Showing papers in "Analytical Chemistry in 1997"

Molecular Imaging of Biological Samples: Localization of Peptides and Proteins Using MALDI-TOF MS

Abstract: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. The high sensitivity of the technique (low-femtomole to attomole levels for proteins and peptides) allows the study of organized biochemical processes occurring in, for example, mammalian tissue sections. The mass spectrometer is used to determine the molecular weights of the molecules in the surface layers of the tissue. Molecules desorbed from the sample typically are singly protonated, giving an ion at (M + H)+, where M is the molecular mass. The procedure involves coating the tissue section, or a blotted imprint of the section, with a thin layer of energy-absorbing matrix and then analyzing the sample to produce an ordered array of mass spectra, each containing nominal m/z values typically covering a range of over 50 000 Da. Images...

Integrated capillary electrophoresis on flexible silicone microdevices: analysis of DNA restriction fragments and detection of single DNA molecules on microchips

Abstract: Microchips for integrated capillary electrophoresis systems were produced by molding a poly(dimethylsiloxane) (PDMS) silicone elastomer against a microfabricated master. The good adhesion of the PDMS devices on clean planar surfaces allows for a simple and inexpensive generation of networks of sealed microchannels, thus removing the constraints of elaborate bonding procedures. The performance of the devices is demonstrated with both fast separations of φX-174/HaeIII DNA restriction fragments labeled with the intercalating dye YOYO-1 and fluorescently labeled peptides. Detection limits in the order of a few zeptomoles (10-21 mol) have been achieved for each injected DNA fragment, corresponding to a mass detection limit of ∼2 fg for the 603 base pair fragment. Single λ-DNA molecules intercalated with YOYO-1 at a base pair-to-dye ratio of 10:1 could be detected with an uncomplicated laser-induced fluorescence detection setup. High single-molecule detection efficiency (>50%) was achieved under electrophoretic...

Liquid-Phase Microextraction in a Single Drop of Organic Solvent by Using a Conventional Microsyringe

Abstract: Two modes of liquid-phase microextraction (LPME) were developed for capillary gas chromatography. Both methodologies, i.e., static LPME and dynamic LPME, involve the use of very small amounts of organic solvent (<2 μL) in a conventional microsyringe. The performance of the two techniques is demonstrated in the determination of two chlorobenzenes extracted into a single drop of toluene by the use of a 10-μL syringe. Static LPME provided some enrichment (∼12-fold), good reproducibility (9.7%), and simplicity but suffered relatively long extraction time (15 min). Dynamic LPME provided higher (∼27-fold) enrichment within much shorter extraction time (∼3 min), and relatively poorer precision (12.8%), primarily due to repeated manual manipulation. Both methods allow the direct transfer of extracted analytes into a gas chromatograph.

Microchannel electrophoretic separations of DNA in injection-molded plastic substrates

Abstract: Microfabricated electrophoretic separation devices have been produced by an injection-molding process. The strategy for producing the devices involved solution-phase etching of a master template on a silicon wafer, followed by electroforming more durable injection-molding masters in nickel from the silicon master. One of the nickel electroforms was then used to prepare an injection mold insert, from which microchannel chips in an acrylic substrate were mass-produced. The microchannel devices were used to demonstrate high-resolution separations of double-stranded DNA fragments with total run times of less than 3 min. Run-to-run and chip-to-chip reproducibility was good, with relative standard deviation values below 1% for the run-to-run data and in the range of 2-3% for the chip-to-chip comparisons. Such devices could lead to the production of low-cost, single-use electrophoretic chips suitable for a variety of separation applications, including DNA sizing, DNA sequencing, random primary library screening, and rapid immunoassay testing.

Fabrication of plastic microfluid channels by imprinting methods.

Abstract: Microfluidic devices have been fabricated on poly(methyl methacrylate) substrates by two independent imprinting techniques. First-generation devices were fabricated using a small-diameter wire to create an impression in plastics softened by low-temperature heating. The resulting devices are limited to only simple linear channel designs but are readily produced at low cost. Second-generation devices with more complex microchannel arrangements were fabricated by imprinting the plastic substrates using an inverse three-dimensional image of the device micromachined on a silicon wafer. This micromachined template may be used repeatedly to generate devices reproducibly. Fluorescent analtyes were used to demonstrate reproducible electrophoretic injections. An immunoassay was also performed in an imprinted device as a demonstration of future applications.

Ultrahigh-pressure reversed-phase liquid chromatography in packed capillary columns.

Abstract: The use of extremely high pressures in liquid chromatography can improve the efficiency and reduce analysis time for columns packed with small particles. In this work, fused-silica capillaries with inner diameters of 30 μm are slurry packed with 1.5 μm nonporous octadecylsilane-modified silica particles. These columns are prepared in lengths up to 66 cm with packing pressures as high as 4100 bar (60 000 psi). Near the optimum flow rate, columns generate as many as 300 000 theoretical plates for lightly retained compounds (k‘ < 0.5) and over 200 000 plates for more retained compounds (k‘ ≈ 2). These translate to plate heights (Hmin) as low as 2.1 μm. The pressures required to run at optimum flow rates are on the order of 1400 bar (20 000 psi). Analysis times at these pressures are on the order of 30 min (k‘ ≈ 2) and can be reduced to less than 10 min at higher than optimum flow rates. Capacity factors are observed to increase linearly with applied pressure.

Identification, Quantification, and Relative Concentrations of Carotenoids and Their Metabolites in Human Milk and Serum

Abstract: Thirty-four carotenoids, including 13 geometrical isomers and eight metabolites, in breast milk and serum of three lactating mothers have been separated, identified, quantified, and compared by high-performance liquid chromatography (HPLC)−photodiode array (PDA) detection−mass spectrometry (MS). Among the metabolites were two oxidation products of lycopene and four of lutein/zeaxanthin. In addition, two metabolites of lutein, formed as a result of dehydration of this dihydroxycarotenoid under acidic conditions similar to those of the stomach, have also been identified in plasma and breast milk. The oxidative metabolites of lycopene with a novel five-membered-ring end group have been identified as epimeric 2,6-cyclolycopene-1,5-diols by comparison of their HPLC−UV/visible−MS profiles with those of fully characterized (1H- and 13C-NMR spectroscopy) synthetic compounds. The HPLC procedures employed also detected vitamin A, two forms of vitamin E (γ- and α-tocopherol), and two non-carotenoid food components, ...

Transport, manipulation, and reaction of biological cells on-chip using electrokinetic effects.

Abstract: A microfluidic system was fabricated on a glass chip to study mobilization of biological cells on-chip. Electroosmotic and/or electrophoretic pumping were used to drive the cell transport within a network of capillary channels. Whole cells such as Saccharomyces cerevisiae, canine erythrocyte, and Escherichia coli were employed in this work. Photographs are presented to illustrate how cells are selected and transported from one location to another within the capillary network, with velocities up to about 0.5 mm/s in capillaries with a 15- × 55-μm cross section. The mixing of canine erythrocytes with the lysing agent sodium dodecyl sulfate, at an intersection within the chip, was performed to demonstrate that cell selection and subsequent reaction can be accomplished within the microchip.

Mass Transfer Characteristics of Solvent Extraction into a Single Drop at the Tip of a Syringe Needle

Abstract: The amount of a sample compound extracted into a 1-μL drop of n-octane suspended in a stirred aqueous solution from the tip of a microsyringe needle is measured by gas chromatography (GC) as a function of time. The observed extraction rate curve is first order and yields the overall mass transfer coefficient for the sample compound, βo. For a given compound, βo varies linearly with stirring rate. Among the four compounds malathion, 4-methylacetophenone, 4-nitrotoluene, and progesterone, at a given stirring rate, βo is linearly proportional to the diffusion coefficient of the compound (Daq). This supports the film theory of convective−diffusive mass transfer, as opposed to the penetration theory. The relative standard deviation of the GC signal for 4-methylacetophenone after a 1.00 min extraction at 1500 rpm is 1.5%, which suggests that the system exhibits excellent potential as a tool for rapid analysis by solvent extraction/GC.

UV Laser Machined Polymer Substrates for the Development of Microdiagnostic Systems

Abstract: This report describes a UV laser photoablation method for the production of miniaturized liquid-handling systems on polymer substrate chips. The fabrication of fluid channel and reservoir networks is accomplished by firing 200 mJ pulses from an UV excimer laser at substrates moving in predefined computer-controlled patterns. This method was used for producing channels in polystyrene, polycarbonate, cellulose acetate, and poly(ethylene terephthalate). Efficient sealing of the resulting photoablated polymer channels was accomplished using a low-cost film lamination technique. After fabrication, the ablated structures were observed to be well defined, i.e., possessing high aspect ratios, as seen by light, scanning electron, and atomic force microscopy. Relative to the original polymer samples, photoablated surfaces showed an increase in their hydrophilicity and rugosity as a group, yet differences were noted between the polymers studied. These surface characteristics demonstrate the capability of generating electroosmotic flow in the cathodic direction, which is characterized here as a function of applied electric field, pH, and ionic strength of common electrophoretic buffer systems. These results show a correlation between the ablative changes in surface conditions and the resulting electroosmotic flow. The effect of protein coatings on ablated surfaces is also demonstrated to significantly dampen the electroosmotic flow for all polymers. All of these results are discussed in terms of developing liquid-handling capability, which is an essential part of many μ-TAS and chemical diagnostic systems.

Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level.

Abstract: A method to directly identify proteins contained in mixtures by microcolumn reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) is studied. In this method, the mixture of proteins is digested with a proteolytic enzyme to produce a large collection of peptides. The complex peptide mixture is then separated on-line with a tandem mass spectrometer, acquiring large numbers of tandem mass spectra. The tandem mass spectra are then used to search a protein database to identify the proteins present. Results from standard protein mixtures show that proteins present in simple mixtures can be readily identified with a 30-fold difference in molar quantity, that the identifications are reproducible, and that proteins within the mixture can be identified at low femtomole levels. Based on these studies, methodology has been developed for direct LC/MS/MS analysis of proteins enriched by immunoaffinity precipitation, specific interaction with a protein−protein fusion product, a...

Real-Time Analysis of Individual Atmospheric Aerosol Particles: Design and Performance of a Portable ATOFMS

Abstract: Two portable aerosol time-of-flight mass spectrometers (ATOFMS) of identical design are described. These instruments are powerful new tools for providing temporal and spatial information on the origin, reactivity, and fate of atmospheric aerosols. Each is capable of analyzing the size and composition of individual particles from a polydisperse aerosol in real-time. Particles are introduced into the instrument through a particle beam interface, sized by measuring the delay time between two scattering lasers, and compositionally analyzed using a dual-polarity laser desorption/ionization time-of-flight mass spectrometer. These are the first dual-ion TOFMS instruments to utilize a dual reflectron design. The instruments measure 72 in. long × 28 in. wide × 60 in. high and weigh ∼500 lb. Pneumatic tires allow them to be transported through standard doorways, elevators, and handicap ramps, granting access to virtually any location. Furthermore, because of rugged construction they will be able to operate during t...

Automated In-Tube Solid-Phase Microextraction Coupled to High-Performance Liquid Chromatography

Abstract: Recently, solid-phase microextraction (SPME) was successfully coupled to high-performance liquid chromatography. However, the efficiency of this analytical method, in terms of manpower, still suffers from its manual operation technique. Furthermore, the selectivity obtained for the analysis of very polar compounds is still poor because of a limited selection of commercially available fiber coatings that can withstand the aggressive HPLC conditions (solvents). This paper describes the first approach to developing an automated SPME-HPLC system. A mixture of polar thermally labile analytes, phenylurea pesticides, was selected for microextraction directly from an aqueous sample. A piece of a ordinary capillary GC column with its coating (Omegawax 250) was used for the absorption of analytes from the aqueous sample (in-tube solid-phase microextraction). A needle hosts the capillary when it is pierced through the septum of the vial containing the spiked aqueous sample. The aqueous samples were stored in 2 mL vi...

Current Rectification at Quartz Nanopipet Electrodes

Abstract: Ag/AgCl reference electrodes fabricated from pulled quartz tubes with orifice radii of 20 nm to 20 μm were characterized in KCl solutions of different concentrations by cyclic voltammetry. Linear current−voltage (i−V) dependence was observed with micropipet electrodes (with micrometer-sized tips) with the same concentration (0.01−1 M) of KCl inside and outside the pipet, but current rectification was found at nanopipet electrodes at KCl concentrations of ≤0.1 M (with nanometer-sized tips). This is attributed to formation of a diffuse electrical double layer within the tip orifice. The effects of electrode size, electrolyte concentration, and solution pH on the nonlinear i−V behavior of these electrodes were investigated. A model for the observed behavior shows the rectification to be caused by the permselectivity in the tip region and the geometric asymmetry of the tip orifice. This phenomenon may be important in studies of ion transport in charged channels and porous membranes.

Quartz Crystal Microbalance Study of DNA Immobilization and Hybridization for Nucleic Acid Sensor Development

Abstract: The immobilization of two 30-mer oligonucleotides, one biotinylated (biotin-DNA) and the other having a mercaptohexyl group at the 5‘-phosphate end (BS1-SH), onto modified gold surfaces has been examined using a quartz crystal microbalance (QCM). Both single-layer and multilayer DNA films were prepared. The single-layer films of biotin-DNA were constructed by binding to a precursor layer of avidin, which had been attached to the QCM either covalently using a water-soluble carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) or via electrostatic interaction with poly(allylamine hydrochloride) (PAH). Single-layer films of BS1-SH were also formed on PAH via the electrostatic attraction between the amine groups on PAH and the negatively charged phosphate backbone of DNA. Multilayer films of DNA were fabricated by the successive deposition of avidin and poly(styrenesulfonate) (PSS), up to a total of nine avidin/PSS layers, followed by DNA adsorption. DNA immobilization and hybridization of the immob...

Molded Rigid Polymer Monoliths as Separation Media for Capillary Electrochromatography

Abstract: Rigid, monolithic capillary columns for reversed-phase electrochromatography have been prepared within the confines of untreated fused-silica capillaries in a single step by a simple copolymerization of ethylene dimethacrylate, butyl methacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid in the presence of a porogenic solvent. The composition of the specifically designed ternary porogenic solvent allows fine control of the porous properties of the monolithic material over a broad range. While the electroosmotic flow through these capillary columns increases with both increasing pore size of the monolith and content of charged functionalities, better chromatographic properties have been observed for monoliths with larger surface area and hydrophobicity. Using this technique, monolithic capillary columns with an efficiency higher than 120 000 plates/m have been easily obtained.

Generating electrospray from microchip devices using electroosmotic pumping.

Abstract: A method of generating electrospray from solutions emerging from small channels etched on planar substrates is described. The fluids are delivered using electroosmotically induced pressures and are sprayed electrostatically from the terminus of a channel by applying an electrical potential of sufficient amplitude to generate the electrospray between the microchip and a conductor spaced from the channel terminus. No major modification of the microchip is required other than to expose a channel opening. The principles that regulate the fluid delivery are described and demonstrated. A spectrum for a test compound, tetrabutylammonium iodide, that was continuously electrophoresed was obtained by coupling the microchip to an ion trap mass spectrometer.

Microchip Device for Performing Enzyme Assays

Abstract: An automated enzyme assay was performed within a microfabricated channel network. Precise concentrations of substrate, enzyme, and inhibitor were mixed in nanoliter volumes using electrokinetic flow. Reagent dilution and mixing were controlled by regulating the applied potential at the terminus of each channel, using voltages derived from an equivalent circuit model of the microchip. The enzyme β-galactosidase (β-Gal) was assayed using resorufin β-d-galactopyranoside (RBG), a substrate that is hydrolyzed to resorufin, a fluorescent product. Reaction kinetics were obtained by varying the concentration of substrate on-chip and monitoring the production of resorufin using laser-induced fluorescence. Derived Michaelis−Menten constants compared well between an on-chip and a conventional enzyme assay. Bias in the derived Km and kcat was primarily due to the limited solubility of RBG and the associated lack of measurements at substrate concentrations exceeding the Km. A Ki of 8 μM for the inhibitor phenylethyl β...

A Predictive Model for Matrix and Analyte Effects in Electrospray Ionization of Singly-Charged Ionic Analytes

Abstract: In electrospray ionization (ESI), droplets with a surface excess charge are created. The rate of production of surface excess charge is a constant and is equal to the rate of ion production. The ions appearing in the mass spectrum are postulated to be those that formed the surface excess charge at the time of droplet formation (or their collision products). An equilibrium model based on competition among the ions in the solution for the limited number of excess charge sites has been developed. This model accurately predicts the response curves of singly-charged ionic analytes as a function of the concentration of electrolyte and other analytes and provides an explanation for the selective effectiveness of ESI. At low concentrations of total analyte (micromolar and less), the response curves are linear, indifferent to the presence of other low concentration analytes, and suppressed by electrolyte concentrations in excess of the minimum required. At higher analyte concentrations, the response becomes independent of analyte concentration but highly affected by the presence of other analytes.

Sol-gel coating technology for the preparation of solid-phase microextraction fibers of enhanced thermal stability.

Abstract: A novel sol-gel method is described for the preparation of solid-phase microextraction (SPME) fibers. The protective polyimide coating was removed from a 1-cm end segment of a 200 μm o.d. fused-silica fiber, and the exposed outer surface was coated with a bonded sol-gel layer of poly(dimethylsiloxane) (PDMS). The chemistry behind this coating technique is presented. Efficient SPME-GC analyses of polycyclic aromatic hydrocarbons, alkanes, aniline derivatives, alcohols, and phenolic compounds in dilute aqueous solutions were achieved using sol-gel-coated PDMS fibers. The extracted analytes were transferred to a GC injector using an in-house-designed SPME syringe that also allowed for easy change of SPME fibers. Electron microscopy experiments suggested a porous structure for the sol-gel coating with a thickness of ∼10 μm. The coating porosity provided higher surface area and allowed for the use of thinner coatings (compared with 100-μm-thick coatings for conventional SPME fibers) to achieve acceptable stationary-phase loadings and sample capacities. Enhanced surface area of sol-gel coatings, in turn, provided efficient analyte extraction rates from solution. Experimental results on thermal stability of sol-gel PDMS fibers were compared with those for commercial 100-μm PDMS fibers. Our findings suggest that sol-gel PDMS fibers possess significantly higher thermal stability (>320 °C) than conventionally coated PDMS fibers that often start bleeding at 200 °C. This is due, in part, to the strong chemical bonding between the sol-gel-generated organic-inorganic composite coating and the silica surface. Enhanced thermal stability allowed the use of higher injection port temperatures for efficient desorption of less-volatile analytes and should translate into extended range of analytes that can be handled by SPME-GC techniques. Experimental evidence is provided that supports the operational advantages of sol-gel coatings in SPME-GC analysis.

Multichannel microchip electrospray mass spectrometry.

Abstract: Microfabricated multiple-channel glass chips were successfully interfaced to an electrospray ionization mass spectrometer (ESI-MS). The microchip device was fabricated by standard photolithographic, wet chemical etching, and thermal bonding procedures. A high voltage was applied individually from each buffer reservoir for spraying sample sequentially from each channel. With the sampling orifice of the MS grounded, it was found that a liquid flow of 100−200 nL/min was necessary to maintain a stable electrospray. The detection limit of the microchip MS experiment for myoglobin was found to be lower than 6 × 10-8 M. Samples in 75% methanol were successfully analyzed with good sensitivity, as were aqueous samples. The parallel multiple-channel microchip system allowed ESI-MS analysis of different samples of standard peptides and proteins in one chip.

Microchip-Based Capillary Electrophoresis for Immunoassays: Analysis of Monoclonal Antibodies and Theophylline

Abstract: A microchip capillary electrophoresis device has been used to separate the reaction products of homogeneous, immunological reactions within ∼40 s. Determination of monoclonal mouse IgG in mouse ascites fluid, via a direct assay, and the drug theophylline in serum samples, via a competitive assay, was demonstrated on-chip. The mouse anti-bovine serum albumin IgG assay gave a linear calibration curve up to at least 135 μg/mL, with ±3% precision. The theophylline assay gave a threshold for detection of 1.25 ng/mL in diluted serum. A calibration curve of signal vs undiluted log[theophylline] is linear from 2.5 to 40 μg/mL, which includes the therapeutically useful range. Theophylline recoveries in spiked samples were 100%, within an experimental error of ±5%. A buffer system consisting of 0.05 M tricine adjusted to pH 8.0, 0.01% (w/v) Tween 20, and ∼40 mM NaCl was used. This buffer allowed for adequate separation (40 000 plates for theophylline; 1000 plates for theophylline−antibody complex and for human IgG)...

Chromatographic Hydrophobicity Index by Fast-Gradient RP-HPLC: A High-Throughput Alternative to log P/log D.

Abstract: A new chromatographic hydrophobicity index (CHI) is described which can be used as part of a protocol for high-throughput (50−100 compounds/day) physicochemical property profiling for rational drug design. The index is derived from retention times (tR) observed in a fast gradient reversed-phase HPLC method. The isocratic retention factors (log k‘) were measured for a series of 76 structurally unrelated compounds by using various concentrations of acetonitrile in the mobile phase. By plotting the log k‘ as a function of the acetonitrile concentration, the slope (S) and the intercept (log k‘w) values were calculated. The previously validated index of hydrophobicity φ0 was calculated as −log k‘w/S. A good linear correlation was obtained between the gradient retention time values, tR and the isocratically determined φ0 values for the 76 compounds. The constants of this linear correlation can be used to calculate CHI. For most compounds, CHI is between 0 and 100 and in this range it approximates to the percent...

A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide: Combination of Marfey's Method with Mass Spectrometry and Its Practical Application

Abstract: The “advanced Marfey's method” proposed in our preceding paper has been developed to nonempirically determine the absolute configuration of constituent amino acids in a peptide using liquid chromatography/mass spectrometry (LC/MS). For the establishment of this method, we had to resolve the following three problems: (1) elucidation of the limitation of Marfey's method, which is chosen as the chromatography technique, and its separation mechanism, because this proposed method relies on the elution order of an amino acid derivatized with 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (l-FDAA) to determine its absolute configuration; (2) how to effectively combine Marfey's method with mass spectrometry in order to detect and identify a desired amino acid without a standard sample; and (3) how to obtain the corresponding enantiomer from either the l- or d-amino acid in a peptide sample. In a preceding paper, we investigated problem 1 and finally described the rational application guideline for Marfey's method to...

Net analyte signal calculation in multivariate calibration

Abstract: Net analyte signal plays an important role in the calculation of figures of merit for characterizing a calibration model. Until now, its computation has only been feasible for the direct calibration model, which requires knowledge of pure spectra or concentrations of all contributing species in the calibration samples. An increasingly important calibration model is the inverse calibration model, which also allows for quantitation if the knowledge about the interferents is incomplete. This paper shows that net analyte signal computation is possible for the inverse calibration case. Application to the determination of protein content in wheat samples by near-infrared spectrometry is presented. Net analyte signal calculation was used to estimate selectivities (ratio of signal available for quantitation to the total measured signal). The selectivities were found to range between 0 and 2% of the measured reflectance signal. A new measure for outlier diagnosis based on the correlation of the net analyte signal ...

High-speed DNA genotyping using microfabricated capillary array electrophoresis chips.

Abstract: Capillary array electrophoresis (CAE) chips have been designed and fabricated with the capacity to rapidly (<160 s) analyze 12 different samples in parallel. Detection of all lanes with 0.3 s temporal resolution was achieved using a laser-excited confocal-fluorescence scanner. The operation and capabilities of these CAE microdevices were first determined by performing electrophoretic separations of pBR322 MspI DNA samples. Genotyping of HLA-H, a candidate gene for the diagnosis of hereditary hemochromatosis, was then performed to demonstrate the rapid analysis of biologically relevant samples. Two-color multiplex fluorescence detection of HLA-H genotypes was accomplished by prelabeling the standard pBR322 MspI DNA ladder with a red emitting bis-intercalation dye (butyl TOTIN) and on-column labeling of the HLA-H DNA with thiazole orange. This work establishes the feasibility of using CAE chips for high-speed, high-throughput genotyping.

Capillary Electrochromatography with Predetermined Selectivity Obtained through Molecular Imprinting

Abstract: This article presents a novel approach to capillary electrochromatography by which predetermined selectivity is achieved. A simple and quick method for the in situ preparation of monolithic molecularly imprinted flow-through polymers inside fused silica capillaries is described. The superporous structure of the polymers permits rapid solvent and electrolyte exchange, as well as easy regeneration of the capillaries by hydrodynamic pumping. Chiral stationary phases were prepared by molecular imprinting of the β-adrenergic antagonists propranolol and metoprolol. The separation systems were operational within 3 h of the start of capillary preparation. Chiral separations with baseline resolution could be carried out in less than 120 s.

Solid Phase Microextraction for Quantitative Analysis in Nonequilibrium Situations

Abstract: Solid phase microextraction (SPME) is a convenient and efficient extraction method that involves using a thin polymer film coating on a fine silica fiber to adsorb analytes of interests from a sample matrix. A theoretical model is proposed to deal with the dynamic adsorption process of SPME. In this model, mass diffusion from the matrix to the SPME polymer film is considered as the rate-determining step in reaching an adsorption equilibrium, and a steady-state diffusion is assumed for SPME in an effectively agitated sampling medium. Mathematical treatment of the adsorption process generates an expression that can describe experimental adsorption time profiles of the SPME process. The expression also provides a directly proportional relationship between the amount of analyte adsorbed by the SPME fiber and its initial concentration in the sample matrix. This relationship indicates that SPME quantification is feasible before an adsorption equilibrium is reached, once the agitation condition and the sampling ...

Analysis of Oligonucleotides by HPLC-Electrospray Ionization Mass Spectrometry.

Abstract: A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates...

Determination of Unbiased Selectivity Coefficients of Neutral Carrier-Based Cation-Selective Electrodes

Abstract: A new procedure for the determination of selectivity coefficients of neutral carrier-based cation-selective electrodes is established that avoids exposure to the preferred ion prior to the measurement of discriminated ions. The method is, therefore, unbiased by the presence of preferred ions in the membrane that otherwise could mask the response to discriminated ion solutions. It is generally applicable as long as a series of considerations are met and can only be applied once for a given membrane. Careful studies with a series of sodium-, silver-, and calcium-selective electrodes reveal that Nernstian response slopes can now be obtained for even highly discriminated cations. Specifically, a 1,3-bridged calix[4]arene derivative as introduced by Yamamoto and Shinkai indeed yields an extraordinary sodium selectivity of log = −4.9, with potassium showing Nernst response as well. Analogous measurements with two different silver carriers, a bisthioether-functionalized calix[4]arene and methylenebis(diisobutyld...