Help with Superdex200 increase Column - (May/30/2015 )

Hello all, 

Our lab has an AKTA 25L which we have started to use for protein purification. Our FPLC is stored and used in our cold room. 

This our first time using our Superdex200 Increase 10/300 GL column, so we wanted to wash it. 

However, we had a hard time finding a suitable flow rate. In the end we found that we had to use a flow rate of 0.025mL/min. Is this a correct flow rate ? Or is this too slow? It basically took up 30 hours just to wash with 1 CV. Now we are worried just to do any run, it'll take us forever. 

Also, how do we know if our connectors are correct? There was a time we were able to do 5mL/min but there was solution coming out at the connectors that were leading to the column. Along with this, how tight do you know to tighten the connectors. We do finger tight, but we are worried this may be too loose/or too tight. 

Thanks for the help! 

Something is wrong with your setup...those are really slow flow rates. You should have a flow rate with that column of about a mL/min.

I hope you were not running 5 ml/min through that column...if that is the case, then you have likely crushed your resin beads, which would explain why you can't run anything through it anymore.Max flow rate for that column is 1.8 m/min. Always read the manufacturer's product handling instructions.  Assuming that is not the problem:

Has this column been used by anyone prior to you? If it is brand new out of the box, return it. If it has been used in the past, then likely it is plugged. You will need to locate the plug by disconnecting one junction at a time to see where the pressure builds up. Most likely it is the column itself, in which case:

Try running water through it backwards. Also try replacing the filter at the top of the column. If those don't work, then try some of the recommended cleaning in place procedures. 1 column volume 0.5M NaOH followed by several column volumes of water kicks a lot of stuff off. You could also run 8M urea through it, followed by water.

Always remember to filter all buffers that go onto the FPLC.

Connectors should be finger tight. Over tightening could cause them to break and pinch the connection. Under tightening is obvious to identify; they will leak of course.