problems in size sxclusion column

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sohaty
sohaty's picture
problems in size sxclusion column

hi everyone
any one can help me in the size exclusion HPLC column, i think my old one loses its effeciency because of backpressure and peak asymmetry, it also shows no positive peaks for my protein of interet, i only see the negative peak corrresponds to ligandboumnd to protein.
i am trying to improve it by washing with hot water around 50 celious, i am working on method optimization and this realy happend when i ues 100 mmolar buffer concentration.

Dr. Analytical
Dr. Analytical's picture
Dear Sohaty:

Dear Sohaty:

High backpressure usually means that you have contaminated the inlet frit at the entrance of the column.  Have you been filtering your samples and mobile phase?  If not, you must change your procedures to require filtering.

You can attempt to reverse the column and flush solvent into a waste container, starting at a low flow rate and increasing over 10 minutes.

You may also try a chemical cleaning.  Check with the column manufacturer for recommended procedures.  If you have been injecting proteins, then you may try TFA in water or 6 M guanidine.

If these procedures do not correct the problem, then you need a new column.