6 Replies Latest reply on Sep 30, 2018 5:54 PM by soccerdad

    Switching from reversed phase to normal phase for a project

    soccerdad

      Hi there,

      We have a HP1100 system with binary pump that we use for RP separations frequently using 0.1% acetic acid in water and methanol as the mobile phases. We have been asked to do a normal phase separation with Isopropanol and hexane. I am looking for a good set of instructions as how to switch from RP to NP and then after the work is done back to RP. I know that everything has to be flushed with suitable intermediate solvents but has anyone seen a set of instructions for doing this as I am not sure what solvents to use and how long things need to be flushed. How is the sample line of the autosampler flushed as well?

       

      Hope that this makes sense

      Kevin

        • Re: Switching from reversed phase to normal phase for a project
          jhagel

          I would flush all channels with IPA between going between Normal and Reverse Phase.

          With reverse. phase solvents, make sure no buffer or additive in it before switching.

          Change out the pump seals for the normal phase seals pn 0905-1420... for both the G1311A and G1312A

          Then when you are done go back to the reverse phase seals pn 5063-6589

          If you have an autosampler, make sure you switch the valve to bypass while flushing.

           

           

           

           

           

           

            • Re: Switching from reversed phase to normal phase for a project
              soccerdad

              Thanks Jhagel and for especially mentioning the seals as I hadn't considered them. Are there any "rules" for how long to flush with the IPA each time.

               

              Kevin

                • Re: Switching from reversed phase to normal phase for a project
                  kbusch

                  Hey Kevin,

                   

                  For how long to flush with IPA: This depends on what type of degasser you have.

                  For G1322, the internal volume of each degasser channel is about 12 mL.

                  For G1379, the internal volume of each degasser channel is about 1 mL.

                  If no degasser, then I would still flush a few mL of IPA before switching.

                   

                  Make sure to do these volumes as an absolute minimum with IPA. Not sure why you would need to flush in bypass mode, as you should also make sure that whatever in the sample loop is flushed out. Maybe jhagel can comment to this.

                   

                  Also, when operating in normal phase mode, I would keep a small flow at all times, 0.1mL/min should be enough. This will prevent the valves for electrostatically charging up and thus sticking during normal phase runs. Another option is that we do have normal phase valves available now, but these cannot be used with acqueous solutions, they will be destroyed. The part numbers for the valves are: Inlet: G1312-60166, Outlet:G1312-60167. The valves have a different ball, which will not be electrostatically effected by solvents like hexane. But if you are only doing this for a short project and you take care, it should not be a problem.

                   

                   

                    • Re: Switching from reversed phase to normal phase for a project
                      rse_sara

                      I would agree with the bypass comment; definitely a good idea to have the autosampler in main pass during the flush as well. This way the whole sample flow path including needle, needle seat and sample loop will be flushed through. Another thing to note is that the normal phase seals are limited to 200 bar so I recommend putting a 200 bar pressure limit on any normal phase methods you'll be running. The autosampler metering head uses the same seals as in the pump so the one in there should also be swapped out for a normal phase seal. Finally, after analysis it is best to flush hexane out of the lines and let the HPLC sit in a solvent such as IPA. This is so that hexane vapours do not seep through the degasser tubing and settle in the vacuum chambers.