How long should I equilibrate my Raptor HILIC-Si column between injections?

In addition to the initial conditioning of the column with the mobile phase, it is critical that the column be re-equilibrated between injections. Because the separation mechanism in HILIC methods involves the adsorption of a water layer on the particle surface, it is very important to completely reestablish or “reset” this water layer in between injections to ensure retention time reproducibility. We recommend equilibration with a minimum of 10 column volumes starting when the gradient program returns to initial conditions, or in isocratic separations, at least 10 column volumes starting from the retention time of the last peak. The number of column volumes required for complete re-equilibration can be very analyte dependent, especially for isocratic separations, so make sure you investigate retention time reproducibility during HILIC method development.