-    If you plan to inject more than 50 µL,  put some buffer in a glass vial and load it into the autosampler of the HPLC system

-   Stop the HPLC pump, and connect your buffer bottle to the SEC system (default is Port A).

-   If you plan to inject more than 50 µL, connect the external valve. Install an appropriate loop (either your own or the beamline's).
 
-    Choose the flow rate depending on the column (e.g. Superpose 6 increase 3.2/100 with  a flow of 0.1 mL/min) and define the maximum pressure for the column. 
 
-    Flush the pumps “auto-purge” tool. Pumping will continue after the auto-purge at the set flow rate. Note the back-pressure and adjust maximum pressure and/or flow rate as necessary.
 
-    Wait until the system is in your buffer. For most buffers, the conductivity  (black line in LC tab) is clearly different from water, so this provides a good method to confirm that the system is in buffer.
 
-  Connect your column:

  • For small injection volumes (up to 50 µL), the inlet of the column gets connected to the tubing coming from the autosampler, the outlet of the column goes to the UV-detection. This implies that the column will be mounted with the inlet below the outlet. Be careful that you can still open the door of the autosampler.
  • If you want to inject more then 50 µL, the inlet of the column gets connected to the tubing coming from the external valve, the outlet of the column goes to the UV-detection.

 
- Based on the flow rate and column volume, calculate the time necessary for the experiment in minutes. Change the "End Time" settting accordingly. Press "Apply to all acquisition times" to ensure that all LC detectors collect the complete run.

- Load sample:

  • For small injection volumes (up to 50 µL), put your sample in a glass vial and place it into the auto-injector of the HPLC system
  • If you want to inject more then 50 µL, move the external injection valve to the "LOAD" position by entering the command "hplc_load" in the spec OH session running on nela. Load your sample with an appropriate syringe.