Chromatography Forum

Hi folks,
I am working on a protein adduct characterization project. The RP HPLC seems straight forward to isolate the protein of my interest after the protein was alkylated. However, I found out that the adduct is not stable under acidic condition. (The mobile phase I am using right now is Water/MeCN with 0.1% TFA.) If I replaced 0.1%TFA with 0.1 formic acid, most proteins was stuck on the column. Therefore I wonder if there is any neutral solvent system for this kind of work.

Thanks.

I will recommend HIC.
Anion exchange would be a feasible option as well.

Best Regards

There are numerous examples where people played with different organic modifyers, chaotropes, detergents.

If you must use reversed-phase chromatography, then try running a gradient of acetonitrile in 10 mM ammonium or sodium phosphate, pH 7.0, containing 100 mM NaClO4. Chaotropes are necessary for really good RPC, so if you're not going to use an unbuffered acid for the purpose, then NaClO4 works nicely. Ref: D. Guo et al., J. Chromatogr. 359 (1986) 499. Incidentally, do NOT use K2HPO4 as the buffering salt. KClO4 is virtually insoluble, and you'll lose your potassium ions through precipitation as the perchlorate salt.

Why do you have to use RPC instead of HIC or ion-exchange, both of which are generally regarded as being more suitable for applications involving intact proteins? Whatever you use, get a material with wide pores (say, 1000 Ã…).