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A home-built stopped-flow apparatus is interfaced to a Hadamard transform time-of-flight mass spectrometer, which permits study of reaction kinetics with a time between reaction initiation and observation as short as about 100 ms and a sampling rate of chemical change that can approach 1 ms. This technique is applied to the trypsin-catalyzed hydrolysis of several peptides and is validated by comparing the results with literature values as well as to optical data obtained with the present stopped-flow apparatus. In addition, we report a kinetic study of the action of trypsin on a peptide having more than one cleavage site. 10.1021/ac101899n

The sampling rate and imaging capabilities of desorption electrospray ionization (DESI) are examined using a rotating sample platform combined with Hadamard transform time-of-flight mass spectrometry (HTTOFMS), a multiplexed time-of-flight technique that allows for millisecond acquisition of full mass-to-charge ratio scans. DESI-compatible dyes are used to produce spatially defined sample patterns on poly(methyl methacrylate) discs. Control of disk rotation rate sets the residence time of the sample spots in the DESI plume, and thus the sampling rate. Surface patterns of alternating analytes are spectrally resolved up to 80 samples/s and single-analyte spots up to 50 samples/s. The rapid movement of the surface under the DESI plume allows for high DESI solution flow rates without blurring the chemical information on the surface. Data from multiple rotations can be additively combined, generating a chemical image of the surface with improved signal-to-noise characteristics. This multipass data enables analysis of the rising and falling edges of the analyte signal, placing a lower limit on both the temporal resolution of DESI and the maximum achievable sampling rate. Multipass analysis is proposed as a method for DESI surface imaging. 10.1021/ac901668a

We have designed and constructed a continuous imaging reflectron time-of-flight mass spectrometer (TOFMS) that provides a mass spectrum at every pixel of a two-dimensional image with a 100% duty cycle. The technique is based on pseudorandom ion beam modulation and three-dimensional (x, y, t) ion imaging. We use a multi-channel plate detector with a delay-line anode that provides x, y positions and flight times t of every ion arrival event. The precision of the peak heights in the 100% duty cycle mass spectra is shown to be enhanced even at short (10 ms) acquisition times, which should prove useful for the study of solution kinetics or fast chromatographic separations. As a demonstration of the system's capability, we have imaged the fragmented ions that underwent surface-induced dissociation inside the reflectron and the ions that fragmented spontaneously through postsource decay.

Inflammatory bowel disease (IBD) is a chronic debilitating disorder that is thought to have both genetic and environmental contributors. Commensal microflora have been shown to play a key part in the disease process. Metabolomics, the study of large numbers of small molecule metabolites, has demonstrated that disease and/or changes in gut microbial composition modulate mammalian urine metabolite fingerprints. The aim of this project was to associate the development of IBD with specific changes in a mouse urinary metabolic fingerprint. Interleukin-10 (IL-10) gene-deficient mice were raised alongside age-matched 129/SvEv controls in conventional housing. Urine samples (22 h) were collected at ages 4, 6, 8, 12, 16, and 20 weeks. Metabolite concentrations were derived from analysis of nuclear magnetic resonance spectra, and both multivariate and two-way analysis of variance (ANOVA) statistical techniques were applied to the resulting data. Principal component analysis and partial least-squares-discriminant analysis of urine derived from the control and IL-10 gene-deficient mice revealed that while both groups initially had similar metabolic profiles, they diverged substantially with the onset of IBD as assessed through external phenotypic changes. Several metabolites, including trimethylamine (TMA) and fucose, changed dramatically in the IL-10 gene-deficient mice following 8 weeks of age, concomitant with the known timeline for development of severe histological injury. This study illustrates that metabolomics is effective at distinguishing IBD using urinary metabolite profiles.

[alpha]-Zirconium phosphate nanoplatelets ([alpha]-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of [alpha]-zirconium phosphate were incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice with water followed by an aqueous solution of 80% acetonitrile to form the binding agent. [alpha]-ZrPN were able specifically to capture phosphoserine-containing peptides from a tryptic digest of a complex peptide mixture in which its abundance was only 0.05%. [alpha]-ZrPN also bound peptides containing phosphothreonine and phosphotyrosine. The limit of detection for phosphopeptides is ~2 fmol, based on using matrix-assisted laser desorption/ionization mass spectrometry. [alpha]-ZrPN were applied for the analysis of tryptic digests of mouse liver and leukemia cell phosphoproteomes and succeeded in identifying 158 phosphopeptides (209 phosphorylation sites) from 101 phosphoproteins in mouse liver lysate and 78 phosphopeptides (104 phosphorylation sites) from 59 phosphoproteins in leukemia cell extract. For these two tryptic digests, the [alpha]-ZrPN approach is able to capture more phosphopeptides than that obtained from Ti[O.sub.2] particles or from [Fe.sup.3+]-IMAC beads, but each method is able to bind some phosphopeptides that the others do not.

Bradbury-Nielsen gates (BNGs) are a standard way for gating or steering beams of charged particles in ion mobility spectrometry and time-of-flight mass spectrometry. They consist of a pair of interleaved electrodes that when at the same potential allow ions to pass through the electrodes undeflected and, when a voltage is applied, cause the ions to be deflected from their propagation axis. Previous efforts to construct such devices have relied on mechanical assembly by winding wires across an aperture. We describe a micromachining method for making monolithic BNGs using deep reactive ion etching of silicon-on-insulator wafers. This method enables the creation of electrodes with spacings ranging from 25 to 100 [micro]m with a thickness of 20 [micro]m, covering a 5 mm by 5 mm active area. We characterize the performance of these micromachined BNGs by ion imaging in a pseudorandom time-of-flight mass spectrometer.

Dynamic modification of poly(dimethylsiloxane) channels using a mixture of n-dodecyl-[beta]-D-maltoside (DDM) and sodium dodecyl sulfate (SDS) is able to suppress analyte adsorption and control electroosmotic flow (EOF). In this mixed surfactant system, the nonionic surfactant DDM functions as a surface blocking reagent, whereas the anionic surfactant SDS introduces negative charges to the channel walls. Changing the DDM/SDS mixing ratio tunes the surface charge density and the strength of EOF. Using 0.1% (w/v) DDM and 0.03% (w/v) SDS, Alexa Fluor 647 labeled streptavidin can be analyzed according to the charges added by the fluorophores. Protein molecules with different numbers of fluorophores are well resolved. DDM and SDS also form negatively charged mixed micelles, which act as a separation medium. The low critical micellar concentration of DDM/SDS mixed micelles also allows the use of SDS at a nondenaturing concentration, which enables the analysis of proteins in their native state. The immunocomplex between a membrane protein, [[beta].sub.2] adrenergic receptor, and anti-FLAG antibody has been fully separated using 0.1% (w/v) DDM and 0.03% (w/v) SDS. We have also analyzed the composition of lightharvesting protein-chromophore complexes in cyanobacteria.

A collinear-beam thermal lens detector has been constructed and its properties were characterized. Its application to the high-performance liquid chromatography (HPLC) separation of a mixture of five anthraquinone dyes dissolved in water shows a linear response over 3.5 orders of magnitude and a detection limit that is subnanomolar in the dye concentrations. These results are compared with those obtained previously using cavity ring-down spectroscopy (CRDS) in a Brewster's angle flow cell (Bechtel, K. L.; Zare, R. N.; Kachanov, A. A.; Sanders, S. S.; Paldus, B. A. Anal. Chem. 2005, 77, 1177-1182). The peak-to-peak baseline noise of the thermal lensing detection is 3.5 x [10.sup.-8] absorbance units (AU) with a path length of 200 [micro]m, whereas the peak-to-peak baseline noise of CRDS detection is ~2 x [10.sup.-7] AU with a path length of 300 [micro]m. Both of these figures of merit should be compared to the peak-to-peak baseline noise of one of the best commercial UV-vis HPLC detection systems, which is ~5 x [10.sup.-6] AU with a path length of 10 mm (1-s integration time). Therefore, the thermal lensing technique has a demonstrated sensitivity of subnanomolar detection that is ~140 times better than that of the best commercial UV-vis detector and ~5 times better than that of CRDS.

Evanescent-wave cavity ring-down spectroscopy has been applied to a planar fused-silica surface covered with crystal violet (C[V.sup.+]) cations to characterize the silanol groups indirectly. A radiation-polarization dependence of the adsorption isotherm of C[V.sup.+] at the C[H.sub.3]CN/silica interface is measured and fit to a two-site Langmuir equation to determine the relative populations of two different types of isolated silanol groups. C[V.sup.+] binding at type I sites yields a free energy of adsorption of -29.9 [+ or -] 0.2 kJ/mol and a saturation surface density of (7.4 [+ or -] 0.5) x [10.sup.12] [cm.sup.-2], whereas the values of -17.9 [+ or -] 0.4 kJ/mol and (3.1 [+ or -] 0.4) x [10.sup.13] [cm.sup.-2] are obtained for the type II sites. The C[V.sup.+] cations, each with a planar area of ~120 [[Angstrom].sup.2], seem to be aligned randomly while lying over the SiO-type I sites, thereby suggesting that this type of site may be surrounded by a large empty surface area (> 480 [[Angstrom].sup.2]). In contrast, the C[V.sup.+] cations on a type II sites are restricted with an average angle of ~400 tilted off the surface normal, suggesting that the C[V.sup.+] cations on these sites are grouped closely together. The average tilt angle increases with increasing concentration of crystal violet so that C[V.sup.+] cations may be separated from each other to minimize the repulsion of nearby C[V.sup.+] and SiOH sites.

We designed, constructed, and tested a surface plasmon resonance (SPR) microscope using a high numerical aperture objective from a commercially available inverted optical microscope. Such a configuration, combined with various methods to shorten the surface plasmon propagation length, achieves diffraction-limited spatial resolution in the transverse direction and near-diffraction-limited resolution in the longitudinal direction. A virtue of the objective-type SPR imaging is that we achieve distortionfree angle-resolved SPR imaging, allowing the angle-dependent reflectivity of the sample to be examined on a pixel-by-pixel basis, thus offering high-resolution information about surface properties

The electroosmotic flow (EOF) in a poly(dimethylsiloxane) (PDMS) separation channel can be altered and controlled by adding a carboxylic acid to the prepolymer prior to curing. When the prepolymer is doped with 0.5 wt % undecylenic acid (UDA), the electroosmotic mobility in a modified PDMS channel rises to (7.6 [+ or -] 0.2) x [10.sub.-4] [cm.sub.2] [V.sup.-1] [s.sup.-1] (in HEPES buffer at pH 8.5), which is nearly twice that in the native PDMS channel. Because this modification does not significantly change the hydrophobicity of the PDMS surface, it is possible to combine the modified PDMS with a dynamic coating of n-dodecyl [beta]-D-maltoside (DDM), which prevents protein sticking (see Huang, B.; Wu, H. K.; Kim, S.; Zare, R. N. Lab Chip 2005, 5, 1005-1007). The modified PDMS channel with a dynamic coating of DDM generates an electroosmotic mobility of (5.01 [+ or -] 0.09) x [10.sup.-4] [cm.sup.2] [V.sup.-1] [s.sup.-1], which shows excellent reproducibility both in successive runs and during storage in water. Combining this surface modification and the dynamic coating of DDM is an effective means for both providing stable EOF in the PDMS channels and preventing protein adsorption on the channel walls. To demonstrate these effects, we show that the electrophoretic separation of immunocomplexes in free solution can be readily accomplished in a microfluidic chip made of UDA-doped (0.5 wt %) PDMS with a dynamic coating of DDM.

Trypsin is covalently linked to a photopolymerized sol-gel monolith modified by incorporating poly(ethylene glycol) (PSG-PEG) for on-column digestion of [N.sub.[alpha]]-benzoyl-L-arginine ethyl ester (BAEE) and two peptides, neurotensin and insulin chain B. The coupling of the enzyme to the monolith is via room-temperature Schiff chemistry in which an alkoxysilane reagent (linker) with an aldehyde functional group links to an inactive amine on trypsin to form an imine bond. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of [N.sub.[alpha]]-benzoyl-L-arginine (BA), the digestion product of BAEE. The BA is separated from BAEE by capillary electrophoresis and detected downstream (18.5 cm from the microreactor) by absorption (254 nm). Using the Bradford assay, we determined that 97 ng of trypsin is bound to the 1-cm microreactor located at the entrance of capillary column. The bioactivity of the trypsin-PSGPEG microreactor at 20 [degrees]C for the digestion of BAEE was found to be 2270 units/mg of immobilized trypsin. The bioactivity of trypsin bound to the capillary wall in the open segment upstream from the monolith was 332 units/ mg of immobilized trypsin under the same conditions. In contrast, the activity of free trypsin could not be observed for the digestion of BAEE at 20[degrees]C after 16 h of incubation time.

We describe the use of liquid-phase continuous-wave cavity ring-down spectroscopy for the detection of an HPLC separation. This technique builds on earlier work by Snyder and Zare using pulsed laser sources and improves upon commercially available UV--visible detectors by a factor of up to 50. The system employs a compact doubled-diode single-mode continuous-wave laser operating at 488 nm and a previously described Brewster's-angle flow cell. Ring-down time constants as long as 5.8 [micro]s were observed with liquid samples in a 0.3-mm path length cell. The baseline noise during an HPLC separation was only 2 x [10.sup.-7] absorbance units (AU) peak to peak, as compared to 1 x [10.sup.-5] AU for a state-of-the-art commercial UV--visible detector.

Microprobe laser desorption/laser ionization mass spectrometry ([micro][L.sup.2]MS) is a sensitive and selective technique that has proven useful in the qualitative and semiquantitative detection of trace organic compounds, particularly polycyclic aromatic hydrocarbons (PAHs). Recent efforts have focused on developing [micro][L.sup.2]MS as a quantitative method, often by measuring the ratio of signal strength of an analyte to an internal standard. Here, we present evidence of factors that affect these ratios and thus create uncertainty and irreproducibility in quantification. The power and wavelength of the desorption laser, the delay time between the desorption and ionization steps, the power of the ionization laser, and the ionization laser alignment are all shown to change PAH ratios, in some cases by up to a factor of 24. Although changes in the desorption laser parameters and the delay time cause the largest effects, the ionization laser power and alignment are the most difficult parameters to control and thus provide the most practical limitations for quantitative [micro][L.sup.2]MS. Variation in ratios is seen in both synthetic poly(vinyl chloride) membranes and in 'real-life' samples of Murchison meteorite powder. Ratios between similar PAHs vary less than those between PAHs that differ greatly in mass and structure. This finding indicates that multiple internal standards may be needed for quantification of samples containing diverse PAHs.

We have made surface plasmon resonance (SPR) measurements of yeast iso-1-cytochrome e (Cyt c) on a gold surface. Angle-resolved SPR curves are recorded as a function of urea concentration before and after self-assembly of the Cyt c. Exposure to a urea solution causes denaturation of Cyt c, which shifts the minimum in the SPR curve to a larger angle and decreases the signal amplitude. The Gibbs free energy change for denaturation of the protein on Au is calculated from the change of the SPR signal amplitude with urea concentration. We find that (1) Cyt c can be reversibly denatured and renatured, depending on the urea concentration, and (2) the Gibbs free energy change for denaturation of Cyt c on Au surface in water, [[DELTA]G[degrees].sub.water], is 1.5 kcal/mol, which is ~4 times less than that in bulk solution.

A miniaturized pepsin reactor was prepared inside a fused-silica capillary (i.d. 75 [micro]m) by coating a pepsin-containing gel on a photopolymerized porous silica monolith. The pepsin-encapsulated film was prepared by a solgel method. The sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its activity. The large surface area of the PSG monolith enabled the immobilized pepsin to achieve a high catalytic turnover rate, and the porous nature of the PSG promotes penetration of large molecular proteins into the column. The immobilized pepsin-digested peptides and proteins, and the resulting mixture of peptide fragments, could be directly separated in the portion of the capillary where no PSG monolith exists. The durability and repeatability of the fabricated pepsin-coated column was tested and found to be satisfactory. An acidic solution consisting of 0.5 M formic acid was used as the running buffer, because it suppresses the adsorption of proteins or peptides on the inner surface of the capillary as well as enables direct connection of the output of the capillary electrophoresis column to a mass spectrometer. The on-line digestion of insulin chain [beta] and lysozyme provides identification of the proteolytic peptides. Recovery was achieved for 100% of the insulin chain [beta] amino acid sequence and 73% of the lysozyme amino acid sequence.

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 [Ca.sup.2+] flux measurements, and multistep receptor-mediated [Ca.sup.2+] measurements. These assays, and others, are achieved with significant improvements in reagent consumption, analysis time, and temporal resolution over macroscale alternatives.

We have demonstrated the use of cavity ring-down spectroscopy (CRDS) as a detector for high performance liquid chromatography (HPLC). For this use, we have designed and implemented a Brewster's angle flow cell such that cavity ring-down spectroscopy can be performed on microliter volumes of liquids. The system exhibits a linear dynamic range of 3 orders of magnitude (30 nM to 30 [micro]M quinalizarin at 470 nm) for static measurements and 2 orders of magnitude (0.5 [micro]M to 50 [micro]M) for HPLC measurements. For the static measurements, the baseline noise is 2.8 x [10.sup.-6] AU rms and 1.0 x [10.sup.-5] AU peak-to-peak, and for the HPLC separations, it is 3.2 x [10.sup.-6] AU rms and 1.3 x [10.sup.-5] AU peak-to-peak. The baseline noise is determined after the data are smoothed by an 11-point boxcar average. The peak areas detected from HPLC separations are reproducible to within 2-3%. The HPLC mass detection limit for a molecule with [member of] = 9 x [10.sup.3] [M.sup.-1] [cm.sup.-1] in a 300-[micro]m path length cell (illuminated volume, 0.5 [micro]L) is reported as 2.5 x [10.sup.-8] g/mL. These results were obtained using a simple pulsed CRDS system and are comparable to, if not better than, a high-quality commercial UN-vis absorption detector for the same path length.

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.

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.

We show that coupled electrorotation (CER) of microscopic particles using microfabricated electrodes can be used for localized sensing and mixing. The effective use of microelectromechanical systems and micro total analysis systems requires many types of control. These include the ability to (1) manipulate objects within microchannels by noncontact means, (2) mix fluids, and (3) sense local chemical parameters. Coupled electrorotation, in which the interactions between induced electric dipoles of adjacent particles lead to particle rotation, addresses aspects of all three challenges simultaneously. CER is a simple means of controlling the rotation of dielectric objects using homogeneous external radio frequency electric fields. CER is sensitive to several chemical and physical parameters such as the solution conductivity, pH, and viscosity. As a step toward integrating CER devices into microfluidic systems, a simple chip was designed to induce local mixing and to detect local changes in salt concentration, pH, and viscosity.

A commercially available miniaturized surface plasmon resonance sensor has been investigated for its applicability to biological interaction analysis. The sensor was found to exhibit excellent repeatability and linearity for high-refractive index solutions and good reproducibility for the binding of proteins. Its detection limit for the monoclonal antibody M1 was found to be 2.1 fmol, which corresponds to a surface concentration of 21 pg/[mm.sup.2]. Simple surface immobilization procedures relying on biotin/avidin or glycoprotein/lectin chemistry have been explored. Equilibrium dissociation constants for the binding of the FLAG peptide to its monoclonal antibody (M1) and for the binding of concanavalin A to a glycoprotein have been determined. The close agreement of these measurements with values obtained by surface fluorescence microscopy and fluorescence correlation spectroscopy helps to validate the use of this device. Thus, this sensor shows promise as an inexpensive, portable, and accurate tool for bioanalytical applications in laboratory and clinical settings.

We have constructed a cavity ring-down spectrometer employing a near-IR external cavity diode laser capable of measuring [sup.13]C/[sup.12]C isotopic ratios in C[O.sub.2] in human breath. The system, which has a demonstrated minimum detectable absorption loss of 3.2 x [10.sup.-11] [cm.sup.-1] [Hz.sup.-1/2], determines the isotopic ratio of [sup.13]C[sup.16]0[sup.16]0/[sup.12]C[sup.16]0[sup.16]0 by measuring the intensities of rotationally resolved absorption features of each species. As in isotope ratio mass spectrometry (IRMS), the isotopic ratio of a sample is compared to that of a standard C[O.sub.2] sample calibrated to the Pee Dee Belemnite scale and reported as the sample's [delta][sup.13]C value. Measurements of eight replicate C[O.sub.2] samples standardized by IRMS and consisting of 5% C[O.sub.2] in [N.sub.2] at atmospheric pressure demonstrated a precision of 0.22 [per thousand] for the technique. [delta][sup.13]C values were also obtained for breath samples from individuals testing positive and negative for the presence of Helicobacter pylori, the leading cause of peptic ulcers in humans. This study demonstrates the ability of the instrument to obtain [delta][sup.13]C values in breath samples with sufficient precision to serve as a useful medical diagnostic.

This work demonstrates that with an intrinsic duty cycle of 50% and spectral storage speeds up to 277 spectra [s.sup.-1] Hadamard transform time-of-flight mass spectrometry (HT-TOFMS) is a promising detector for any capillary-format separation that can be coupled to MS by electrospray ionization. Complete resolution of the components of a nine-peptide standard was achieved by coupling pressurized-capillary electrophoresis (pCE) to HT-TOFMS. The addition of pressure to the separation capillary decreased analysis times and stabilized the electrospray ionization source. Pulsed-pressurized injection of reserpine was used to experimentally simulate narrower peaks than those obtained in the pCE. HT-TOFMS was able to sample peaks having widths in the millisecond range.

Preconcentration effects of solvent gradient and sample stacking are investigated on a photopolymerized sol--gel (PSG) in capillary electrochromatography. The porous PSG monolith has a high mass-transfer rate. This characteristic promotes preconcentration of dilute samples. Plugs of samples more than 2 cm in length prepared in the separation solution (nongradient condition) are injected onto the PSG column. The extent of preconcentralion is quite significant, showing up to a 100-fold increase in peak heights of the separated analytes. Even larger preconcentrations are achieved under gradient conditions by dissolving the sample in a matrix with a higher concentration of noneluting solvent (water). For eight alkyl phenyl ketones and four polycyclic aromatic hydrocarbons that serve as neutral test analytes, improvements in peak heights obtained under gradient conditions can be more than a 1000-fold. Indeed, injection of a 91.2-cm plug, which is more than 3 times the total length of the capillary, was possible with only a minor loss in resolution. Five peptides serve as charged test analytes. Nongradient conditions in which the sample is hydrodynamically injected onto the PSG column show sizable preconcentration because of sample stacking. The use of a solvent gradient with the same ionic strength, however, does not appear to have practical value because of destacking caused by the changing organic composition that affects the conductivity. As an alternative preconcentration method, we demonstrate that electric field-enhanced sample injection on the PSG yielded up to a 1000-fold improvement in detection sensitivity for the test peptides.

In chromatographic separations, the heights of peaks are proportional to the concentrations of sample components present in an injected mixture. In general, an increase in the peak height cannot be achieved by simply increasing the injection time or the sample plug length. An exception occurs if some form of on-line preconcentration is possible. We present a new strategy for achieving on-line preconcentration by the use of a porous chromatographic material that acts as a solid-phase extractor as well as a stationary-phase separator. We are able to realize significant on-line preconcentration using capillary columns filled with a photopolymerized sol--gel (PSG). More than 2-cm plugs of sample solution can be loaded into the capillary and concentrated using a running buffer that is the same as the injection buffer (to avoid solvent gradient effects). As a demonstration, mixtures of three different polycyclic aromatic hydrocarbons, eight different alkyl phenyl ketones, and five different peptides in solutions of aqueous acetonitrile have been injected onto the PSG column and separated by capillary electrochromatography. The preconcentration is marked in terms of peak heights, with up to 100-fold increase for the PAH mixture, 30-fold for the alkyl phenyl ketone mixture, and 20-fold for the peptide mixture. Preconcentration takes place because of the high mass-transfer rates possible in the highly porous structure, and the extent of preconcentration follows the retention factor k for a given analyte.

Unknown compounds in polar fractions of Arabidopsis thaliana crude leaf extracts were identified on the basis of calculations of elemental compositions obtained from gas chromatography/low-resolution quadrupole mass spectrometric data. Plant metabolites were methoximated and silylated prior to analysis. All known peaks were used as internal references to construct polynomial recalibration curves of from raw mass spectrometric data. Mass accuracies of 0.005 +/- 0.003 amu and isotope ratio errors of 0.5 +/- 0.3% (A + 1/A), respectively, 0.3 +/- 0.2% (A + 2/A), could be achieved. Both masses and isotope ratios were combined when the elemental compositions of unknown peaks were calculated. After calculation, compound identifies were elucidated by searching metabolic databases, interpreting spectra, and, finally, by comparison with reference compounds. Sum formulas of more than 70 peaks were determined throughout single GC/MS chromatograms. Exact masses were confirmed by high-resolution mass spectrometric data. More than 15 uncommon plant metabolites were identified, some of which are novel in Arabidopsis, such as tartronate semialdehyde, citramalic acid, allothreonine, or glycolic amide.

Macroporous polymer flits have been fabricated in fused-silica capillaries by the UV photopolymerization of a solution of glycidyl methacrylate and trimethylolpropane trimethacrylate. This in situ preparation is a simple, rapid, and reproducible process. The flit can be placed at any desired position along the column. Photopolymer flits can withstand the short exposure to a high pressure (over 6000 psi). Bubble formation is not observed to occur with these flits under our experimental conditions. By choice of porogens, it is possible to control the porous properties. The use of such flits in capillaries to retain particles of chromatographic packing has been demonstrated to be stable and robust with continuous operation over 3 days.

Using sol-gel technology, a porous glass matrix (xerogel) is formed in a capillary column and acts as a support for a stationary phase of chromatographic particles used in capillary electrochromatography. Preparation of the sol-gel matrix and immobilization of the octadecylsilica (ODS) stationary phase occur in a single step. The presence of the particles in the column greatly reduces matrix cracking caused by internal pressure differentials within the pores of the sol-gel matrix. Good electroosmotic flow is achieved in part because of the inherent negative charge of both the particles and the sol-gel matrix. The performance of these sol-gel/ODS capillary columns was evaluated with a mixture of aromatic and nonaromatic organic compounds. Efficiencies of up to 80 000 plates/m were observed in columns with immobilized 3-[[micro]meter] ODS particles. The efficiency and resolution are enhanced when 3-[[micro]meter] ODS particles are used in place of the 5-[[micro]meter] particles.

Capillary columns packed electrokinetically with 1.5-[[micro]meter] nonporous octadecylsilica particles are used to achieve rapid separations with high efficiencies. A mixture containing five polycyclic aromatic hydrocarbons (PAHs) as test compounds is separated in less than 5 s with 28 kV applied to a column with a packed length of 6.5 cm (10 cm total length). A sample containing 16 PAHs (classified as priority pollutants by the U.S. Environmental Protection Agency) is isocratically separated in under 10 min by using longer columns (15-30 cm). Separation efficiencies greater than 700 000 theoretical plates/m are obtained when detection (via UV-excited laser-induced fluorescence) is performed on the packed portion of the capillary. The outlet frit is observed to play an important role in determining column efficiency. The optimum flow rate for best separation efficiency occurs at [approximately]2 mm/s. The run-to-run reproducibility of the peak retention times on a single column is better than 1% (relative standard deviation).

Several amine-containing components of individual vesicles from the atrial gland of Aplysia californica were identified with capillary electrophoresis (CE). On-line derivatization with naphthalene-2,3-dicarboxaldehyde was performed, and the derivatized amine-containing components were detected with laser-induced fluorescence (LIF). Amino acids, including taurine, that had not been determined previously in atrial gland vesicles were observed by using CE-LIF, and their identities were confirmed with CE, HPLC, NMR, and electrospray ionization mass spectrometry. The finding that taurine is packaged and stored into secretory vesicles supports the hypothesis that taurine may exhibit neuromodulatory activity. The bioactive peptides, well-known to be in atrial gland vesicles, were detected in lysed vesicle samples fractionated with HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These peptides were also observed in single-vesicle runs with CE-LIF. The atrial gland vesicles (ranging from 0.5 to 2 [[micro]meter] diameter and 65 aL to 4 fL volume, respectively) studied in this work represent the smallest biological entities to be analyzed chemically on an individual basis.

We present a capillary electrophoresis-patch clamp detection system optimized for screening of antagonists and inhibitors of ligand-gated ion channels. In this system, highly selective receptor agonists are delivered through the electrophoresis capillary to the cell surface where they continuously activate a receptor, resulting in increased steady-state transmembrane currents. Thus, receptor selection and biosensor functionality is simply achieved by selection of an appropriate agonist. The antagonists are fractionated in the same electrophoresis capillary and inhibit the agonist-evoked response, resulting in transiently decreased steady-state transmembrane currents. Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly blocks a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-spectrum glutamate receptor antagonist, were separated and detected by kainate-activated patch-clamped interneurons freshly dissociated from rat brain olfactory bulb. In addition, [Mg.sup.2+] that reversibly blocks the N-methyl-D-aspartate receptor in a voltage-dependent way was detected using the same cell detector system when activated by N-methyl-D-aspartate and the co-agonist glycine. The presented method offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor system of interest.