I use biosep SEC s2000 column which has a column volume of 15 ml a peak is observed at 47 min in all runs, even when I pass BSA through the column. I have tried washing the column with 0.1 M sodium phosphate buffer, pH 3.0 and and 0.5% SDS follwed by an extendes water wash. However, the peak still appears at 47.min. How do I proceed?
You can not use phosphate buffer at pH 3 . For pH 3 you most use another buffers like acetate or citrate buffers .Try to change the buffer and try to add NaCl to it .you can add 0.2 M NaCl.
Follow the manufacturer's procedure for cleaning the column. This may involve acid and base washes.
The peak may not be protein. Try collecting the fraction, concentrating it, and testing for protein.
Definitely check the manufacturers recommendation, as your resin is silica-based. It will likely not like basic buffers.
Check if you can use 6M urea or 6M guanidine.HCl.
Thank you for the response. I tried wahing the column with 30 ml (2 column volumes) 6 M guanidine thyocyanate, while injecting it too, as per the manufacturers' instructions. Now the profile shows many peaks at random. Could it mean the injection loop/port is contaminated?If so, how do I flush it?
You should purge injector, at first, using 50 ml of solvent. Then regenerate column according to manufacturers recommendation. This peak may be buffer of protein ( for example, glicerol or glutathione). i had the same problem and i didn't consider such non-specific interaction in column
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