RESUMEN
The performance of a particle packed column will inevitably degrade through use or misadventure. 'Active flow technology' (AFT) is known to greatly improve the performance of pristine columns, but is as of yet untested when used on columns that have degraded significantly. In this study AFT was used to regenerate a degraded column, where the reduced plate height and asymmetry values were 3.5 and 1.25 respectively. Once the AFT fittings were fitted to the column outlet and the flow segmentation ratio adjusted to 28% from the radial central exit port, the reduced plate height decreased to 2.0, and the bands were almost perfectly symmetrical with asymmetry factors equal to 1.04. Subsequently, the performance of the degraded column with AFT fittings provided performance that was comparable to that of a new conventional column fitted with traditional end fittings. The separation power of the degraded conventional column and that of the same column fitted with the AFT end fittings was then tested using the separation of oligostyrenes. In AFT mode, detection was undertaken at both the radial central exit port of the column and the peripheral exit port. The resulting separation that was achieved from the radial central exit port was superior to that observed on the conventional column, whereas, the separation observed from the peripheral port was very poor. It was subsequently determined that the reason for the degraded performance of the conventional column was a result of increased heterogeneity associated with the packing material in the wall region of the column.
Asunto(s)
Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Poliestirenos/análisis , Diseño de Equipo , Tamaño de la PartículaRESUMEN
Conventional techniques for identifying antioxidant and phenolic compounds in native Australian food plants are laborious and time-consuming. Here, we present a multiplexed detection technique that reduces analysis time without compromising separation performance. This technique is achieved using Active Flow Technology-Parallel Segmented Flow (AFT-PSF) columns. Extracts from cinnamon myrtle (Backhousia myrtifolia) and lemon myrtle (Backhousia citriodora) leaves were analysed via multiplexed detection using an AFT-PSF column with underivatised UV-VIS, mass spectroscopy (MS), and the 2,2-diphenyl-1-picrylhydrazyl (DPPH(â¢)) derivatisation for antioxidants as detection methods. A number of antioxidant compounds were detected in the extracts of each leaf extract.
Asunto(s)
Antioxidantes/química , Antioxidantes/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Plantas Comestibles/química , Cromatografía Líquida de Alta Presión , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología , Análisis Espectral/métodosRESUMEN
A new type of chromatography column referred to as a parallel segmented flow (PSF) column enables ultra-high-speed high-performance liquid chromatography-MS to be undertaken. This occurs because the separation efficiency obtained on PSF columns has been shown in prior studies to be superior to conventional columns, and the flow stream is split radially inside the outlet end fitting of the column, rather than in an axial post-column flow stream split. As a result, the flow through the column can be five times higher than the flow through the MS. In this work, the degradation of amino acids in dilute nitric acid was used to illustrate the process. Separations were obtained in less than 12 s, although the reinjection process was initiated 6 s after the previous injection. The degradation rate constant of tryptophan, in the presence of tyrosine and phenylalanine, was determined.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Aminoácidos/análisis , Aminoácidos/química , Modelos QuímicosRESUMEN
The performance of active flow technology chromatography columns in parallel segmented flow mode packed with 5 µm Hypersil GOLD particles was compared to conventional UHPLC columns packed with 1.9 µm Hypersil GOLD particles. While the conventional UHPLC columns produced more theoretical plates at the optimum flow rate, when separations were performed at maximum through-put the larger particle size AFT column out-performed the UHPLC column. When both the AFT column and the UHPLC column were operated such that they yielded the same number of theoretical plates per separation, the separation on the AFT column was twice as fast as that on the UHPLC column, with the same level of sensitivity and at just 70% of the back pressure. Furthermore, as the flow velocity further increased the performance gain on the AFT column compared to the UHPLC column improved. An additional advantage of the AFT column was that the flow stream at the exit of the column was split in the radial cross section of the peak profile. This enables the AFT column to be coupled to a flow limiting detector, such as a mass spectrometer. When operated under high through-put conditions separations as fast as six seconds, using mobile phase flow rates in the order of 5-6 mL/min have been recorded.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Cromatografía Líquida de Alta Presión/instrumentación , Tamaño de la PartículaRESUMEN
The performance of curtain flow chromatography column technology with MS detection was evaluated for the analysis of labile compounds. The curtain flow column design allows for separations that are faster and/or more sensitive than conventional columns, depending on how exactly the curtain flow column is configured. For example, when mass spectral detection is employed, the curtain flow column can yield separations that are 5-times faster than conventional columns when the curtain flow and the conventional columns have the same internal diameter. Or when the internal diameter of the conventional column is reduced in order to yield the same analytical through-put as the curtain flow column, the sensitivity on the curtain flow column can be as much as 66-fold higher than the conventional column. As a consequence of the higher analytical through-put less standardization is required in the analysis of labile compounds because less sample degradation is apparent. Consequently the sample integrity is preserved yielding data of a higher quality.
Asunto(s)
Aminoácidos/análisis , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodosRESUMEN
Analytical scale active flow technology first generation silica monolithic columns kitted out in curtain flow mode of operation were studied for the first time. A series of tests were undertaken assessing the column efficiency, peak asymmetry and detection sensitivity. Two curtain flow columns were tested, one with a fixed outlet ratio of 10% through the central exit port, the other with 30%. Tests were carried out using a wide range in inlet flow segmentation ratios. The performance of the curtain flow columns were compared to a conventional monolithic column. The gain in theoretical plates achieved in the curtain flow mode of operation was as much as 130%, with almost Gaussian bands being obtained. Detection sensitivity increased by as much as 250% under optimal detection conditions. The permeability advantage of the monolithic structure together with the active flow technology makes it a priceless tool for high throughput, sensitive, low detection volume analyses.
Asunto(s)
Cromatografía Liquida/normas , Dióxido de Silicio/química , Cromatografía Liquida/instrumentación , Permeabilidad , Sensibilidad y EspecificidadRESUMEN
Active flow technology (AFT) columns are designed to minimise inefficient flow processes associated with the column wall and radial heterogeneity of the stationary phase bed. This study is the first to investigate AFT on an analytical scale 4.6mm internal diameter first-generation silica monolith. The performance was compared to a conventional first-generation silica monolith and it was observed that the AFT monolith had an increase in efficiency values that ranged from 15 to 111%; the trend demonstrating efficiency gains increasing as the volumetric flow to the detector was decreased, but with no loss in sensitivity.
Asunto(s)
Cromatografía Liquida/métodos , Dióxido de Silicio/químicaRESUMEN
A preliminary investigation was undertaken to assess the performance of a new chromatography column technology in applications involving liquid chromatography coupled to mass spectrometry. The new column design allows mobile phase and solute to be extracted from the radial central region of the column, which reduces the solvent load to the mass spectrometer and improves separation efficiency. Effectively the column functions as a 'wall-less' column. The advantages of this design is that the analysis through-put can be increased by a factor of five, while at the same time there is a reduction in baseline noise, which results in an increase in the signal to noise response by up to 10-fold in comparison to standard columns with the same internal diameter and approaching 66-fold in comparison to standard columns with the same virtual internal diameter.
Asunto(s)
Aminoácidos Esenciales/aislamiento & purificación , Cromatografía Líquida de Alta Presión/instrumentación , Espectrometría de Masas en Tándem/métodos , Límite de DetecciónRESUMEN
The analysis of antioxidants from complex samples is conveniently achieved using liquid chromatography, which provides sample fraction, coupled with an on-line antioxidant assay, which provides detection. One particularly useful on-line antioxidant assay that has routinely been coupled with HPLC involves the diphenylpicrylhydrazyl radical (DPPH), which provides a positive test for phenolic antioxidants through a decolorisation of the DPPH reagent. A limitation of this assay, however, is the need to employ a reaction coil, which is often large with respect to the peak volume, consequently adding substantial band broadening to the separation. In this study we introduce a new concept that can be employed for systems requiring post column derivatisations, such as the DPPH assay. We have termed this 'reaction flow' chromatography, whereby, the derivatisation reagent can be added directly into one of the outlet ports of a parallel segmented flow column. Subsequently, the mixing between the derivatising reagent and the solute is very efficient removing the need to employ reaction coils. The concept is tested here using the DPPH assay for the analysis of antioxidants in samples derived from natural origin.