High temperature and temperature programming in high-performance liquid chromatography: Instrumental considerations

The successful application of high temperature and temperature programming in high-performance liquid chromatography (HPLC) requires the appropriate configuration of a chromatographic system and selection of proper column dimensions. Knowing the exact column temperature is important, especially if a developed method is to be transferred to another laboratory or is going to be performed on a chromatographic instrument with a different heating system. We have examined the suitability of several columns of different bore diameters in isothermal and temperature programming modes. The effect of different mobile phase flow rates on the overall system performance was also studied. Low volumetric flow rates, which generate high linear velocity in capillary and narrow-bore columns, allow fast heat equilibration between column wall and bulk liquid. The uniformity of the column heating, observed in the narrow-bore columns, is another characteristic that favors small diameter columns over the wide-bore columns. Mobile phase preheating significantly improves system performance, minimizing the effect of axial and radial temperature gradients on separation quality. The use of temperature programming in HPLC can be improved through the appropriate selection of the heating systems and by optimization of operating parameters, such as column dimensions and mobile phase flow rate.