Preparing FPLC

Follow these steps before starting your FPLC run. Please feel free to edit as you see fit.

Check column
If you need to change columns, make sure you perform a wet transfer

• While pumping mobile phase (ideally H2O), remove the bottom connection from the existing column (usually requires taking out the black connector first and then the brown connector, so that the tubes don't get too twisted)
• Wait for mobile phase to drip out of the bottom of the column to assure that the entire column is wet
• Cap the bottom of the existing column with a spring loaded cap
• Unscrew the top connector to the existing column and screw it into the new column
• Unscrew the bottom cap of the new column, wait to see mobile phase drip out, and then screw in the FPLC tube to the bottom of the new column
• Make sure the old column is capped on both sides before storing

Purge hoses

• In Inject Valve module, select the System Pump Waste option, so that mobile phase is going directly to waste
• In the System Pump module, set flow rate to 5mL/min and duration to 5min. Run 100% A and then 100% B for 5min each, isocratic

Purge pumps

• Set flow rate to 0.4mL/min, 100%B (if column is already in H2O) and duration to 15min. Make sure Column Switching Valve is in Bypass mode
• In the Inject Valve module, change to Manual Load Loop
• Make sure that the 20EtOH falcon tube on the right side of the FSEC has at least 10mL, otherwise add more filtered 20EtOH
• Unscrew pump A slightly so that liquid is dripping, then use the big plastic syringe to suck out 1-2mL. Repeat for pump B

Flush injection valve

• Fill 4 beakers: Waste, EtOH, H2O, Buffer
• Remove the syringe from the inject port and rinse 3x with EtOH and then 3x with H2O, injecting into waste each time
• Fill syringe with buffer, making sure to eject bubbles. Inject buffer into valve. Avoid injecting bubbles. Repeat this step again
• In Inject Valve module, change to System Pump Inject Loop. Switch Column Switching Valve to 2 (so that the mobile phase pumps through the column)

Collecting fractions: see this page by Chenlin - FPLC fraction collector

Begin equilibration -> sample application -> elution -> equilibration method

• When you start the method, check the box to clear fraction collection history just to make sure that you start collecting at A1
• Load sample, avoiding any bubbles, before the sample application module begins