Chromatography Forum

I hope someone else has had this problem because it has me baffeled. I'm running an Amino Acid method similar to Agilent's eclipse column method for AA. There is an unknown peak eluting at about the same time as the USP standard Tryptophan amino acid. Originally this peak was so small that it appeared to be a part of or shoulder to the TRP peak. In the past 9 months it has appeared as a significant peak almost non-distinguishable from the TRP. Peak height at about 20mAU , consistent and reproducible. I have replaced just about everything on the system and it is still there. I've flushed till the cows come home with IPA and Acetonitrile/water/MEOH.
Agilent also recommended I flugh with a mild nitric acid solution. I'm reluctant to do this. Has anyone any suggestions?
C. B.

Do you see the peak only when you inject your sample or during blank injections as well?

Thank You Kastas for responding. I see this unknown peak with the blank (water) injections as well as the sample and standard.

In this case I suggest some contaminant from your reagent/solvent or container (vial/plate or whatelse).

Thank You Samuele. I have changed the buffer several times and reagents except the borate buffer. That is my next try. I've switched to another HPLC to see if it is the system. No result yet. Perhaps tonight.
I appreciate all your input.

i would try an injection of the mobile phase alone, this should give some indication as to whether the 'peak' is originating from the actual system or your blank/std diluent.

Greg

Try different equilibration times... if the peak area increases (almost lineary) with increasing equilibration times then you do have an impurity in your mobile phase or HPLC system...