTA solution (0.03 mol L-1, pH 10.5). Three kinds of potential interferences, i.e., pre-concentration interferences from metal ions with an ionic radius similar to that of Pb(II) due to their competition for the cavity of the macrocycle, elution kinetic interferences from ions which form stable complexes with EDTA due to their competition for EDTA, and interferences in the atomizer from residual matrix, were evaluated and compared in view of the read-out mode of the analyte response (peak area vs. peak height), column wash step (with vs. without), column capacity (50 vs. 145 µL), and column shape (conical vs. cylindrical). The results showed that a combination of increase in column capacity, quantitation based on peak area, and use of dilute nitric acid for column wash before elution efficiently avoid the above-mentioned potential interferences. With the use of a 145 µL column the present system tolerated up to 0.1 g L-1 Ba(II), 1 g L-1 Sr(II), and at least 10 g L-1 Fe(III), Cu(II), Ni(II), Zn(II), Cd(II), Al(III), K(I), Na(I), Ca(II), and Mg(II) in the sample digest. Further improvement of the interference tolerance can be achieved by increasing column capacity if more complicated samples need to be analyzed. At a sample loading rate of 3.9 mL min-1 with 30-s pre-concentration, an enrichment factor of 52, a detection limit (3s) of 5 µg L-1 Pb in the digest and a sampling frequency of 63 h-1 were obtained. The precision (RSD, n = 11) at the 200 µg L-1 level was 1.9%. The enrichment factor and the detection limit can be further improved by increasing sample loading rate without degrdn. in the efficiency due to the favorable kinetics and low hydrodynamic impedance of the present system. The anal. results obtained by the proposed method for a number of biological and environmental standard reference materials were in good agreement with the certified and recommended values.
Extraction

"Flow Detection Of Nucleic Acids At A Conducting Polymer-modified Electrode"
Anal. Chem. 1999 Volume 71, Issue 18 Pages 4095-4099
Joseph Wang, Mian Jiang, and Baidehi Mukherjee

Abstract: A new protocol for electrochemical detection of nucleic acids, based on polypyrrole (PPy)-coated electrodes, is demonstrated using flow injection analysis The conducting polymer detection scheme offers a fast, sensitive, universal detection of oligonucleotides, DNA, and RNA. The response is attributed to the adsorption/desorption of the nucleic acids onto the PPy film during the passage of the sample plug over the coated electrode. The influence of various experimental parameters of the film preparation and the electrochemical detection has been investigated. Optimal performance has been achieved using the nitrate film dopant, a detection potential of -0.15 V (vs Ag/AgCl), and a flow rate of 0.6 mL/min. Good linearity and reproducibility (RSD = 2.1%) are observed, along with very low detection limits (e.g., 6.1 x 10^-16 mol of dsDNA in the 20 µL samples). Compared to common detectors based on the electroactivity of DNA, the conducting polymer detection scheme is not limited to purine-containing nucleic acids or to a strongly alkaline media and is less susceptible to interferences from easily oxidizable species (e.g., ascorbic acid). The attractive behavior of the PPy flow detector holds great promise for monitoring nucleic acids in other flow systems, microscale sepns., and on-chip platforms.

"Improving Data Acquisition For Fast-scan Cyclic Voltammetry"
Anal. Chem. 1999 Volume 71, Issue 18 Pages 3941-3947
Darren J. Michael, Joshua D. Joseph, Michaux R. Kilpatrick, Eric R. Travis, and R. Mark Wightman

Abstract: Described is an improved data acquisition system for fast-scan cyclic voltammetry (FSCV). The system was designed to significantly diminish noise sources that were identified in previously recorded FSCV measurements for the detection of neurotransmitters. Minimized noise is necessary to observe the low concentrations of neurotransmitters that are physiol. important. The system was based on a high-speed, 16-bit AD/DA acquisition board that allowed high scan rates and better resolved the small faradaic currents which remained after background subtraction. Irregularities that occur when independent timing sources are used for generation of the voltage waveform and collection of the current can create large noise artifacts near the voltage limits du