Have been facing problem related to the column pressure from quite some time. Started the column clean-up procedure using 0.5M NaOH. Firstly, washed it twice the column volume with milliQ. Then, washed it with 0.5M NaOH. However, during this step, the pressure started increasing with time and reached to 1MPa at flow rate of 0.05ml/min. Before the washing, the column pressure of 1MPa used to be at 0.3ml/min. But, during normal course of work, after sample injection of the protein that I work upon (protein is involved in aggregation diseases), it used to follow the same pattern of high pressure even at low flow rate. Now, the column has been washed using 0.5M NaOH 1.5 times the column volume and now is being washed with milliQ. However, the column pressure is 1MPa at a flow rate of 0.05ml/min. Any suggestions on the issue are more than welcome.
You could repack the column. Get the gel out of the column, give it a good washing, remove any fines. Make sure the filters and lines are clean. Then pack the gel into the column again, following manufacturers instruction (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-gel-filtration.pdf). This will take a whole day and require a gel reservoire that matches the column.
Well just do one thing. Before loading the column with your proteins, make sure that the protein sample has totally solubilized in the buffer. Secondly, after washing and charging the column with alkali, have you noted the pH of the void volume (may be distilled water or buffer) which must be near neutral. If the protein is getting precipitated in the column then to maintain the flow rate, the pressure is bound to increase.
Because the particles have swollen after treatment with strong alkali.
Dear Omar Gonzalez-Ortega,
The online filter was cleaned recently through standard procedure of sonication. However, I am not sure about the outlet filters and their location.
Dear Jai Ghosh,
Yes, the pH value of the void volume was neutral. The pressure is now decreasing but slowly. I am waiting for it to come to the usual 1MPa at a flow rate of 0.3ml/min.
Dear Bruce Neagle,
Thank you for the information. Still, the pressure is 0.9MPa at flow rate of 0.02ml/min. I am waiting for it to get to the usual.
You could repack the column. Get the gel out of the column, give it a good washing, remove any fines. Make sure the filters and lines are clean. Then pack the gel into the column again, following manufacturers instruction (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-gel-filtration.pdf). This will take a whole day and require a gel reservoire that matches the column.
Hi Devanshu,
I had similar problems recently with my Superdex column. Before repacking the column, I would try changing the pre-column filter as has already been suggested (you can find documentation how to do this on the GE web page looking for Tricorn filter kits: https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=12716 ). For me, it also always helps washing the column in 0.5M acetic acid, additional to the wash in NaOH. It often also helps to run the column in the reverse direction for cleaning, which I have experienced will significantly reduce the back pressure.
Bernhard
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