Browsing by Author "Cabooter D."
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- ItemFast method development of rooibos tea phenolics using a variable column length strategy(2011) Cabooter D.; Broeckhoven K.; Kalili K.M.; de Villiers A.; Desmet G.The development of a method for the separation of standard compounds of the 15 main phenolics found in rooibos tea is presented. The separation of these compounds in a single HPLC analysis is particularly challenging due to the similarity of rooibos phenolics. As a result, multiple methods are often required to analyze all major phenolics in rooibos tea samples. The method development process is significantly enhanced in this study by using the recently introduced automated column coupler in combination with the variable column length strategy. This strategy consists of performing the initial scouting runs, wherein the best separation conditions are determined, on a short column and subsequently fine-tuning the separation on longer columns to benefit from their higher separation performance. It is demonstrated that the method development process can further be expedited by operating each column length at the maximum pressure, in this case 1000 bar. Although this holds in general, it is even more the case for the presently considered sample, since the selectivity of the sample is more pressure- than temperature-dependent. Applying the optimized method to unfermented and fermented aqueous rooibos tea extracts in combination with Q-TOF mass spectrometry, some 30 phenolic compounds are tentatively identified. © 2011 Elsevier B.V.
- ItemHigh performance liquid chromatography analysis of wine anthocyanins revisited: Effect of particle size and temperature(2009) de Villiers A.; Cabooter D.; Lynen F.; Desmet G.; Sandra P.The complex anthocyanin fraction of red wines poses a demanding analytical challenge. We have found that anthocyanins are characterised by extremely low optimal chromatographic velocities, and as a consequence generic HPLC methods suffer from limited resolving power. Slow on-column inter-conversion reactions, particularly between carbinol and flavylium species, are shown to occur on the same time scale as chromatographic separation, leading to increased plate heights at normal chromatographic velocities. In order to improve current routine HPLC separations, the use of small (1.7 μm) particles and high temperature liquid chromatography (HTLC) were investigated. 1.7 μm particles provide better efficiency and higher optimal linear velocities, although column lengths of ∼20 cm should be used to avoid the detrimental effects of conversion reactions. More importantly, operation at temperatures up to 50 °C increases the kinetics of inter-conversion reactions, and implies significantly improved efficiency under relatively mild analysis conditions. It is further demonstrated using relevant kinetic data that no on-column thermal degradation of these thermally labile compounds is observed at 50 °C and analysis times of <2 h. © 2009 Elsevier B.V. All rights reserved.
- ItemHigh-efficiency high performance liquid chromatographic analysis of red wine anthocyanins(2011) De Villiers A.; Cabooter D.; Lynen F.; Desmet G.; Sandra P.The analysis of anthocyanins in natural products is of significant relevance in recent times due to the recognised health benefits associated with their consumption. In red grapes and wines in particular, anthocyanins are known to contribute important properties to the sensory (colour and taste), anti-oxidant- and ageing characteristics. However, the detailed investigation of the alteration of these compounds during wine ageing is hampered by the challenges associated with the separation of grape-derived anthocyanins and their derived products. High performance liquid chromatography (HPLC) is primarily used for this purpose, often in combination with mass spectrometric (MS) detection, although conventional HPLC methods provide incomplete resolution. We have previously demonstrated how on-column inter-conversion reactions are responsible for poor chromatographic efficiency in the HPLC analysis of anthocyanins, and how an increase in temperature and decrease in particle size may improve the chromatographic performance. In the current contribution an experimental configuration for the high efficiency analysis of anthocyanins is derived using the kinetic plot method (KPM). Further, it is shown how analysis under optimal conditions, in combination with MS detection, delivers much improved separation and identification of red wine anthocyanins and their derived products. This improved analytical performance holds promise for the in-depth investigation of these influential compounds in wine during ageing. © 2011 Elsevier B.V.
- ItemKinetic optimisation of the reversed phase liquid chromatographic separation of proanthocyanidins on sub-2μm and superficially porous phases(2012) Kalili K.M.; Cabooter D.; Desmet G.; de Villiers A.Phenolic compounds, and proanthocyanidins in particular, are important natural molecules which are of significant importance due to their sensory and biological activities. The analysis of proanthocyanidins in natural products is very challenging due to their complex nature. In this study, the kinetic performance of a range of recently developed C18 columns, including sub-2 μm fully porous and 2.6 μm superficially porous particle-packed columns, was evaluated for improved proanthocyanidin analysis. The kinetic plot method was employed to compare the ultimate performance limits of each column in terms of efficiency and speed for different maximum pressures and temperatures using representative proanthocyanidins comprising a range of molecular weights and functionalities as test analytes. By combining plate height data with relevant parameters such as column permeability and mobile phase viscosity, plots of practically attainable efficiencies as a function of analysis time for specific experimental configurations were obtained and performance limits for all investigated supports could accurately be compared. Both fully- and superficially porous particles provided significant speed and/or efficiency gains compared to conventional 5 μm particle packed columns. Analyte properties, particle size and packing quality as well as analysis temperature were all found to have a significant influence on the performance of the presently investigated chromatographic supports. For smaller compounds, higher optimal linear velocities and better performance in the low-efficiency range were observed, while the lower diffusion coefficients of larger procyanidins resulted in lower optimal linear velocities and better performance in the high-efficiency regime. Faster analyses become possible at higher temperatures due to decreased eluent viscosity and faster mass transfer, which was especially beneficial for larger compounds and resulted in dramatic improvement in efficiency. A possible explanation of the abnormal behaviour of oligomeric procyanidins is presented. Our findings indicate that new column formats, when used under optimal conditions, significantly improve the speed and/or efficiency of reversed phase liquid chromatographic analyses of proanthocyanidins. © 2012 Elsevier B.V.