I accidentally compressed my Superdex 200 10/300 GL column with 3-MPa. Now I can see a small gap at the top of my column whenever I run above 1.0-MPa (recommend limit is 1.5-MPa). Has anybody got experience using a column after it has been compressed? I will test it with BSA but I am not sure if I should run below 1.0-MPa (a bit slow at ~0.3mL/min).
Thanks Agata. I tried that and I did get rid of the gap by reverse flow path, but as soon as I run at around 1.0-MPa (~0.3-mg/mL) the gap will appear again. I think that just because some of the beads were already crushed. I will try to run BSA at 0.3-mg/mL and hope the column still works well.
Consider inverting the column 180 degrees (e.g. the inlet is not the outlet) and running it ii the reverse direction for several runs at elevated flow rates - - it should not hurt the stationary phase
And you can run the column "upside down" forever more....the more compressed beads - now at the bottom - should have little to no impact on your separation.
I do have the problem which the pressure increase slightly over time. I will definitely try to reverse wash and see if it solve the problem.
As for using the column upside down, my colleague mentioned that the top side of the column has a better filter so it may not be ideal to run it upside down forever.
As Agata mentioned, there should be an adapter screw at the top of the column which you can tighten down to eliminate the gap you are observing. This will reduce sample mixing in that gap at the top prior to entering the gel matrix and maintain elution peak resolution.
Another thing worth trying is to take out the top adapter and replace the filter disc above the resin with a new one. GE sell an (overpriced, naturally) set of discs and a tool to remove the old one. This sometimes helps to get rid of the top of the gel bed compressing. Couple that with removing 1-2 mm of the top of the resin and you may rescue the column.
Thanks for the input everyone. I ended up running the column at 0.3-mL/min from now on to keep the pressure at ~1.0-MPa. Although I do still see a very small gap at the top of the column, I ran a BSA standard and the column seems to behave well and the elution profile did not changed because of the compression.
I will keep in mind for the suggestions to replacing the adapter, taking out some damaged resin and readjust the adapter screw. Hopefully I won't need to do this in the future.
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