Chromatography Forum

Hi all,

I don't think this is the typical ghost peak question. I'm evaluating nanogram quantities of a novel protein by RP-HPLC on a C8 column with a water/acetonitrile/0.1% TFA solvent system using UV detection. One day, after many injections of the same material, a very large peak showed up in a chromatogram. The peak remained in all subsequent sample and blank injections and was highly reproducible. After switching to a fresh (new) column, the extra peak will disappear but will eventually resurface. Once it shows up, the extra peak is there to stay (N=3 columns so far ). The phenomenon is completely two-state (i.e., it does not slowly build up, rather it suddenly shows up and once there, it's intensity is very reproducible).

Aside from the tiny amount of protein, the only other sample component is 0.2% (w/v) non-ionic detergent.

Have any of you ever experienced such a thing or have any possible insight into what is going on? TIA.

p.s. This board is a great resource. Kudos to all the participants.

This sounds like a late eluting peak problem.

Most likely the compound giving the extra peak is the detergent, but it has so strong binding to the stationary phase that it does not elute until after a veeery long time. Do you see the extra peak also when injecting a long series of blanks? You can try to raise the acetonitrile concentration and see if you can to wash out the stuff.

Thanks Tobias. I think you are right about the problem coming from the detergent. The extra peak comes off at approximately 85-90% MeCN so, yes, it binds the column strongly. I have not run a long series of blanks but will try that. I've been sidetracked by non-lab duties for the last several days but now I can get back to resolving the problem. Thanks for your help.