Column Pressure too high

[Nofertari]

Unfortunately i cant change column temperature on this machine (((

[ksharp]

I would guess that since it happened right away, that you've crudded up the head of the column.

It probably isn't a lipid problem (your wash cycle would have taken care of that) but more likely you've got proteins and other bio-junk strongly absorbed on the column. You probably need to flush the column with detergents, try SDS or a denaturant like guanidine thiocyanate.

You should re-consider your sample preparation stage - maybe try 0.2um filtration instead of 2um. If the 5um column worked OK then maybe switching to the 3um is like asking for trouble.