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A chiral anion-exchanger stationary phase based on cinchonidine (CD) was developed. Two columns were packed with and without endcapping (EC) treatment (CD-chiral stationary phase[CD-CSP(EC)] and [CD-CSP], respectively) and studied for their ability to separate N-2,4-dinitrophenyl α-amino acids (DNP-amino acids) enantiomers over a temperature range of 10-40 °C with a hydro-organic buffer mobile phase. The more hydrophobic, endcapped stationary phase showed significantly larger retentive capacity than the non-endcapped one. The apparent thermodynamic transfer parameters of the enantiomers from the mobile to both CSPs were estimated from van't Hoff plots within the cited temperature range. Similar studies with two natural quinine-based columns (QN-CSP and QN-CSP(EC)) were previously reported. In this work, a critical comparison in the chiral recognition ability to DNP-amino acids of these cinchonidine and QN-based chiral columns was drawn. It has been found that QN-based CSPs show greater chiral recognition capability towards these derivatives than CD-CSPs. The influence of the QN methoxy group on the equilibrium constants of the enantioselective interaction between these DNP-amino acids with these two cinchona CSPs could be assessed.

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In this report, we demonstrate how chiral liquid chromatography combined with multivariate chemometric techniques, specifically unfolded-partial least-squares regression (U-PLS), provides a powerful analytical methodology. Using U-PLS, strongly overlapped enantiomer profiles in a sample could be successfully processed and enantiomeric purity could be accurately determined without requiring baseline enantioresolution between peaks. The samples were partially enantioseparated with a permethyl-ß-cyclodextrin chiral column under reversed-phase conditions. Signals detected with a diode-array detector within a wavelength range from 198 to 241 nm were recorded, and the data were processed by a second-order multivariate algorithm to decrease detection limits. The R-(-)-enantiomer of ibuprofen in tablet formulation samples could be determined at the level of 0.5 mg L⁻¹ in the presence of 99.9% of the S-(+)-enantiomorph with relative prediction error within ±3%.

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We developed an improved reversed-phase high-performance liquid chromatography (RPLC) assay for the rapid separation and determination of the 2-hydroxyethidium ion. The 2-hydroxyethidium ion is the specific product of the redox reaction between hydroethidine with superoxide radical. High resolution between the chromatographic bands corresponding to ethidium and 2-hydroxyethidium ions was achieved within a practicable analysis time. The RPLC-fluorescence method can reliably detect 2-hydroxyethidium ion concentrations down to 0.12 µM (or 1.2 pmol) and the signal is linear with concentration beyond 50 µM. An application of the method to neutrophil samples demonstrated that intracellular quantification of 2-hydroxyethidium was reproducible, as evidenced by low values of the relative standard deviations: 0.016 for non-stimulated cells, and 0.056 and 0.0125 for neutrophils incubated with agonists phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), respectively. The resulting analytical method combines a rapid separation of the relevant peaks with the degree of sensitivity required for use in routine biological analyses.

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Recent developments in HPLC methods have focused on various strategies in order to increase the speed of analysis. One area of impressive growing is column technology. Today, analytical methods that propose the use of short columns packed with sub-2 µm particles installed in ultra high-pressure LC instruments are not uncommon. Another strategy consisted of heating thermally resistant columns to temperatures well above of 100°C in order to reduce eluent viscosities and, therefore, column backpressure. We discuss experimental conditions for achieving high-throughput analysis using standard instruments with a few simple modifications. The chromatographic performance of two particulated and a silica-based monolithic column operated at moderate temperatures and flow rates are compared. The monolithic column proved to be stable over several thousands column volumes at 60°C. More important, its resistance to mass transfer at this temperature was significantly reduced. Very fast separations of two different mixtures of pharmaceutical compounds, anti-inflammatory drugs and ß-blockers, were achieved with the three columns at 60°C by using ACN/buffer at 5 mL/min. Excellent peak shapes of basic solutes and quite reasonable resolutions were achieved in very short analysis times with columns operated at temperatures moderately higher than the usual room temperature.

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This paper reports the study of poly(oxyethylene) as a solvent for heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (PM-beta-CD) and the potential of these mixtures for constructing enantioselective gas chromatography columns. Enantioseparations of volatile racemic mixtures using capillaries coated with 10-50% PM-beta-CD diluted in Carbowax 20M were evaluated. The influence of the polymer type on retention, separation and reproducibility over a period of time is critically discussed. The poly(oxyethylene)-based columns were also compared to columns prepared by dilution of PM-beta-CD in two polysiloxanes, SE-30 and SE-54, at several temperatures. Finally, we evaluated a new stationary phase prepared by crosslinking the chiral selector to the poly(oxyethylene) matrix.

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