An Evaluation Of UHPLC-MS Technology Encompassing Elevated Temperatures, Low Viscosity Operation and Recently Developed Stationary Phase Materials
There are several approaches to reducing mobile phase viscosity in order to overcome high pressures related to stationary phase permeability. This can be achieved either by elevating column temperature and or by employing organic rich eluents. To begin with, a standard application of UHPLC-MS was developed for the high throughput quantification of quinine and its major metabolite. In attempting to expand the performance capabilities of ultra-pressure systems a high temperature column oven was evaluated. It was found to be mostly unsuitable for incorporating micro-bore columns due to excessive system volume necessary for installation, and due to the effects of frictional heating associated with operation at ultra-high pressures. An attempt to reduce the effect of frictional heating by modifying the column hardware was evaluated, however only slight improvements were observed. This system has benefits to operate at elevated temperatures, however reduced column performance was always observed as shown by the separation of anabolic steroids and neutral test substances. An evaluation of HILIC column efficiency was undertaken by assessing the performance merits of several commercially available packings using the Knox/van Deemter and kinetic plots approaches. This work centred on evaluation using realistic test substances on underivatised silica, highlighting that very high efficiencies could be achieved using these phases, otherwise not achievable with reversed-phase systems due to peak shape improvements. The justification of which was further addressed by comparing the performance merits of ephedrine-substances separated by both reversed-phase and HILIC noting that the latter was far superior. Arguably the biggest disadvantage with HILIC is the reliance on acetonitrile based mobile phases. An in-depth evaluation of acetone as an alternative, aprotic organic modifier for HILIC-ESI-MS was undertaken, showing that this may not be suitable for the analysis of small polar molecules in positive ion electrospray.
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