After a series of 6-7 purifications in SuperdexTM 200 increase 10/300 column, its pressure has increased to 1.08MPa even at a very low flow rate of 0.1ml/min. And I could see collapsing of the gel bed as well. I usually run 0.5M NaOH in a reverse flow during regular cleaning. But now even that is not possible because of the high pressure. I feel like there is some aggregate stuck inside the column and its causing this high pressure and its not coming off in NaOH run also. Is there anyway they can be dissolved and get the column pressure back to normal?
According to GE, you may try with 0,5 M of acetic acid, or you may try adding some detergents. Otherwise, try 0.2 M HCl, 70% acetonitrile or 50% isopropanol
or 6M Guanidine HCl with 0.2M Acetic acid. There are people that combine acetic acid with NaCl and pepsin too, so up to you.
Reverse column and inject 1 mg/ml pepsin in 0.1 M acetic acid, and 0.5 M NaCl and leave overnight. Next day just wash it with water. Again wash with 0.5 M acetic acid in reverse flow, then 5-10 cv water and store it in ethanol. It will be okay to use
I would suggest trying to replace the top filter - you could have some material stuck on there that is impeding flow and increasing back pressure. Cytiva sell filter replacement kits. There are some guidelines on column cleaning and filter replacement here: https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=17491
Dear Sreelaksmi
I thin that as already suggested from Mahesh Chandak and Roger Bradley Dodd
you can try to:
wash the coloumn in the opposite direction, also with buffer, NaoH or urea and replacing the top filter that is prone to be blocked expecially in case you do not filter the samples and buffer at 0.22uM prior loading in the coloumn.
ciao
Manuele
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