Hydrophilic interaction liquid chromatography, or HILIC, is a powerful mode of liquid chromatography (LC) that uses polar stationary phases and high-organic initial mobile phase conditions for the separation of polar analytes. Because of the highly organic initial conditions, it is important to condition the instrument and column to get the best performance. We’ll look at how to do that in this video.
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So, now you get a new HILIC column and are very eager to develop or run a HILIC analysis. What should you do at the very beginning?
As we discussed previously, for HILIC-based LC separation, the initial condition is under highly organic mobile phase to generate a water-rich layer on the surface of the polar stationary phase. Therefore it is necessary to condition and then equilibrate the column between runs to fully reestablish the water layer on the particle surface. When you start using a new HILIC column or change the mobile phase composition, we recommend to flush the column with at least 50 column volumes of the mobile phase. In addition, at least 10 blank injections running the full-time elution program should be performed when you start a new batch run. As for the equilibration time between injections, this could be very analyte dependent, so make sure to observe the retention time reproducibility to determine the time needed for re-equilibration.
Now, other than the column itself, we want to remind you that your LC systems has to be conditioned for HILIC analysis as well. We know most commonly LC instruments are used for reversed-phase LC analysis. So before switching to the HILIC analysis, make sure to flush the system with high organic phase for a period of 30 to 60 minutes. Now that you’ve conditioned your column and your system for HILIC analysis, you’re ready to go.
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