18
August/September 2010
Turning on the Heat in Liquid Chromatography
by Tony Edge*, Luisa Pereira, Thermo Fisher Scientific, Tudor Road, Runcorn,WA7 1TA *Corresponding author:
tony.edge@thermofisher.com
The use of temperature within liquid chromatography (LC) has traditionally been limited to isothermal studies up to 50° or 60°C. However, this does not realise the full potential that temperature can have within a liquid chromatographic system. In particular the ability to run with thermal gradients [1,2]
or to run green LC [3,4] liquid chromatography community.
The advent of ultra-high pressure liquid chromatography (UHPLC) has meant that chromatographers are becoming more aware of the extremes of chromatography, and the benefits that this can have in terms of the separations either due to efficiency gains or reduction in sample analysis times. The advent of the new phases has also led coincidentally to robustness in the column performance at elevated temperature allowing for more extreme chromatography to be tried. The advantages of this form of extreme chromatography are many-fold e.g. a reduction in viscosity leads naturally to a reduction in the operating pressure, allowing for much higher flow rates and thus reducing analysis times. It also leads to substantial changes in selectivity which can benefit the chromatographer. Interestingly the use of HypercarbTM which is an ideally suited stationary phase for these extreme conditions, is seeing an increase in the number of applications [5,6]
.
Experimental Two different experimental arrangements were employed to demonstrate the benefits of high temperature liquid chromatography. The first set of experiments used a UHPLC column (C18 100 x 2.1mm, sub 2µm). The chromatographic systemcomprised of an autosampler and a UHPLC binary pump which was used in conjunction with a GC oven, employed to heat the column, with pre-column heating and post column cooling to ensure optimal performance. This oven was capable of operating at temperatures up to 450°C. The eluent from the column was cooled before it went into a mass spectrometer.
Calibration of the physical parameters associated with the instrumentation has been discussed previously [2]
, but is essential in
determining what experimental flow rates can be used without damage to the instrumentation. It also allows for isobaric studies, where temperature gradients are applied to an isocratic mobile phase to elute components from a HPLC column, but maintaining a constant pressure which is used as a coarse marker to determine the optimum flow for a set temperature.
For the production of the experimental data on Hypercarb there is a change to the
Figure 1: Van’t Hoff plot of a series of polar compounds for a range of temperatures with a mobile phase of 0.1% formic acid (aq), flow rate 0.3 ml/min, injection volume 10 µl.
experimental arrangement. Instead of a GC oven the Selerity Polaratherm™ Series 9000 oven was employed. This has an inbuilt pre- column heater and also a post column cooler. Instead of a mass spectrometer, a UV detector is used.
Results and discussion The initial work investigated the selectivity differences observed with a series of compounds eluting froma column at differing
temperatures. Figure 1 shows a plot of the 1/T vs. Ln k’. The relationship between the capacity factor and the temperature is well defined and derives fromthe Van’t Hofft equation. This relationship is based on some assumptions, primarily that the interaction between the stationary phase and the analyte does not alter, and that the temperaturemerely affects the rate of adsorption and desorption within the column. Clearly, over a wide temperature range itmight be expected that
, where there is no organic solvent used, is something that is not considered as routine within the
,
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