14
August/September 2010
The Use of Temperature for Method Development in LC
by Gerd Vanhoenacker*, Frank David, Pat Sandra Research Institute for Chromatography, Kennedypark 26, B-8500 Kortrijk, Belgium
Corresponding author:
gerd.vanhoenacker@
richrom.com
The use of temperature as a tool for method development is gaining interest. In most of the reversed-phase LC methods the selectivity can be altered significantly by changing the temperature. Since temperature is an instrumental parameter it is easier to change than e. g. buffer pH and is more robust. This note demonstrates the potential of temperature variations for controlling the selectivity in LC method development. Additionally, the features of a silica based and a polymeric stationary phase for elevated temperature LC are highlighted.
Increasing the column temperature has several advantages amongst which the gain in speed and efficiency and the possibility to alter the chromatographic selectivity are some of the most important.
Speed & Efficiency An increase in temperature will generally cause a decrease in retention. The reduced solvent viscosity at elevated temperature will lead to lower back pressure and allows the use of higher flow rates and/or smaller particles to increase the analysis speed using standard HPLC equipment. The low back pressure at elevated temperature also permits to use longer columns combined with higher flow rates to increase the efficiency and resolution without significantly raising the analysis time.
At elevated temperature the solute transfer from the mobile phase to the stationary phase is more efficient (C-term in van
Deemter equation). The result is a flatter van Deemter curve. This enables, and even requires, using higher flow rates without sacrificing efficiency. For reversed-phase separations on silica based columns an increase in temperature will not significantly affect the minimum plate height unless secondary interactions are prominent. Polymeric stationary phases suffer from a significantly slower diffusion rate of the solutes in and out the stationary phase, leading to lower efficiencies compared to their silica equivalent. By using elevated temperature the diffusion rates are improved and the viscosity of the mobile phase is reduced. Therefore, the plate height will decrease and efficiencies at high temperature will be comparable to efficiencies obtained with silica based reversed-phase columns.
This is demonstrated in Figure 1 where the reduced plate height (h) at various
temperatures and flow rates is compared for a silica based column (Blaze200 C18, 15 cm L x 4.6 mm ID, 3 µm particles, Selerity Technologies, Salt Lake City, UT, USA) and a polymeric column (ET-RP1, 15 cm L x 4.6 mm ID, 5 µm particles, Shodex, Munich, Germany). The mobile phase composition was modified to keep the k-value nearly constant at ca. 1.8. It has to be noted that the silica based stationary phase that is used here is specially designed for use at high temperatures and that most commercially available phases should be used at temperatures below 70-80°C. The use of polymeric columns has been described by our group in reference [1]
.
Selectivity Selectivity is the most important factor for resolving compounds in LC. There are numerous ways to alter selectivity. The most common in reversed phase LC are the
Figure 1. Van Deemter curves for acetophenone (200 µg/mL in water/acetonitrile 75/25 v/v ) on a polymeric and a silica based column. Mobile phase: water/acetonitrile, injection volume: 2 µL, detection: DAD, 210 nm. Temperature is controlled by Polaratherm 9000 Series column oven equipped with active mobile phase preheating and effluent cooling (SandraSelerity Technologies, Kortrijk, Belgium).
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