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Why the column pressure of Superdex 75 10/300 GL has increased to...
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+ Column chromatography
+ Protein purification
+ Size exclusion chromatography
+ Liquid chromatography
+ High pressure
+ Gel filtration chromatography
+ Chemistry
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LaToya Boggans
Why the column pressure of Superdex 75 10/300 GL has increased to...
Hi, your problem might to be related to some residuals accumulated in the column run after run, and not only in the filter. This is a common thing, impossible to avoid it with classic cleaning procedure used in the labs. I normally avoid/solve this issue by using a service provided by a company that is specialised in recovering and regenerating chromatography columns. In our case I can say that the service was wonderful and the cost was fair. Please, have a look in this website: http://www.regenfix.eu/. There is everything written there and there their e-mail address and an easy contact form to ask for a quote. In the lab we regenerate all columns once of twice per year using this ReGenFix, and they are never getting stuck anymore. Furthermore, this regeneration service not only cleans the column, but they also provide the calibration, which is a very important thing for standardisation of SEC chromatography. I hope this hint will help you!
Hi, your problem might to be related to some residuals accumulated in the column run after run, and not only in the filter. This is a common thing, impossible to avoid it with classic cleaning procedure used in the labs. I normally avoid/solve this issue by using a service provided by a company that is specialised in recovering and regenerating chromatography columns. In our case I can say that the service was wonderful and the cost was fair. Please, have a look in this website: http://www.regenfix.eu/. There is everything written there and there their e-mail address and an easy contact form to ask for a quote. In the lab we regenerate all columns once of twice per year using this ReGenFix, and they are never getting stuck anymore. Furthermore, this regeneration service not only cleans the column, but they also provide the calibration, which is a very important thing for standardisation of SEC chromatography. I hope this hint will help you!
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.
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.
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.
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.
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
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
Hi, your problem might to be related to some residuals accumulated in the column run after run, and not only in the filter. This is a common thing, impossible to avoid it with classic cleaning procedure used in the labs. I normally avoid/solve this issue by using a service provided by a company that is specialised in recovering and regenerating chromatography columns. In our case I can say that the service was wonderful and the cost was fair. Please, have a look in this website: http://www.regenfix.eu/. There is everything written there and there their e-mail address and an easy contact form to ask for a quote.
In the lab we regenerate all columns once of twice per year using this ReGenFix, and they are never getting stuck anymore. Furthermore, this regeneration service not only cleans the column, but they also provide the calibration, which is a very important thing for standardisation of SEC chromatography.
I hope this hint will help you!
Hi, your problem might to be related to some residuals accumulated in the column run after run, and not only in the filter. This is a common thing, impossible to avoid it with classic cleaning procedure used in the labs. I normally avoid/solve this issue by using a service provided by a company that is specialised in recovering and regenerating chromatography columns. In our case I can say that the service was wonderful and the cost was fair. Please, have a look in this website: http://www.regenfix.eu/. There is everything written there and there their e-mail address and an easy contact form to ask for a quote.
In the lab we regenerate all columns once of twice per year using this ReGenFix, and they are never getting stuck anymore. Furthermore, this regeneration service not only cleans the column, but they also provide the calibration, which is a very important thing for standardisation of SEC chromatography.
I hope this hint will help you!
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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.
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.
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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.
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.
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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.
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.
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Excellent solution suggested by Engelbert.....
Excellent solution suggested by Engelbert.....
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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 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.
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Because the particles have swollen after treatment with strong alkali.
Because the particles have swollen after treatment with strong alkali.
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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.
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.
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Have you changed the inlet and outlet filters?
Have you changed the inlet and outlet filters?
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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 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.
More
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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
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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