Chromatography Forum

I have thought of another possibilty. If you use helium to degas it is vital to use the best grade, baloon gas will not do. I once had a problem with gas introducing an impurity into the mobile phase.

ARM, we still don´t know whether you are seeing a fluorescence or scattering.

Thank you guys for all your suggestions,sorry Ive been away for a couple of weeks...before I left I thought I had solved the problem but still not sure. I think the problem was the filtering device I was using,I filtered the water and buffer with a different filtering device from other lab and the peak went really small,although I couldnt get completely rid of it. It could be that some of the impurity is adsorbed irreversible in the column?To answer your questions, I dont use helium to degass,just vacuum while filtering and the HPLC has an inline degasser. The peak is fluorescent but the spectrum is different than one of my compounds of interest, althought they elute at the same time. It also gives a small signal in the UV. We did a preventative maintenance very recently but it didnt help. I clean the system thoroughly, with water and methanol. My colleagues are in the US and Im in the UK so its not possible to try their HPLC with my buffers. But Im using exactly the same reagents,the same column,same HPLC,same degasser than them,everything is the same. Ill let you know if I manage to completely get rid of the peak...thanks a lot

I'm glad you are getting nearer to solving the problem - the small peak you see now may be because you contaminated the system from your filtering device - hopefully it will go away with time.

I would like to reiterate - if you use the best reagents and water from a system such as Milli-q which has 0.2u terminal filtration you are much
better off not filtering, filtering just introduces contamination.

well maybe I still have a problem! I put a brand new column with new solvents and the peak is still there...

How do you know that the peak is really a fluorescence?