1. Check the following:

   a. The piston seal rinsing system should
      be full of rinsing solution (20%
      ethanol). If not, look for the
      instructions of how to fill it in the ring
      binder called “Components User
      Manuals and Instructions” section 1
      (Pump P-900) page 6.

   b. The flow restrictor that is mounted is:
         FR-904     for    high    performance
         columns FR-902 for low pressure

   c. The On-line filter that is mounted is:
         Titanium for organic solvents
         Glassfibre for everything else.
                                                          to the water-
   d. If you are going to run reverse phase               vacuum.
      columns, the waste bottle should be
      emptied so that no organic/TFA waste
      is mixed with aqueous waste from
      other runs.

   e. All your liquids have been degassed.
      Stir them for 20-30 minutes while

2. Switch on the Äkta and then the computer and log on to Unicorn.

3. Purge the pumps. Pumps should NEVER be run with air in the tubing

      I.     Immerse the inlet tubing in the bottles with the solutions
             that you are going to use:
             a. Use A1 and B1 for reverse phase. A1 for water/TFA and
                B1 for MeCN/TFA

             b. Use A2 and B2 for other buffers. B2 has, if not changed,
                20% ethanol.

     IMPORTANT: Reservoir bottles must always be placed above the
     level of the pumps.

     II.     Top the bottles with Nescofilm

      III.   The default values for the pumps are A1 and B1. If you are
             NOT running reverse phase you have to change the
             Flowpath to A2 and B2:

             From the System Control window, choose Manual in the
             toolbar and choose Flowpath in the dropdown menu.
             Click on PumpAinlet . Choose A2 and click on Execute.
             Repeat for PumpB.

     IMPORTANT: Every time the button END is clicked, the pumps go
     back to A1 and B1. Make sure you change them back to A2 and
     B2 before starting pumping again.

     IV.     Connect a 50-ml syringe to the LEFT purge valve of the LEFT
             pump (PumpA). Turn the valve counterclockwise half a
           turn and draw eluent to the syringe. Continue drawing
           eluent until there are no bubbles in the inlet tubing.
   V.   Connect the syringe to the LEFT purge valve of the RIGHT
        pump (PumpB) and repeat the purging.

4. Choose the sample loop that you are going to use and replace the
   loop that is currently connected. Screw it to ports 2 and 6 on the
   injector valve. There must always be a loop connected.

5. From the System Control window, choose Manual in the toolbar and
   select Pump in the dropdown list. Type a flow rate of 5 ml/min. Allow
   all the tubing to be filled with eluent, in order to remove air and old
   eluent that could have been trapped inside. Click on Flowpath and
   then on Valve, select Inject in the dropdown list, this will get rid of
   the air trapped inside the sample loop. Pump until no air remains in
   the system.

6. Decrease the flow rate to 1 ml/min. Connect the inlet of the column
   to the tubing, open the outlet of the column and connect it to the
   UV cell. The system is ready to use.

NOTE: The default set of the pressure alarm is 1.0 MPa. If you are
working with a column that can support more pressure than this,
change the value before starting to pump.


1. Loosen the injection fill port
2. Insert the injection needle and tighten the port.
3. Make sure the valve is set in the Load position.

DO NOT take the syringe away before the sample is injected, this might
empty the loop.
DO use at least 5 times the volume of the sample loop to flush the loop
in your programmed methods.

Partial filling
It is the method to use when you don’t want to waste any sample.

                   1.   The flow must be off.
                   2.   Inject 5 loop volumes of buffer into the loop.
                   3.   Set the injection valve to the inject position.
                   4.   Take the syringe out and load it with the sample.
                   5.   Insert the syringe and set the injection valve to
                        the load position.
                  6. Load the sample. At maximum, 50% of the loop
                     should be filled.
                  7. LEAVE the syringe in the port until the sample is

Complete filling
If what you need is to make sure you are loading the same volume of
sample each time, this is the method to choose. You need at least 2
times the volume of the sample loop, idealy 5 volumes should be used.

                  1. Load the syringe with 2-5 times the volume of the
                  2. Insert the syringe into the port and load the loop.
                  3. LEAVE the syringe in the port until the sample is


Take into account that the UV lamp suffers both for being turn off and
from remaining running for a long time. If the Äkta is going to be used
the next day DO NOT SWITCH IT OFF. If it is going to remain idle for more
than two days, flush it with water and then fill the tubing with 20%
ethanol before turning it off.


1. The Äkta itself generates a considerable back pressure. If the
   pressure displayed in the pump is bigger than the recommended
   maximum pressure for your column, proceed as following:

            1.    Set the injector to the waste position.
            2.    Start running the system, WITHOUT the column, at the
                  flow rate you intend to use in your runs. Take note of
                  the back pressure generated.
            3.    The actual value for the maximum pressure for the
                  column is the sum of the manufacturer’s maximum
                  pressure plus the pressure obtained above. Update
                  the column specifications in your Methods

2. If you are generating TFA/organic waste, make sure to pour the
   waste into the blue container at the end of the day. DO NOT leave
organic/TFA waste in the waste bottle, because the next user will
assume that the waste is aqueous and pour it down the sink.

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