As there is continued interest in running AccelaTM High Speed LC as a stand alone LC (either conventional or U-HPLC), some users may be working with samples at very sensitive conditions. For these users, minimizing baseline noise is very important. In such instances, we have found a number of measures that are good operating practices and are described in more detail below:

1. Software 

            a. If using ChromQuest 4.2 ensure the Hot Fix described in FAST 2007.043 has been

                installed.

      2.    Accela PDA Detector

             a. For high speed/high pressure applications the 1 cm, 2 μL LightPipe^TM Flowcell must be

                 used to ensure optimum peak shape.

             b.  Install a 100 psi back pressure regulator on the outlet tubing of the PDA detector. This

                 reduces the out-gassing effects eliminating the occurrence of bubbles in the flow cell

                 and is recommended for high pressure applications.

           c.  If there are already bubbles in the flowcell, then follow this procedure for "burping" the

                 flowcell:

                 i.  Pump 100% methanol through the system for five minutes at 1mL/min.

                 ii.  After the five minutes have passed and the pump is still pumping, place your

                      thumb over the end of the outlet tubing of the PDA detector and hold for three

                      seconds then release. Repeat the hold and release procedure two more times.

      3.   Accela Pump Pulse Dampener

              a.   Purge the pulse dampener because there could be a bubble in the pulse dampener

                    tube. The pulse dampener should be purged every 30 days and filled with Isopropyl

                    Alcohol (IPA).

                      i.   Ensure that the connection from the pump outlet (3) to the autosampler

                           injection valve inlet is connected.

                      ii.  Connect a syringe to the pump purge outlet (1)

                     iii.  Connect a syringe to the pulse dampener flush (2)

                     iv.  Open the pump purge outlet (1)

                     v.   Start pumping 100% IPA at 1mL/min.

                    vi.  Wait five minutes

                    vii.  Open the pulse dampener flush (2)

                   viii.  Close the pump purge outlet (1)

                    ix.   Wait until air bubbles are no longer visible (at least 5 minutes)

                     x.   Close the pulse dampener flush (2) and open the pump purge outlet (1)

                     xi.  Wait five minutes

                     xii.  Close the pump purge outlet (1)

      4. Additional Mixing

             a. For complex gradients such as those using formic acid it may be required to have

                 additional mixing capacity. Below are a list of mixers you can choose from:

                        i. 25 μL in-line static mixer P/N 00109-99-00031

                       ii.  50 μL in-line static mixer P/N 00109-99-00032

                       iii. 150 μL in-line static mixer P/N 00109-99-00033

For other great technical tips and helpful information designed to assist you in optimizing your LC methods, visit our Chromatography Resource Center.