Investigation of poly(styrene-divinylbenzene-vinylsulfonic acid) as retentive and electroosmotic flow generating phase in open-tubular electrochromatography.

In this work, a new sulfonated polystyrene based porous layer was synthesized on the wall of a capillary by a single step in situ polymerization process. To obtain a capillary suited for electrochromatography, vinylsulfonic acid (VSA) was, next to divinylbenzene (DVB), copolymerized to induce charges for the electroosmotic flow (EOF) generation. The VSA ratio in the monomer mixture and the polymerization time were optimized while the chromatographic characteristics of the obtained open tubular columns were investigated in electrochromatography. To allow unambiguous study of only chromatographic processes, evaluations were performed with a mixture of sufficiently retained and electrophoretically neutral parabens. Comparison of SEM pictures and chromatograms revealed that the polymerization time had a great influence on the polymer layer morphology and on the chromatographic performance. An increase in the VSA ratio, led to an increase in the mobile phase velocity but simultaneously lowered paraben retention. The novel optimized stationary phase could generate a stable and significant electro-osmotic flow (EOF) of 1.1mm/s over a wide pH range which could be produced in a reproducible manner. Minimal plate heights of 10µm, equivalent to the capillary internal diameter, were obtained. The open-tubular character of this optimized porous layer column allowed successful analyses at elevated temperature, resulting in a maximum efficiency of 85,500 plates for a 75cm capillary and linear velocities up to 1.4mm/s. Finally, a thermal gradient was successfully applied, leading to artificial sharpened peaks with a peak capacity of 55 in a 20min time span.

Antioxidant components of Viburnum opulus L. determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methods.

Antioxidant activity of the juice and seed and skin extracts prepared with methanol, acetonitrile, and water of Viburnum opulus L. grown in Eastern Black Sea Region were studied with an on-line HPLC-ABTS method and off-line antioxidant methods, among which a linear positive correlation was observed. The fruit extracts were analysed with the HPLC-UV method optimised with 14 standard phenolics. Identification of the phenolic components in the juice was made using an HPLC-UV-ESI-MS method. Nineteen phenolic compounds in juice were identified by comparing the retention times and mass spectra with those of the standards and the phenolics reported in the literature. The major peaks in the juice belonged to coumaroyl-quinic acid, chlorogenic acid, procyanidin B2, and procyanidin trimer. Quite different antioxidant composition profiles were obtained from the extracts with the solvents of different polarities. The antioxidant activities of the seed extracts were higher than those of the skin extracts in general.

Optimization of a high-resolution radical scavenging assay coupled on-line to reversed-phase liquid chromatography for antioxidant detection in complex natural extracts.

This paper reports the optimization of the on-line coupling of 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid based radical scavenging assays with reversed-phase high-performance liquid chromatography. The residence time in the reactor was reduced to 6.4 s to ensure minimal peak broadening and loss of separation. Peak capacity losses between compound detection and measurement of the radical scavenging potential were reduced to 10% and lower on coupled column systems. The methodology was successfully applied for the detection of the scavenging activity of molecules encompassing a broad hydrophobicity range. The method shows promise for the assessment of low-molecular-weight polyphenols in red wine by coupled-column high-resolution high-performance liquid chromatography with mass spectrometry analysis.

Kinetic performance evaluation and perspectives of contemporary packed column capillary electrochromatography.

Capillary electrochromatography (CEC) is in essence a highly efficient and fast separation technique but practical constraints limit the current performance, robustness and routine implementation of the technique. In this work the kinetic performance limit (KPL) curve was used to evaluate commercial packed column CEC; this firstly in order to assess the broader applicability of the kinetic plot approach in electrodriven chromatographic techniques, and secondly to allow a more general unbiased comparison with HPLC performance. Evaluations were performed with a mixture of well retained and electrophoretically neutral phenones, to allow the observation of only chromatographic processes. Initial CEC retention time irreproducibility issues were solved by applying high acetonitrile content (80%) in the mobile phase, and solute retention was increased by increasing the phenone chain length. Comparison was performed with HPLC, with a column packed with an identical stationary phase to allow measurement of the performance under optimal conditions, and not with µ-LC on the CEC column as extra column peak broadening phenomena would thereby negatively affect the µ-LC performance. This comparison demonstrated that current HPLC performance largely outcompetes what is achievable with contemporary packed column CEC. Interestingly, significantly improved CEC performance could be obtained at lower temperatures (10°C) indicating a persistent degree of joule heating phenomena taking place in the contemporary packed column (100µm) CEC approach. Effective suppression of the latter opens possibilities for increasing the applicable voltage and outperforming HPLC and UHPLC.

Comprehensive two-dimensional liquid chromatography coupled to the ABTS radical scavenging assay: a powerful method for the analysis of phenolic antioxidants.

The on-line combination of comprehensive two-dimensional liquid chromatography (LC × LC) with the 2,2'-azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid (ABTS) radical scavenging assay was investigated as a powerful method to determine the free radical scavenging activities of individual phenolics in natural products. The combination of hydrophilic interaction chromatography (HILIC) separation according to polarity and reversed-phase liquid chromatography (RP-LC) separation according to hydrophobicity is shown to provide much higher resolving power than one-dimensional separations, which, combined with on-line ABTS detection, allows the detailed characterisation of antioxidants in complex samples. Careful optimisation of the ABTS reaction conditions was required to maintain the chromatographic separation in the antioxidant detection process. Both on-line and off-line HILIC × RP-LC-ABTS methods were developed, with the former offering higher throughput and the latter higher resolution. Even for the fast analyses used in the second dimension of on-line HILIC × RP-LC, good performance for the ABTS assay was obtained. The combination of LC × LC separation with an on-line radical scavenging assay increases the likelihood of identifying individual radical scavenging species compared to conventional LC-ABTS assays. The applicability of the approach was demonstrated for cocoa, red grape seed and green tea phenolics.