Obstruction Superdex 200 in FPLC system
Posted: Sun May 21, 2017 1:30 pm
Good morning,
I have been using a Superdex 200 in the last step of protein purification. I use a very old fplc system (Pharmacia Biotech). After 8 injections of 1 mililiter (10mg protein) the pressure was high even at low flow rate. I tryed to clean the column inverting it and following the cleaning in place protocol from GE Healthcare user manual: 25ml 0,5M sodium hydroxide at a flow rate of 0,5ml/min. I charged an old 10ml superloop to inject. As the pressure was high I had to lower the flow rate to 0,1ml/min. I suspect that the hydroxide degraded part of the loop and all that entered the column as I observe the pressure does not lower. I washed with 3 column volumes of MiliQ water. Pressure its still very high, reaching the max permited at a flow rate of 0,1ml/min.
Please, what do you recommend doing? We cannot afford buying a new one.
Thank you very much in advance for your help.
Luciana from Argentina
I have been using a Superdex 200 in the last step of protein purification. I use a very old fplc system (Pharmacia Biotech). After 8 injections of 1 mililiter (10mg protein) the pressure was high even at low flow rate. I tryed to clean the column inverting it and following the cleaning in place protocol from GE Healthcare user manual: 25ml 0,5M sodium hydroxide at a flow rate of 0,5ml/min. I charged an old 10ml superloop to inject. As the pressure was high I had to lower the flow rate to 0,1ml/min. I suspect that the hydroxide degraded part of the loop and all that entered the column as I observe the pressure does not lower. I washed with 3 column volumes of MiliQ water. Pressure its still very high, reaching the max permited at a flow rate of 0,1ml/min.
Please, what do you recommend doing? We cannot afford buying a new one.
Thank you very much in advance for your help.
Luciana from Argentina