The increasing need for higher speed and resolution in analytical separations drives the trend towards moving from HPLC (ca. 400 bar max. pressure) to UHPLC (>1000 bar).

This tutorial will cover the basic principles of UHPLC and discuss various aspects that need to be considered to successfully apply this technology in method development and routine work.

In the first section the basic theory of mass transfer in columns packed with different particle types will be discussed.   It will be demonstrated how the increase in operation pressure and temperature can result in enhanced resolution and/or speed of separation.

In the second section optimization strategies for UHPLC separations for different types of applications will be discussed using the concept of kinetic plots. The kinetic plot (also known as Poppe plot) model uses column efficiency along with column permeability to determine the number of plates generated per unit time. This is a much more useful measure for column performance than using column efficiency or plate number alone. The kinetic plot model will be explained in detail and examples for different particle sizes and morphologies will be shown.

The third section will focus on instrumental aspects of UHPLC such as dwell volumes and system dispersion, where the requirements are much more stringent as in HPLC instruments due to the smaller column diameters used in UHPLC. The impact of extra-column volume on the efficiency for small bore columns will be demonstrated for isocratic and gradient separations. Another topic is the transfer of methods between HPLC and UHPLC instruments where differences in system properties (dwell volume and mixing behavior) can lead to changes in retention times and selectivity.

Finally some of the side effects of high pressure separations will be discussed such as frictional heating, possible selectivity changes and changes of physical properties of eluents at high pressures.