Hi,
I am wondering if anyone has any good suggestions for washing of a reversed phase HPLC column when doing intact protein analysis to reduce carry over between runs? I am currently using a diphenyl column, with water and acetonitrile with 0.1% formic acid as my respective mobile phases.
I have tried different procedures for cleaning but the best is so far just to run the gradient program a few times (even so it is difficult to lose the last few percent of a previously injected sample).
I would appreciate any helpful suggestions. I am curious if anyone is using any washing mixtures. I have played around with DMSO/acetonitrile mixtures but have so far not seen any positive effect of these (washing by doing an injection of the mixture following sample injection). I realize that proteins are notoriously sticky and assume that carry-over is one of the problems everyone who is analyzing intact proteins are facing.
This is the first time I am asking a question on Research Gate so I would be happy with any response :)
Best regards,
Henrik Carlsson
Hi there,
I would suggest high concentration of chaotropic agents like urea (up to 8M) or guanidinium chloride (up to 6M) to solubilize sticky protein contaminants.
Thanks for your replies. These suggestions (mostly) involve pumping of a solution through the column. It would be more practical to do an injection of a "cleaning solution" while running the same gradient program as for samples (with the same mobile phases). The article recommended by Dr Ghosh above suggests a plug injection of trifluoroethanol. Do anyone have any other suggestions for different solvents/solvent mixtures?
Dominique, your answer is excellent. However, I would not advice this as we do not know the user and he might damage the column at the end of the washing. Your method is the classical one but then it should be carried out by experienced handler only and not by any "Tom-Dick and Harry".
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