Gel filtration Chromatography - (Apr/04/2011 )
I am doing gel filtration chromatography(GFC) to seperate a 12 kDa protein and its dimer approx 24 kDa. I am using Sephadex G75 resin from GE Healthcare. I have packed my column and started running it using gravity flow(as we donot have an automated system like FLPC). I find that the volume of bed changes from 46 cm to 44cm if I keep the flow rate 1ml/min and the volume gets back to its original volume as I stop the flow. I thought may be the flow rate was very high thats why it got compressed. Even with 0.75 ml/min i find the same thing happening again. I calculated the Void volume using blue dextran which came with the LMW calibration kit, with 1ml/ min Vo=26 ml while with 0.75ml/min Vo=27.5 ml. However in both the experiments only flow rate was changed. I did not repack the column.
Why is my gel bed getting compressed is it beacuse of the flow rate. Is it normal? Or should I reduce the flow rate further.(As I can see the literature people go with a flow rate of 1ml/min and i am working in the same range.)
I understand that void volume would change every time you pack a new column but if the same packed column is used then does Vo change with the flow rate?
Any help would be appreciated. My questions might sound naive... as i am new to GFC and there is nobody around my place to help me with this...
are you pumping your column (peristaltic pump)? if so are you pumping at the inlet or outlet?
most gel filtration matrices will compress under flow, especially if pumped.
after packing and prior to running a sample the column should be thoroughly equilibrated under the same conditions under which it will be run.
if not pumped then you should use a mariotted buffer well (see this wikipedia page: mariotte bottle) to ensure a constant pressure on the system for the entire run.
in case you don't already have it i am attaching the gel filtration handbook from ge healthcare.