For the LC part of the instrument method, consider the following options:

  • Maintain the autosampler at a cool temperature, for example, at 6 °C. Enable temperature control for the column chamber. Set the column chamber thermostatting mode to StillAir and set the temperature to your preferred setting, for example, 25 °C.
  • NOTE

    Lower column temperatures tend to give higher reproducibility.

  • You can specify an equilibration stage before or after the run stage. The run stage begins at retention time RT = 0 min. If you equilibrate before the run, the RT to begin the equilibration stage must be the negative value equal to the equilibration duration.
  • For reducing carryover into the next injection, it is advised to us a double gradient in the run stage. A typical method run time would be, for example, 115 minutes, with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), provided at a fixed rate of 0.250 mL/min, with the gradient shown in the following figure.
LC double gradient
LC double gradient

For more details on how to set up a gradient for peptide separation and regarding the settings of your Vanquish UHPLC system, refer to the Chromeleon CDS Software User Help.