PubMed Health⌕ Search

Biomedical subjects

J W Jorgenson

Publications and source records attributed to J W Jorgenson.

At least 19 recordsLinked to original sources
PubMed1992

Quantitative determination of catecholamines in individual bovine adrenomedullary cells by reversed-phase microcolumn liquid chromatography with electrochemical detection.

A microcolumn liquid chromatographic method for the determination of norepinephrine and epinephrine in single bovine adrenomedullary cells is described. A single cell is isolated from a culture plate, (3,4-dihydroxybenzyl)amine is added as an internal standard, and the cell is lysed with perchloric acid. After centrifugation, the supernatant is injected directly into a 42 or 50 microns Inner diameter C18 reversed-phase column operated with electrochemical detection. Detection limits for norepinephrine and epinephrine were 46 and 75 amol, respectively. Of the 22 cells examined, 8 contained predominantly norepinephrine, 10 contained predominantly epinephrine, and at least 4 cells contained significant amounts of each. On average, a single cell contained approximately 150 fmol of catecholamine.

Adrenal Medulla↗
PubMed1991

Nanoscale separations combined with tandem mass spectrometry.

High-efficiency separations of peptide mixtures, tryptic digest and other biological compounds have been achieved using nanoscale packed capillaries and capillary zone electrophoresis (CZE). The coaxial continuous-flow fast atom bombardment design is an excellent interface for coupling these separation techniques with mass spectrometry (MS). In addition, this interface is very useful for the acquisition of MS-MS data from compounds separated by nanoscale packed capillary liquid chromatography and CZE. Structurally informative daughter-ion spectra can be obtained at the low picomole to femtomole level.

Amino Acid Sequence↗
PubMed1991

Nanoscale packed-capillary liquid chromatography coupled with mass spectrometry using a coaxial continuous-flow fast atom bombardment interface.

Nanoscale packed-capillary liquid chromatography (LC) columns have been coupled with mass spectrometry (MS) using a coaxial continuous-flow fast atom bombardment interface. The combined system has been applied to the analysis of mixtures of peptides, including synthetic mixtures of bioactive peptides and tryptic digests of proteins. Nanoscale packed-capillary columns offer two principal advantages for LC/MS analysis--high chromatographic separation efficiencies and low mobile-phase flow rates. The high separation efficiencies facilitate the separation of complex mixtures, and the low mobile-phase flow rates reduce problems with coupling the LC effluent with the high-vacuum, high-voltage environment of sector MS ion sources. The columns used in this work were 50- or 75-micron i.d., 1-2 m long, packed with 10-micron C18 particles, using mobile-phase flow rates of 50-350 nL/min.

Animals↗
PubMed1991

On-column sample gating for high-speed capillary zone electrophoresis.

High-speed zone electrophoresis in a fused-silica capillary is described. Elevated electric fields and short capillary lengths allow a mixture of fluorescein isothiocyanate (FITC) labeled amino acids to be separated in times as short as 1.5 s. Formation of the analyte zone at the head of the capillary is controlled by laser-induced photolysis of a tagging reagent. This gating procedure allows rapid and automated introduction of sample into the capillary. Ultimately, Joule heating of the buffer limits the speed and efficiency of the separation.

Automation↗
PubMed1991

Sample gating in open tubular and packed capillaries for high-speed liquid chromatography.

A new liquid chromatography instrument is described that employs small-diameter capillary columns and an optically controlled sample gating procedure to separate a mixture of fluorescein isothiocyanate (FITC) labeled amines in as few as 6 s. Efficiency of the separation, expressed as the number of theoretical plates, is observed to increase linearly with column length and thus analysis time. The combination of short analysis time and automated sample introduction allows signal averaging to be used to enhance the precision of the measurement.

Automation↗
PubMed1991

Determination of bioactive peptides using capillary zone electrophoresis/mass spectrometry.

Mixtures of bioactive peptides have been analyzed by capillary zone electrophoresis/mass spectrometry (CZE/MS) using an on-line coaxial continuous-flow fast atom bombardment interface. High separation efficiencies (up to 410,000 theoretical plates) were obtained from low femtomole levels of peptides. The analysis of basic peptides was accomplished by using aminopropyl-silylated CZE columns to minimize zone broadening due to adsorption effects. CZE/MS/MS data were acquired from femtomole levels of peptides in electrophoretic real time.

Absorption↗
PubMed1990

Capillary zone electrophoresis-mass spectrometry using a coaxial continuous-flow fast atom bombardment interface.

Mixtures of peptides have been analyzed by capillary zone electrophoresis in conjunction with mass spectrometry (MS) using an on-line coaxial continuous-flow fast atom bombardment interface. MS and MS-MS spectra have been acquired in electrophoretic real time from femtomole levels of the peptides, while maintaining separation efficiencies in excess of 100,000 theoretical plates.

Amino Acids↗
PubMed1990

Quantitative amino acid analysis of individual snail neurons by open tubular liquid chromatography.

A method is described for the determination of amino acids in individual cells. The amino acids are derivatized with naphthalene-2,3-dicarboxaldehyde and then analyzed by open tubular liquid chromatography with amperometric detection. The total volume present after derivatization is approximately 25 nL. It was possible to quantitatively determine 17 amino acids in three different neurons of the land snail Helix aspersa. Quantitation was accomplished through the use of two internal standards and a calibration curve. Alanine was found to be the most abundant amino acid by about a factor of 2 over glutamine in all three types of neurons. This method has the advantages of sensitivity in the attomole range (5 X 10(-9) M) and selectivity for a specific class of compounds and is at least as reliable as other methods used for single-cell analysis.

Amino Acids↗
PubMed1990

Quantitative amino acid analysis of subnanogram levels of protein by open tubular liquid chromatography.

A method is described for the hydrolysis and quantitative amino acid analysis of as little as 0.1 ng (4 fmol) of protein. Hydrolysis is performed by using a gas-phase method. The resulting amino acids are derivatized with naphthalene-2,3-dicarboxaldehyde and then analyzed by open tubular liquid chromatography with amperometric detection. The total volume present after derivatization is approximately 25 nL. To our knowledge this is currently the lowest level of protein to be quantitatively analyzed, as well as the smallest volume in which derivatization has been accomplished. It was possible to quantitatively determine 14 amino acids with an error of 5.0% for known protein and 8.5% for a protein whose identity was unknown to the researcher.

Amino Acids↗
PubMed1990

Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins.

A comprehensive two-dimensional (2-D) liquid chromatographic separation system is presented. The system uses a microbore cation exchange column, operated under gradient conditions, as the first dimension separation. Effluent from this first column alternately fills one of two loops on a computer-controlled eight-port valve. A second pump then forces loop material onto a second column, a size exclusion column. UV detection is used, and the system is applied to the separation of protein standards and serum proteins. The 2-D system has a higher resolving power and peak capacity than either of the two columns used alone. The entire first column effluent is analyzed on the second column in virtually the same time it takes to complete the first column separation, without the use of stopped flow methods. The entire system is automated and operated under computer control. Three-dimensional (3-D) data representation provides a means of viewing peak profiles in either separation dimension and contour mapping of the 3-D data provides a more reliable means of peak identification from run to run than that provided by single-column elution times.

Autoanalysis↗
PubMed1989

Coaxial continuous flow fast atom bombardment in conjunction with tandem mass spectrometry for the analysis of biomolecules.

The capability of interfacing coaxial continuous flow fast atom bombardment (CF-FAB) with tandem mass spectrometry (MS/MS) is demonstrated. The goal of this research is to demonstrate the ability of obtaining on-the-fly (i.e. chromatographic real time) MS/MS spectra of biomolecules and to demonstrate the feasibility of using open tubular CF-FAB as a means of introducing and maintaining a constant flux of analyte into the mass spectrometer over long periods of time. On-the-fly MS/MS spectra of a tripeptide, Met-Leu-Phe, were obtained on a 220-pg injection and a 22-pg injection. With a total acquisition time of 2 s, fragment ions resulting from common backbone cleavages were observed. With a 50 microns i.d. packed microcapillary column, the separation of a mixture was obtained and the MS/MS spectra were acquired as the analytes were eluting from the column. Through the use of the coaxial CF-FAB interface to deliver a constant flow of analyte, MS/MS spectra of a variety of compounds, including peptides, sugars, fatty acids, phospholipids, and steroids, were obtained as well as an MS/MS/MS spectrum of a tetrapeptide.

Carbohydrates↗
PubMed1989

Characterization of a post-column reaction-laser-induced fluorescence detector for capillary zone electrophoresis.

Several modifications have been made to a post-column labeling system for use with capillary zone electrophoresis. Fluorescence excitation is now performed with a helium-cadmium laser rather than an arc lamp. The focusability of the laser beam allows the use of larger diameter capillaries in the post-column reactor without the excessive band broadening observed previously. These larger capillaries can be assembled in the reactor much more easily. Another improvement is that the flow-rate of the labeling reagent can now be accurately controlled and determined. Incorporating these changes, the performances of two reactors with capillaries of the same dimensions are compared.

Animals↗
PubMed1989

Coupling of capillary zone electrophoresis and capillary liquid chromatography with coaxial continuous-flow fast atom bombardment tandem sector mass spectrometry.

The coaxial continuous-flow fast atom bombardment (FAB) system has proven to be very useful for interfacing capillary liquid chromatography and capillary zone electrophoresis (CZE) with sector mass spectrometry (MS). The interface can be used for the acquisition of both MS and MS-MS spectra from femtomole levels of non-volatile and/or thermally labile analytes while maintaining separation efficiencies of hundreds of thousands of plates. The use of coaxial fused-silica capillary columns to independently deliver the microcolumn effluent and the FAB matrix to the tip of the FAB probe offers the following advantages: the composition and flow-rates of the two liquid streams can be independently optimized; the FAB matrix does not effect the microcolumn separation process; peak broadening is minimized since the two liquid streams do not mix until they reach the tip of the FAB probe where ion desorption occurs; and, with CZE, active electrophoretic transport delivers the analytes directly to the FAB probe tip. These features combine to make this coaxial continuous flow fast atom bombardment interface particularly well suited for use with microcolumn separation methods.

Chromatography, Liquid↗
PubMed1989

Capillary electrophoresis of proteins in buffers containing high concentrations of zwitterionic salts.

A method for improving protein separations in capillary zone electrophoresis utilizing high concentrations of zwitterionic buffer additives was examined. Lysozyme and alpha-chymotrypsinogen A were used as test proteins in untreated fused-silica capillaries in buffers of pH ca. 7.0 and 9.0 The zwitterion-containing buffers were compared with buffers containing high ionic salt concentrations and a buffer containing a combination of high ionic salt and high zwitterion concentrations. Over 100,000 theoretical plates were obtained in less than 30 min. for both test proteins in a pH 7 buffer containing both trimethylglycine and potassium sulfate. The advantages and disadvantages of this technique compared with those of other methods used to prevent protein adsorption are discussed.

Animals↗
PubMed1989

Microcolumn separations and the analysis of single cells.

Capillary zone electrophoresis and open tubular liquid chromatography are two examples of an emerging area of analytical instrumentation known as microcolumn separations. The high resolution and small sample requirements of these methods make them suitable for the quantitative, multicomponent chemical analysis of single cells. Appropriate instrumentation for the analysis of nanoliter and subnanoliter samples is discussed. Data from the analysis of individual neurons are presented, including amino acid and neurotransmitter content.

Amino Acids↗
PubMed1989

Determination of naphthalene-2,3-dicarboxaldehyde-labeled amino acids by open tubular liquid chromatography with electrochemical detection.

Naphthalene-2,3-dicarboxaldehyde (NDA) has been investigated as a new derivatizing reagent for the electrochemical detection of tagged amino acids. Gradient elution allowed for the separation of 18 NDA-derivatized amino acids on an open tubular liquid chromatography column in less than 50 min. Gradient elution and electrochemical detection were found to be compatible. A detection limit of 36 amol was obtained for the asparagine-NDA derivative. The usefulness of this technique for quantitation was demonstrated by the analysis of the NDA-tagged hydrolysis products from bovine chymotrypsinogen.

Amino Acids↗
PubMed1989

Quantitative analysis of individual neurons by open tubular liquid chromatography with voltammetric detection.

The ability to analyze individual cells is often important in biology because of the heterogeneity of tissue; this is especially true in the area of neurobiology. A method is described for the determination of trace levels of organic compounds in individual cells by open tubular liquid chromatography with voltammetric detection. In the method, a cell is isolated, an internal standard is added, the cell is homogenized and centrifuged, and the supernatant is injected directly onto the chromatography column. Since data are collected in both the electrochemical and chromatographic domains, the resolution of the method is better than that obtained by using amperometric detection. The combination of voltammetry and chromatography also aids in the identification of compounds. By use of this method three different neurons, D2, E4, and F1, from the land snail Helix aspersa are analyzed. The data show that the cells give certain unique and repeatable chemical profiles. Dopamine, serotonin, tyrosine, and tryptophan were identified and quantified in two of the cells at the femtomole level. In the third cell, only the two amino acids were observed and measured. The quantitative data indicate that the method is at least as reliable as other methods that have been applied to single cells and considerably more sensitive. The combination of qualitative and quantitative information allows for the chemical mapping of cells.

Animals↗