Chromatography Forum

Hello,

I am currently working on an SEC method for a protein that will have Pluronic F68 (about 0.01% which is above the CMC) in the sample buffer. I think that the detergent is causing over pressure issues on the system over time. I am currently using Agilent Bio SEC-5 columns which worked great before the pluronic was added. Does anyone have a suggestion of an SEC column that will not deteriorate as quickly in the presence of pluronic? We are currently only getting about 50 injections from one column.

Thanks a lot!!

what is your mobile phase?
i saw an ESA application on this pluronic F68. it was dissolved in THF
is the pluronic added to the sample in solution simply or is it connected to the protein
if only in the sample solution then you might need to consider sample clean up.
maybe like for triton-X this stuff will actually bind to a PVDF filter and be absorbed at 100% or close,making it easy for you.

Hello,

I am currently working on an SEC method for a protein that will have Pluronic F68 (about 0.01% which is above the CMC) in the sample buffer. I think that the detergent is causing over pressure issues on the system over time. I am currently using Agilent Bio SEC-5 columns which worked great before the pluronic was added. Does anyone have a suggestion of an SEC column that will not deteriorate as quickly in the presence of pluronic? We are currently only getting about 50 injections from one column.

Thanks a lot!!

I think it is because of the presence of pluronic. Why not trying some special surface coated columns? Either http://www.sepax-tech.com/Zenix-C.php or http://www.sepax-tech.com/Nanofilm.php may let you get more injections.

Hi Ting,

Actually, I already tried the Sepax SRT 1000 column and still had an over pressure issue. Does Sepax have any other columns that I might want to try?

Thanks unmgvar for the suggestion.. I will try the PVDF filter and let you know how it goes.

Hi Ting,

Actually, I already tried the Sepax SRT 1000 column and still had an over pressure issue. Does Sepax have any other columns that I might want to try?

Thanks unmgvar for the suggestion.. I will try the PVDF filter and let you know how it goes.

Yes, SRT-C may be more suitable to try. The 1000A is quit large pore size, I have to double check if we can customize this for you. You can contact your local sales rep or me about this issue you mentioned, so we can have our application scientists to work with you.

PVDF may also absorb your protein - it is used for Western-blotting for this particular reason after all. Ion exchange should allow you to get rid of Pluronic (it's non-ionic and would elute, while protein can be captured).

(edit: clarification)

PVDF and PES are used for filtration for HPLC actually because they are low binding for proteins

PTFE and Nylon are the best to catch proteins in those cases, they stick to the filters like crazy
but then again not all filter are equal, we do not use Pall PVDFs for this reason, they have a very bad recovery of proteins

I was bit surprised by what you wrote and did little search. Yes, it seems PVDF filters are prepared to have low protein binding, while it's completely opposite for PVDF membranes.

Is it possible to elute protein from Nylon or PTFE filter? After all a17thony wants to keep his protein for SEC and get rid of pluronic.