15
of 20 herbicides (phenylurea and triazines) is analyzed on a Zorbax StableBond C18 (Agilent Technologies, Waldbronn, Germany) at 50 and at 90°C. Major selectivity changes are seen in the chromatogram. A graph showing the retention time in function of temperature for the central region of the chromatogram is inserted in the figure.
Figure 2. Influence of temperature on the analysis of a mixture herbicides on a Zorbax StableBond C18 column (150 mm L x 4.6 mm ID, 1.8 µm particles). Flow rate: 1 mL/min, gradient: water/ACN 80/20 to 45/55 in 30 min, detection: DAD, 230 nm. Temperature is controlled by Polaratherm 9000 Series column oven equipped with active mobile phase preheating and effluent cooling (SandraSelerity Technologies, Kortrijk, Belgium).
nature of the stationary phase and the composition of the mobile phase (buffer pH and composition, organic modifier type, additives etc). Since the ionization equilibria of analytes and mobile phase are temperature dependent, the retention behavior of polar and ionizable compounds, and consequently the selectivity, will be significantly affected by temperature variations. Although reported more than one decade ago and recently re-emphasized in the literature [2-5]
,
temperature is often overlooked in this respect and is rarely investigated in depth during method development. Very often, temperature screening will be the last resort if the previously described variables do not deliver or it will be evaluated at the end of the method development process to fine-tune the resolution between critical pairs.
In order to fully exploit the influence of temperature on a separation, larger temperature windows (e.g. between 20 and 150 °C) have to investigated. When doing so, very significant selectivity changes and
often reversal elution orders can be accomplished. Even so, at this moment high temperature LC is not a common approach in daily practice. One reason is that performing high temperature LC and temperature programmed LC on an analytical scale necessitates the use of dedicated equipment to preheat the incoming mobile phase and column and to cool down the column effluent before detection [5]
. A second reason is that the
range of columns that is compatible with high temperature is considerably smaller than the number of traditional columns. Very often, the high temperature rated columns (e.g. graphitized carbon, zirconium oxide based phases, polymers) are also very different in nature compared to the more common phases. This gives rise to very different selectivities which makes it much harder for the chromatographer to translate existing methods to high temperature methods.
However, several typical LC columns tolerate relatively high temperatures. An example is shown in Figure 2 where a set
The highest temperature in the previous analysis corresponds with the upper limit that is supported for this particular stationary phase. If higher temperatures or larger temperature windows need to be investigated, alternative column material has to be used. When using some polymeric stationary phases (e.g. ET-RP1), the temperature can be increased up to 150°C. Additionally, polymeric stationary phases often are chemically more resistant and inert. The combination of a large temperature and mobile phase pH window is extremely powerful in
method development. Figure 3 shows an example of the analysis of a mixture of a pharmaceutical (Metoclopramide) and its documented impurities (European Pharmacopoeia, EP) on a polymeric ET- RP1 column (Shodex, Munich, Germany). The analysis was carried out at different temperatures with a mobile phase at pH 9. The temperature was varied between 50 and 125°C during method development and the final temperature was set at 105°C. It is clear that the performance of most traditional silica based columns would deteriorate fast when applying these conditions. This was not the case for the polymeric column where the performance was maintained after the complete validation of the high temperature method [1]
.
Conclusion This technical note demonstrates that temperature is a useful parameter for method development in LC. Temperature can be actively employed as a selectivity tool and not merely as a final fine-tuning parameter.
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