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Biomedical subjects

R N Zare

Publications and source records attributed to R N Zare.

At least 19 recordsLinked to original sources
PubMed2003

Use of broadband, continuous-wave diode lasers in cavity ring-down spectroscopy for liquid samples.

Cavity ring-down spectroscopy (CRDS) is an extremely sensitive absorption technique that has been applied primarily to gas samples, which are characterized by having narrow absorption features. Recently, CRDS has also been applied to liquid samples, which have broad absorption features. The use of small inexpensive diode lasers as light sources for liquid samples is demonstrated. The low cost coupled with the ease and technical straightforwardness of application gives this technique wide appeal.

Acetonitriles↗
PubMed2001

Strategy for on-line preconcentration in chromatographic separations.

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.

Journal Article↗
PubMed2001

On-line preconcentration in capillary electrochromatography using a porous monolith together with solvent gradient and sample stacking.

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 preconcentration 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.

Angiotensin II↗
PubMed2001

Photopolymerized sol-gel monoliths for capillary electrochromatography.

A solution of methacryloxypropyltrimethoxysilane in the presence of an acid catalyst, water, toluene, and a photoinitiator was irradiated at 365 nm for 5 min in a 75-microm i.d. capillary to prepare a porous monolithic sol-gel column by a one-step, in situ, process. The photopolymerized sol-gel (PSG) column shows reversed-phase behavior. Using this column, a variety of low-molecular-weight neutral compounds, including polycyclic aromatic hydrocarbons, alkyl benzenes, alkyl phenyl ketones, and steroids are separated from mixtures. Various different operational parameters, such as buffer composition, field strength, and column temperature, were varied to assess their influence on column performance. Use of PSG as a stationary phase for a pressure-driven separation is also demonstrated.

Journal Article↗
PubMed2001

Photopolymerized sol-gel frits for packed columns in capillary electrochromatography.

Porous sol-gel frits are fabricated in a capillary column by filling it with a solution of 3-(trimethoxysilyl)propyl methacrylate, hydrochloric acid, water, toluene (porogen), and a photoinitiator (Irgacure 1800) and exposing it to UV light at 365 nm for 5 min. The separation column (30 cm x 75 microm I.D.) contains between the inlet and outlet frits a 15-cm packed segment filled with 5-microm silica particles modified with the chiral compound (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine. A detection window (1 mm long) is placed immediately after the outlet frit. To demonstrate the performance of this chiral separation column, mixtures of 16 different amino acids (three of which are not naturally occurring) derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole were separated by capillary chromatography. The enantiomeric separation of the column results in a resolution ranging from 1.21 to 8.29, and a plate height ranging from 8.7 to 39 microm.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2001

Electrophoretron: a new method for enhancing resolution in electrokinetic separations.

Two capillaries, each of which have different surface preparations on their inside walls, are joined together to form a closed loop, and electrodes are placed inside the two capillaries. When the loop is filled with liquid and a potential difference is applied between the two electrodes, a circulating flow of liquid is established inside the loop because the resistance to flow is unequal in going from one electrode to another in a clockwise versus a counterclockwise direction. Consequently, a sample injected into this device, which we call an electrophoretron, repeatedly circulates between the two electrodes and the capillary separation column becomes effectively one of unlimited length. On each cycle the separation between analytes with different mobilities increases, thus enhancing resolution of analytes having nearly the same mobilities. The operation of a prototype electrophoretron is demonstrated.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2001

Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein.

Single-molecule studies of the conformations of the intact beta(2) adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the beta(2) adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

Animals↗
PubMed2001

Semipreparative capillary electrochromatography.

Capillaries with inner diameters of 550 microm have successfully been packed with 1.5-microm octadecyl silica particles using frits made of macroporous polymers by the UV photopolymerization of a solution of glycidyl methacrylate and trimethylolpropane trimethacrylate. This type of frit is found superior to one made of low-melting point poly(styrene-co-divinylbenzene) beads. Bubble formation is not observed to occur within these capillary columns under our experimental conditions. Separations can be achieved with sample injection volumes as high as 1 microL. To demonstrate its semipreparative use, a mixture of 500 nL of taxol (20 mM) and its precursor, baccatin III (30 mM), is separated using such a column with a Tris buffer.

Alkaloids↗
PubMed2001

Nanoengineered structures for holding and manipulating liposomes and cells.

We describe the fabrication of nanoengineered holding pipets with concave seating surfaces and fine pressure control. These pipets were shown to exhibit exceptional stability in capturing, transporting, and releasing single cells and liposomes 1-12 microm in diameter, which opens previously inaccessible avenues of research. Three specific examples demonstrated the utility and versatility of this manipulation system. In the first, carboxyrhodamine was selectively incorporated into individual cells by electroporation, after which nearly all the medium (hundreds of microliters) surrounding the docked and tagged cells was rapidly exchanged (in seconds) and the cells were subsequently probed by laser-induced fluorescence (LIF). In the second study, a single liposome containing carboxyrhodamine was transported to a dye-free solution using a transfer pipet, docked to a holding pipet, and held firmly during physical agitation and interrogation by LIF. In the third study, pairs of liposomes were positioned between two microelectrodes, held in contact, and selectively electrofused and the resulting liposomes undocked intact.

Cells↗
PubMed2001

Effect of sequence length, sequence frequency, and data acquisition rate on the performance of a Hadamard transform time-of-flight mass spectrometer.

Various factors influencing the performance of a Hadamard transform time-of-flight mass spectrometer (HT-TOFMS) have been investigated. Using a nitrogen corona discharge to produce an ion stream of N2+, N3+, and N4+, it is found for spectra containing only N4+ that the signal-to-noise ratio (SNR) closely approaches the value calculated from the ion background by assuming that the ion background follows a Poisson distribution. In contrast, for a more intense beam containing N2+, N3+, and N4+, the SNR is less than its theoretical value because of the appearance of discrete spikes in the mass spectrum caused by deviations in the actual modulation sequence from the ideal one. These spikes can be reduced, however, by decreasing the modulation voltage. Under these optimized conditions, the pseudo-random sequence length is varied to understand how it alters SNR, mass resolution, and scan speed. When the length of the pseudo-random sequence is doubled, the SNR increases by the square root of 2 while the time necessary to record a mass spectrum also doubles. Mass resolution can be varied between 500 and 1200 at m/z = 609 as the sequence length, modulation speed (10 MHz, 25 MHz), and acquisition rate (up to 50 MHz) are changed. Scan speeds of 6000 passes per s can be obtained using a sequence containing 4095 elements modulated at 25 MHz. The capability to tailor the HT-TOFMS to increase the scan speed and resolution with a constant 50% duty cycle makes the technique extremely appealing as a mass analyzer for measuring rapid changes in the composition of an ion stream.

Algorithms↗
PubMed2000

Deuterium enrichment of polycyclic aromatic hydrocarbons by photochemically induced exchange with deuterium-rich cosmic ices.

The polycyclic aromatic hydrocarbon (PAH) coronene (C24H12) frozen in D2O ice in a ratio of less than 1 part in 500 rapidly exchanges its hydrogen atoms with the deuterium in the ice at interstellar temperatures and pressures when exposed to ultraviolet radiation. Exchange occurs via three different chemical processes: D atom addition, D atom exchange at oxidized edge sites, and D atom exchange at aromatic edge sites. Observed exchange rates for coronene (C24H12)-D2O and d12-coronene (C24D12)-H2O isotopic substitution experiments show that PAHs in interstellar ices could easily attain the D/H levels observed in meteorites. These results may have important consequences for the abundance of deuterium observed in aromatic materials in the interstellar medium and in meteorites. These exchange mechanisms produce deuteration in characteristic molecular locations on the PAHs that may distinguish them from previously postulated processes for D enrichment of PAHs.

Deuterium↗
PubMed2000

Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion.

A method for cell-cell and cell-liposome fusion at the single-cell level is described. Individual cells or liposomes were first selected and manipulated either by optical trapping or by adhesion to a micromanipulator-controlled ultramicroelectrode. Spatially selective fusion of the cell-cell or cell-liposome pair was achieved by the application of a highly focused electric field through a pair of 5-micrometer o.d. carbon-fiber ultramicroelectrodes. The ability to fuse together single cells opens new possibilities in the manipulation of the genetic and cellular makeup of individual cells in a controlled manner. In the study of cellular networks, for example, the alteration of the biochemical identity of a selected cell can have a profound effect on the behavior of the entire network. Fusion of a single liposome with a target cell allows the introduction of the liposomal content into the cell interior as well as the addition of lipids and membrane proteins onto the cell surface. This cell-liposome fusion represents an approach to the manipulation of the cytoplasmic contents and surface properties of single cells. As an example, we have introduced a membrane protein (gamma-glutamyltransferase) reconstituted in liposomes into the cell plasma membrane.

Animals↗
PubMed2000

Separation of related opiate compounds using capillary electrochromatography.

Capillary electrophoretic separations have been investigated for six controlled narcotic analgesic compounds having related structures. Owing to the similar charge-to-mass ratios of these compounds, capillary zone electrophoresis failed to provide a satisfactory separation, whereas a baseline-resolved separation was achieved in 10 min using micellar electrokinetic chromatography. Column efficiencies of 40,000-150,000 plates/m were obtained with a 50 cm long, 50 microm inner diameter (ID) capillary using 50 mM sodium dodecyl sulfate (SDS) in a 50 mM borate solution containing 12% isopropanol. In contrast, separation of this mixture by capillary electrochromatography proved to be significantly superior. The capillary was 15 cm long, with an ID of 75 microm, and was packed with 1.5 microm nonporous octadecyl silica (ODS) particles. The mobile phase consisted of 80% 10 mM tris(hydroxymethyl)aminomethane (Tris) and 20% acetonitrile, and contained 5 mM SDS. A complete separation was obtained in 2.5 min with an efficiency of 250,000-500,000 plates/m.

Analgesics, Opioid↗
PubMed2000

Softening of fused-silica capillaries during particle packing.

When a semipreparative capillary electrochromatography (CEC) capillary is packed with silica particles and exposed to solvent, its mechanical strength is markedly reduced. In our studies, a fused-silica capillary (internal diameter > 200 microm and wall thickness < 150 microm) was packed under pressure (approximately 200 psi) with spherical silica particles (1.5-5 microm) suspended in water or various common organic solvents. After one hour of exposure, the capillary can be readily deformed, and it keeps its deformed shape upon release of the force causing deformation. It is suggested that capillary softening is promoted through the propagation of internal microcracks that have been caused by action of the particles during packing in the presence of solvent. Application of a protective coating to the inside of the capillary is found to reduce or eliminate capillary softening.

Chromatography, Liquid↗
PubMed2000

Enantiomeric separation of amino acids and nonprotein amino acids using a particle-loaded monolithic column.

A solution is prepared of 5 microm silica particles modified with (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine (particle 1) or (S)-N-3,5-dinitrophenylaminocarbonyl-valine (particle 2) suspended in liquid tetraethylorthosilicate, ethanol, and aqueous hydrochloric acid. This solution is injected under pressure into a 30 cm long, 75 microm inner diameter capillary column and heated for 1 h at 120 degrees C after which the modified particles are embedded in a monolithic column of sol gel. The packed column measures approximately 15 cm from the inlet to the window used to view the laser-induced fluorescence. Thirteen different amino acids and three nonprotein amino acids are derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) before injection onto the column for capillary electrochromatographic separation. The enantiomeric separation of the monolithic column packed with particle 1 results in a resolution ranging from 1.14 to 4.45, whereas that packed with particle 2 results in a resolution ranging from 0.79 to 1.17. On the basis of resolution and amount of chiral packing material the enantiomeric separation obtained by capillary electrochromatography is judged to be superior to that obtained previously with high performance liquid chromatography (HPLC).

Amino Acids↗
PubMed2000

Molecular tennis--flat smashes and wicked cuts.

Molecular photodissociation typically produces open-shell photofragments that have angular momentum. This Account explores how the measurement of the directional nature of photofragment angular momentum can provide us with a new viewpoint for seeing and understanding the process whereby molecules fall apart under irradiation. Although the photodissociation process is more complicated than might be imagined at first, the measurements reveal detail that approaches conventional bound-state spectroscopy, enhancing our understanding of the chemical bond and even allowing us to manipulate the ultimate fate of a photofragment by selecting the wavelength and the polarization of the photolysis source.

Chemical Phenomena↗
PubMed1999

Chemical transformations in individual ultrasmall biomimetic containers.

Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of a short (10-microsecond), intense (20 to 50 kilovolts per centimeter) electric pulse delivered across ultramicroelectrodes. Product formation was monitored by far-field laser fluorescence microscopy. The ultrasmall characteristic of this reaction volume led to rapid diffusional mixing that permits the study of fast chemical kinetics. This technique is also well suited for the study of reaction dynamics of biological molecules within lipid-enclosed nanoenvironments that mimic cell membranes.

Alkaline Phosphatase↗