Retention time precision is extremely important in being able to accurately compare results. Traditionally, retention time precision was always attributed to flow characteristics, such as consistency in flow rate and gradient precision, in an LC system. However, with UHPLC a point has now been reached where other factors, such as pressure and speed, play major roles. At injection the sample is pressurized from one bar to pressures above 1000 bar with the switch of a valve and peak widths can be so narrow that the data collection rate can become insufficient to accurately profile the peak. Finally, there are issues with gradient formation at pressures above a 1000 bar. In this work, we will look at all the aspects of a UHPLC system and discuss how chosen parameters can influence retention time.

　　We will show that more factors are important than just pump flow and gradient stability, and that it is possible to generate very accurate retention time precision at the top of the UHPLC operating range.