Can you try fulvic acid

Perform a run with 100% acetonitrile in 2.5% acetic acid solution at 40 ° C

Make a run at 1ml per minute with 100% acetonitrile (in 2.5% acetic acid solution) at 40 ° C

Hi Nadia,

one easy way is to look up the website of the company that supplied the column. They will have the recommended regeneration procedure. The exact method may vary with column used. I think you'll find things like isopropanol are a popular method given it's very high efficiency at removing peptides from C18. It is very viscous, so you will see an increase in back pressure.

Acetonitrile and methanol may not work alone since they may not dissolve peptides and protein. A mixtures of organic solvents with buffer, acids, and sometimes, ion-pairing reagents can be effective.Aqueous trifluoroacetic acid and trifluoroacetic

acid–propanol can regenerate contaminated columns.However, consult the column manual or the manufacturer to ensure that these solvents are compatible with the packing material.

If you have a quternary solvent system run through a range of water miscible solvents all containing say 15% water like methanol to acetonitrile, isopropanol and finally a mix of water:IPA and as much butanol as to maintains miscibility in the mixture. You will have to have either acid present (formic or acetic) or, since you have already loaded your column up with ion paring reagent, then you might stick with IP. It may be possible to extract more sensitivity if you remove the IP from your method, though for the chromatography to be reproducible you may need to start with a fresh column that has not been exposed to IP reagents. IP reagents are notoriously difficult to remove from a RP column once exposed to it!! So retention times will vary from a non-IP method. Good luck!

It is sometimes very hard, if not imposible, to remove proteins from a C18 column. Many proteins are very hydrophobic in nature and can litarally stick to your column. Gradually flushing you column with methanol or acetonitrile might not remove the proteins from the column. Organic solvents can often act as denaturants. In this case the hydrophobicity of the denatured protein usually increases and as a consequence it can stick to the column even stronger.

An elegant solution could maybe be the use of hyperbranched monolithic columns. If I am not mistaken these could be flushed with a NaOH solution, which should remove (hydrolize) any protein from the column.

Thanks a lot for all the answers, what I can say for now from my experience:

organic solvens like AcN or iPrOH do not work alone 'cause (I believe) they are useless in dissolving proteins.

The thing that worked previously was the AcN gradient in trifluoroacetic acid (as indicated in the manual proposed by Shulamit, thank you!), but I ran out of TFA and I'm not sure it does not affect the column effectivity.

I also tried flushing the column with 6M urea, but that was something crucial because it got stuck deadly with literally no way of flushing it even with water or so, and I had to buy a new one.

Would it help if I only injected the loop volume insted of flushing the column?

Thank you again for all the pieces of advice!

Just in case someone finds this thread many years after it was posted, as I have, I wanted to add one suggestion.

To try and regenerate a fouled RP HPLC column, you may find this short article, entitled "Notes on Cleaning bound Protein from RP HPLC columns" useful to solve the problem. Article Link: http://hplctips.blogspot.com/2016/10/notes-on-cleaning-bound-protein-from-rp.html

http://hplctips.blogspot.com/2016/10/notes-on-cleaning-bound-protein-from-rp.html

Dear Nadia Seniavina,

Go through according to Bill Letter suggestions. I believe it will works.