Reversed-phase Microcolumn Liquid Chromatography Research Articles

Determination of hydrolysis products of sulfur mustards by reversed-phase microcolumn liquid chromatography coupled on-line with sulfur flame photometric detection and electrospray ionization mass spectrometry using large-volume injections and peak compression

On-line coupling of reversed-phase microcolumn liquid chromatography (micro-RPLC) and sulfur-selective flame photometric detection (S-FPD) was studied for the selective and direct determination of thiodiglycol, bis(2-hydroxyethylthio)methane, 1,2-bis(2-hydroxyethylthio)ethane, 1,3-bis(2-hydroxyethylthio)propane, and 1,4-bis(2-hydroxyethylthio)butane, which are breakdown products of the chemical warfare agents called sulfur mustards. Both isocratic and gradient elution were used. To improve sensitivity, large-volume injections were applied together with peak compression by displacement for late-eluting analytes. With S-FPD, detection limits of 1 microgram/mL were obtained for all compounds. Using the same approach, the target analytes as well as various oxidation products could be identified by micro-RPLC with electrospray ionization mass spectrometry (ESI-MS) and ESI-MS/MS. The optimized micro-RPLC-S-FPD system was successfully used for the analysis of a spiked soil sample.

Determination of the sulfur mustard hydrolysis product thiodiglycol by microcolumn liquid chromatography coupled on-line with sulfur flame photometric detection using large-volume injections and peak compression

A selective, direct and relatively rapid method has been developed for the determination of thiodiglycol (TDG) in aqueous samples. TDG is the main hydrolysis product of the chemical warfare agent sulfur mustard. The method of analysis is based on the on-line coupling of reversed-phase microcolumn liquid chromatography and sulfur-selective flame photometric detection. To improve sensitivity and efficiency, peak compression by displacement was used in combination with large-volume injections. A concentration of 1% n-propanol was added to the sample to obtain the best sensitivity and efficiency after a 10 μl injection. Detection limits of 0.25 μg/ml were achieved with efficiencies of 4·10 5 plates per meter. The method was successfully applied during the Fourth Official Proficiency Test organized by the Technical Secretariat of the Organization for Prohibition of Chemical Weapons for the determination of TDG in a soil sample.

Retention behavior of large polycyclic aromatic hydrocarbons on cholesteryl 10-undecenoate bonded phase in microcolumn liquid chromatography

The retention behavior of large polycyclic aromatic hydrocarbons (PAH) in reversed-phase microcolumn liquid chromatography has been found to depend primarily on the inherent planarity of the molecules, the degree of orderliness of the bonded phase, and the concentration of solvent. The planarity recognition capability of cholesteryl 10-undecenoate bonded phase for large PAH is comparable with that of monomeric ODS phases. During solvation, however, the phase characteristics seemed to suggest a fairly rigid structure (an ordered phase), i.e. increased selectivity for non-planar molecules which approaches that of polymeric ODS rather than that of other phases with similarly bulky groups, e.g. naphthylethyl and pyrenylethyl bonded phases.

Retention behavior of small polycyclic aromatic hydrocarbons with cholesteryl 10-undecenoate bonded phase in microcolumn liquid chromatography

The retention behavior of large polycyclic aromatic hydrocarbons (PAH) in reversed-phase microcolumn liquid chromatography has been found to depend primarily on the inherent planarity of the molecules, the degree of orderliness of the bonded phase, and the concentration of solvent. The planarity recognition capability of cholesteryl 10-undecenoate bonded phase for large PAH is comparable with that of monomeric ODS phases. During solvation, however, the phase characteristics seemed to suggest a fairly rigid structure (an ordered phase), i.e. increased selectivity for non-planar molecules which approaches that of polymeric ODS rather than that of other phases with similarly bulky groups, e.g. naphthylethyl and pyrenylethyl bonded phases.

Cerebrospinal fluid dynorphin-converting enzyme activity is increased by voluntary exercise in the spontaneously hypertensive rat

The activity in cerebrospinal fluid (CSF) of dynorphin-converting enzyme (DCE) has been studied after voluntary exercise in the spontaneously hypertensive (SH) rat. The exercise consisted of spontaneous running in wheels for 4–5 weeks and the average running distance during the last two weeks was 4.0 km/24h. CSF samples were obtained under anaesthesia from the cisterna magna after penetration of the atlanto-occipital membrane. DCE transforms the members of the dynorphin family of opioid peptides into Leu-enkephalin-Arg 6. In the present investigation a radioimmunoassay was used for quantitation of Leu-enkephalin-Arg 6 release from dynorphin A 1–17 and dynorphin B 1–13. The rats that were running had a DCE activity (vs. both substrates) in CSF that was approximately 6–12 times higher than in animals not given the running opportunity. A statistically significant correlation between the two prodynorphin-derived substrate peptides was found in terms of DCE activity. We therefore propose that a single enzyme activity may be responsible for the hydrolysis of dynorphin B 1–13 and dynorphin A 1–17. Furthermore, a significant correlation was also found between running activity and DCE activity 12–14 h before the CSF was withdrawn. Besides measurement of DCE activity by radioimmunoassay, the formation of Leu-enkephalin-Arg 6 was identified by reversed-phase micro-column liquid chromatography and plasma desorption mass spectrometry. The experiment shows that voluntary exercise affects opioid peptidergic mechanisms.

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.

Simultaneous flow rate and temperature programming based on computer-assisted retention prediction for the separation of PTH-amino acids in reversed-phase microcolumn liquid chromatography

The separation of phenylthiohydantoin (PTH) amino acid derivatives by flow rate and temperature programming in reversed-phase micro-column liquid chromatography (LC) has been investigated. For both programming modes a microcomputer-assisted retention prediction system (REPRES) was constructed. The performance of this system has been evaluated by comparing actually measured retention data to those predicted. Excellent agreements between those data were found. It is clearly indicated that simultaneous flow rate and temperature programming can improve the separation of PTH-amino acids in reduced analysis time.

Microcolumn liquid chromatography of small nucleic acid constituents

Mixtures of small nucleic acid fragments were separated by reversed-phase microcolumn liquid chromatography. By using a miniaturized UV detector, a detection limit of 10 pg with a signal-to-noise ratio of 2 for individual nucleosides was achieved. Various mixtures of nucleobases, nucleosides, nucleotides, and cyclic nucleotides were separated. Using tetrabutylammonium hydrogensulfate as an ion-pairing reagent, the negatively charged mono-, di-, and triphosphate nucleotides were also resolved. As an application for nucleic acid research, the separation of the enzymatic hydrolysis products from a 100-ng sample of tRNA-Phe is shown.

Retention prediction of large and condensed polycyclic aromatic hydrocarbons in reversed‐phase microcolumn liquid chromatography with diphenylsilica stationary phase

AbstractThe retention behavior of condensed large polycyclic aromatic hydrocarbons has been investigated with diphenylsilica stationary phases in reversed‐phase microcolumn liquid chromatography. The results were correlated with two parameters which indicate size and shape of the molecules. Since the resulting equation can be used for retention prediction of large polycyclic aromatic hydrocarbons, computer‐assisted “standardless” identification is accomplished for “unknown” compounds contained in the standard.

Amperometric detector with a manganin electrode for microcolumn liquid chromatography

The response of manganin and copper microelectrodes in amino acid solution was evaluated by using slow-scanning voltammetry. Enhancement of the complexation rate of the manganin electrode in relation to the copper electrode is explained on the basis of the properties of the electrode surface. Based on voltammetric measurements, manganin is suggested as a working electrode material for the confined wall-jet microflow-cell of an amperometric detector. The detector performance was tested under the conditionsof reversed-phase microcolumn liquid chromatography using selected amino acids and dipeptidees as the solutes. Employing flow-rates up to 1 μ1/s, an increase in response corresponding to the 1 3 power of the flow-rate was found, which indicates that a diffusion-limited electrode reaction occurs. The minimum detectable concentrations of the amino acids and some dipeptides in the ranges 0.04–0.08 and 0.1–0.25 μM, respectively, appear promising for application of this detector to trace analysis.

Optimization of fat soluble vitamin separation in reversed-phase microcolumn liquid chromatography

Separation behavior of fat soluble vitamins on various chemically bonded materials in non-aqueous reversedphases microcolumn liquid chromatography has been investigated. The best performance was found on a polymer-based octadecyl bonded phase. Optimization of separation for vitamins D2, D3, E and E acetate has been tried using this polymer-based column and it has been found that a binary mobile phase consisting of acetonitrile and methanol gave complete separation of fat soluble vitamins.