HOW TO USE THE ÄKTA AND KEEP IT HAPPY BEFORE CONECTING YOUR COLUMN 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 columns. c. The On-line filter that is mounted is: Titanium for organic solvents Glassfibre for everything else. connected to the water- driven 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. INJECTION OF THE SAMPLE 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 injected. 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 loop. 2. Insert the syringe into the port and load the loop. 3. LEAVE the syringe in the port until the sample is injected. STORAGE 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. OTHERS 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 accordingly. 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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