hello

i was wondering if you can tell me what i can use to remove BSA or other proteins from a reverse phase symmetry C18 4.6x75mm column, 3.5um. i am looking for other answers besides dmso washing. i currently was my column with 100% ACN. there seems to be impurities coming out at the end of the chomatogram and are repetitive in multiple samples of differing purity. these impurities do wash away eventually over time. I would like to find an instant wash protocol to use right after every injection to completely remove these protein impurities.

thanks

i donīt work with proteins, but a friend of mine yess. I think that sds helps to solubilizate them ; AcN helps to precipitation.Ask to someone else about SDS...

SDS can also precipitate proteins, depending on the concentration and maybe other factors. Chaotropic substances, like 4-6M urea, are generally better. I have mentioned a "hammer" in the form of lithium dodecylsulfate + dithiothreitol, but especially the latter takes a long time to get rid of again. Others have mentioned some possibly useful methods above.
Ah, one more thing, even protein precipitants like MeOH, ACN,.... can, apparently, get rid of SMALL amounts of proteins, it takes time though (monitor it with a UV detector). But donīt do this if you have a large amount of protein on the column!

To get rid of proteins, it is best to do what protein chemists do when they inject proteins onto a C18 column: a few gradients (5 minutes) of 10% acetonitrile to 90% acetonitrile with 0.1% TFA in the water and the acetonitrile. BSA elutes with little difficulties (some residual BSA can be found in blank runs after the first injection, so I expect that you will need to run several gradients until your column is clean).

Uwe, your suggestion only works for proteins that chromatograph, or nearly do so, under these conditions. I should have mentioned that above when mentioning cleaning with MeOH, ACN.

Hans

I agree with you, Hans. BSA is a simple, run-of-the-mill protein though. However, as I said before, some BSA can be found in blank runs after the first run. This phenomenon has been attributed to folding, and unfolding. Don't know if this is correct, but it does come off the column in multiple runs.

You can run gradients from H3PO4 25mM/water to 25mM H3PO4/2-propanol, until BSA is not present. During the run pressure will increase due to the viscosity of 2-propanol, run at low flow rate and increase temperature if possible. Do not use TFA, it increases the retention. Inject at the beginning of the running 200-500ul GuHCl sat/CH3COOH 70% to facilitate the elution of BSA during the gradient. Why you use a so hydrophobic stationary phase for BSA ? Can't you use RPC4? It is more adequate. C18 is used for peptides.