5


versus 1/T) yields a straight line. In reversed phase HPLC, as the temperature is increased, retention will usually decrease [2,14]


. Since the


selectivity of the separation also depends on retention as is shown in Equation 6, temperature can be actively used to change the selectivity of the phase system and hence optimize resolution.


Equation 6


A very nice example to demonstrate the potential of optimising resolution and analysis time has been given by Giegold et al. [1]


. In


Figure 2, an optimized separation of a mixture containing sulfonamides and trimethoprim is shown using a simultaneous temperature and solvent gradient. Neither the isothermal separation at 70 °C nor the isothermal separation at 90 °C were successful to achieve both a fast analysis time and a high resolution (data not shown here). In the case of the lower temperature, the last peak pair was not fully resolved while at the higher temperature, a co-elution of the first peaks was observed. In order to optimize the separation, a temperature gradient from 70 to 90 °C was performed simultaneously to solvent gradient programming. This example clearly shows that temperature is a powerful tool to adjust the selectivity and can be complementary to solvent gradient programming. Although this optimization was done by trial and error, work is currently carried out in the author’s own laboratory to implement temperature programming in commercially available software designed for structured method development. With this tool, which will be incorporated into the DryLab®


software (Molnar Institut, Berlin), the


user will be able to use temperature programming in reversed phase HPLC to optimize chromatographic methods.


or the direct gustatory evaluation of separated species by a human being known as LC taste® [18]


Although it was stated 30 years ago that temperature programming should yield the same results as solvent programming, solvent gradient elution is by far the most often used mode for method optimisation in HPLC. However, there are some special hyphenation techniques which could play an ever greater role in the near future. These comprise isotope ratio monitoring mass spectrometry (IRMS) [3,17]


. This system uses the


advantage of separation based on high- temperature HPLC and combines it with an in vivo detection of taste active compounds by a sensory tester or sensory panel. While for IRMS any addition of carbon to the mobile phase is strictly forbidden, LC taste®


allows


the use of mobile phase additives which are non-toxic and can be swallowed by a human being. Both techniques heavily depend on temperature programming. Here, the effect of decreasing the polarity of the mobile phase by increasing the temperature is exploited [7]


.


Hence, temperature programming is not only a concomitant tool to influence the selectivity of a separation, but it is detrimental if solvent programming cannot be used.


A final comment should be made to the heating system and the instrumental requirements. As was also outlined above, the heating system should be able to generate temperatures as high as 200 °C. A very important prerequisite to use high eluent temperatures is that the mobile phase can be adequately preheated to the temperature of the column. Otherwise, a severe band broadening or even peak splitting will be observed, leading to a complete loss of efficiency at higher temperatures. Meanwhile, most ovens are equipped with a device for eluent preheating and thus, the efficiency can be


Figure 3. Modular oven for high-temperature HPLC based on contact heating.


maintained. However, additional requirements must be met if the oven is used in temperature programmed mode. While air-based ovens might be used successfully for isothermal separations, block heating ovens have a much better heat transfer [19]


. In some cases it might be


necessary to apply fast temperature ramps of about 20 to 30 °C/min which cannot be achieved with air-based heating systems. An oven which is based on contact heating has recently been introduced to the market and offers the full capability of temperature programming, also including a fast cool- down of the mobile phase after a temperature programme (HT-HPLC 200, SIM Scientific Instruments Manufacturer, see Figure 3). The system is modular and allows for an independent adjustment of the preheating and the column temperature as well as a post column cooling of the eluent. This guarantees that the sensitivity is not compromised if a detector is used which is very sensitive to changes of the eluent’s temperature.


A last word should be addressed to the HPLC system.


In principal, any HPLC system can be


used for high-temperature operation. The only modification is that a back-pressure regulator should be installed behind the column to keep the mobile phase in the liquid state. The highest vapour pressure is observed for the water-methanol system and is around 40 bar when pure methanol is used at 200 °C[20]


.


Figure 2. Chromatogram of the separation of a sulfonamide mixture and trimethoprim using a simultaneous solvent and temperature gradient on a Zorbax StableBond C-18 column. For experimental information, see reference [1]


.


Conclusion It can be concluded that temperature has a huge influence on all chromatographic parameters which are used to optimize a


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