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Biomedical subjects

J W Jorgenson

Publications and source records attributed to J W Jorgenson.

At least 37 records · Page 2Linked to original sources

Preparation and evaluation of slurry-packed liquid chromatography microcolumns with inner diameters from 12 to 33 microns.

Fused silica capillary liquid chromatography columns with inner diameters between 12 and 33 microns were slurry packed with 5 microns octadecylsilane-modified silica particles. Column efficiencies and van Deemter coefficients were compared. A linear decrease of the A term as column diameter was decreased was the most significant contributor to a lower overall plate height at the optimum velocity.

Chemical Phenomena↗

Electrochemical detection of histamine and 5-hydroxytryptamine at isolated mast cells.

The electrochemical oxidation of histamine has been investigated as an analytical tool. In a physiological buffer, histamine is oxidized at carbon fiber microelectrodes at potentials close to the background in a chemically irreversible process. Cylindrical carbon fiber electrodes were used as amperometric detectors for histamine separated with a reversed-phase capillary column, and detection limits of 240 amol were achieved. Electrodes with beveled tips were used as real-time sensors by monitoring with repetitive cyclic voltammograms at a scan rate of 800 V/s with a 16.7-ms repetition rate, and detection limits of 1.4 microM were achieved. Both techniques were used to probe histamine and 5-hydroxytryptamine (5-HT) stored in rat peritoneal mast cells. The content in single cells was measured by capillary HPLC, and both substances were found in single cells. Although the analysis revealed a large cell-to-cell variation in the amount of histamine and 5-HT, the average amount was 150 and 4 fmol of histamine and 5-HT, respectively. Release of histamine and 5-HT was measured with the electrode placed 1 microm from the cell surface. Release was observed as a series of sharp concentration spikes, consistent with corelease of the two substances from individual vesicles following exocytosis.

Animals↗

Rapid comprehensive two-dimensional separations of peptides via RPLC-optically gated capillary zone electrophoresis.

Coupled-column two dimensional (2D) separation systems offer potentially high peak capacity and are amenable to automation. Reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) are complementary techniques well suited for use in a 2D system. Optically gated CZE is a means of performing rapid CZE analyses. In a 2D system, these rapid CZE analyses enable more frequent sampling of the RPLC separation and thus more freedom in control of the RPLC analysis conditions. Complete 2D separations can be done in the time usually required to do the RPLC analysis alone. With the present system, a complete 2D analysis can be done in under 10 min. Because the peak capacity of a 2D method is inherently high, some of the available peak capacity can be exchanged for speed of analysis. Acceleration of the RPLC elution gradient will decrease analysis time, but RPLC resolution will suffer. However, because it is a 2D system, some of the resolution lost in the RPLC can be regained in the second dimension CZE analysis. Here, the RPLC gradient was done over only 2 min, but CZE analyses done every 2.5 s restore some of the resolution lost in the rapid RPLC gradient.

Chromatography, Liquid↗

Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis.

Multidimensional separation methods are attractive because of their potentially high peak capacities. Coupled-column systems in particular offer the advantages of on-line detection and automation. With the development of rapid two-dimensional (2D) analysis, it becomes possible to consider three-dimensional (3D) separation systems. In such a 3D system, effluent from a slow first dimension is repetitively sampled into a rapid 2D system. In the resultant data, each sample component has been subjected to three separative displacements, and the overall peak capacity is the product of that of each of the three dimensions. This paper demonstrates a comprehensive coupled-column 3D analysis of peptides. Size exclusion chromatography (SEC) is used as the first dimension to separate sample components by molecular weight, over an analysis time of several hours. The SEC effluent is repetitively sampled on-line into a rapid 2D reversed phase liquid chromatography/capillary zone electrophoresis (CZE) system with an analysis time of 7 min. Detection of sample zones is done only after the final CZE separation, by laser-induced fluorescence detection. Analysis data from this system consist of a series of 2D "slices" of the SEC effluent, which when stacked together give the 3D separation "volume".

Chromatography, Gel↗

Resolution of cis and trans isomers of peptides containing proline using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) is known for its high separation efficiency. Optically gated CZE is a form of CZE that allows rapid analyses while high separation efficiency is maintained. Analysis of peptides containing proline with CZE and optically gated CZE gives two peaks for dipeptides and multiple peaks for oligopeptides containing more than one proline. The multiple peaks are believed to be due to the cis and trans forms possible in peptide bonds containing proline. Isomerization between the cis and trans forms takes place at room temperature with a lifetime of seconds to minutes. Resolution of these peaks in CZE is found to be a function of separation efficiency and time of analysis. Rapid analyses with relatively lower separation efficiency give resolution equal to or better than that of longer analyses with much higher efficiency. This confirms the relationship between the multiple peaks and isomerization between the cis and trans forms. To extract kinetic information from the experimental data, a digital simulation is used to model the separation obtained experimentally. The equilibrium constant and rate constants that give the best fit of the simulated data to the experimental data are considered estimates of the actual values for the given experimental conditions.

Amino Acid Sequence↗

Separation of nanoliter samples of biological amines by a comprehensive two-dimensional microcolumn liquid chromatography system.

A two-dimensional liquid chromatography system based on the combination of a charge separation mechanism, provided by anion exchange chromatography, with a hydrophobic separation mechanism, provided by reversed phase chromatography, is presented. A 90 cm long anion exchange microcolumn is coupled to a 3 cm long reversed phase microcolumn. Both microcolumns are interfaced by two electronically controlled valves. Effluent from the anion exchange microcolumn is collected in a sample loop and then concentrated onto the head of the reversed phase microcolumn. Analyte peaks consisting of tagged amines elute from the second column and are detected by laser-induced fluorescence. The entire operation is controlled by a personal computer. The resolving power of this system is demonstrated with a two-dimensional chromatogram of the peptides obtained from a tryptic digest of porcine thyroglobulin. The sensitivity of the system is demonstrated with a two-dimensional chromatogram of the contents of a single bovine chromaffin cell.

Amines↗

Electrospray-condensation particle counter: a molecule-counting LC detector for macromolecules.

A new detector for macromolecular separations is described. The detector counts individual macromolecules (molecular weights greater than about 10,000) and reports counts per second. The chromatographic effluent is electrosprayed, neutralized, and swept to the detector by a stream of air. The detector is a condensation particle counter that detects individual particles by light scattering from droplets condensed on the particles. When used as the detector for a size exclusion separation of proteins, the detector has a linear range of 4 orders of magnitude with detection limits as low as 0.1 microgram/mL. The detector can be directly interfaced (no makeup flow) with effluent flows as low as 10 nL/min. A Monte Carlo model based on size measurements of the electrosprayed droplets correctly predicts the observed detector behavior.

Biopolymers↗

Quantitation of epinephrine and norepinephrine secretion from individual adrenal medullary cells by microcolumn high-performance liquid chromatography.

The cellular response of individual adrenal medullary cells to a prolonged stimulation with carbamylcholine (carbachol) was studied using microcolumn high-performance liquid chromatography (MHPLC) with electrochemical detection. Chromaffin cells will not release all of their catecholamine stores when continuously exposed to a secretagogue. The process of desensitization prevents the continued release of catecholamines after a certain percentage of chemical stores have been depleted. The percentage of a cell's chemical stores released upon stimulation was determined, and whether there is a difference in release between the cell types was examined. A single cell was exposed to a stimulation solution for 5.0 min at 25 degrees C. Approximately 80% of the solution was removed and analyzed on a microcolumn reversed-phase chromatography column. A lysing solution was immediately added to the miniaturized vial which still contained the cell, and this solution was also analyzed chromatographically. The first run was used to determine the amount of catecholamine the cell released upon stimulation. The second run determined the amount of catecholamine that remained in the cell. From these values the percentage of catecholamine released was calculated. Twenty-seven individual cells were studied by this method, and 22 could be classified as norepinephrine or epinephrine dominant. There was no statistical difference in the amount of catecholamine the norepinephrine dominant cells released (14 +/- 5%, n = 11) as compared to the epinephrine dominant cells (12 +/- 4%, n = 11).

Adrenal Medulla↗

Study of zone broadening in optically gated high-speed capillary electrophoresis.

Fluorescein isothiocyanate (FITC) labeled compounds are separated by capillary zone electrophoresis (CZE) in seconds rather than minutes while high separation efficiency is maintained. These rapid analysis times are achieved through high electric fields applied over short separation distances and the use of a unique optical-gating injection system. The optical-gating injection is based on photodecomposition of FITC induced by an argon ion laser beam. A fraction of this same laser beam is also used for detection of the analyte zones by on-column laser-induced fluorescence. Four FITC-labeled amino acids and fluorescein dye were analyzed to compare system performance to that predicted by theory. To do so, the total variance for each analyte zone was measured experimentally. This was compared to the sum of the theoretically expected variance contributions from the injection and detection systems and longitudinal diffusion. The variance due to longitudinal diffusion was calculated from diffusion constants measured experimentally with a traditional CZE system. After accounting for these three known sources of variance, this method is found to achieve better than 80% of the performance predicted by theory in analysis times as short as 2-3 s.

Amino Acids↗

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↗

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↗

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↗

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↗

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↗

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↗