Bumpy baseline only at certain section in gradient.

I run a method on UPLC Binary, its a gradient method and the column is a 1.7micron 100 x 1.2 length. I use as high a quality of water an acetonitrile as possible for the run but about 20minutes into the gradient and its a 50 minutes run the baseline gets really bumpy as if the check valve is going but then it evens out only to repeat the jagged bumps the next injection about 20mins in, it lasts for about 3 minutes and makes the baseline very ugly. Thing is, the condition at that time is 75% Water and 25% Organic and the jaggedness is only in my samples not standards. I work at a low wavelength if 215nm too. 

Could it be dirt in the solvents or just the samples? How would I troubleshoot this to determine the cause? Thanks for any help. 

Best Answer

  • jeanette
    Answer ✓

    Such a low wavelength is always difficult - we are experiencing similar. In this case it seems that something in the sample matrix is eluting at 20 min.

    I think that if this is 100% always only the samples then I would say that you already found the reason - the hard thing can be determine what in the sample solution causes this and then is it possible to get rid of it?

    To dig more into this knowledge on the sample solution or the sample itself is needed.


  • Well I zoomed in on the baseline of all samples, blanks and standards and its very jagged and bumpy there too, definitely bad enough to be an issue for reliable peak detection. That rules out the standards and samples. The column is about 8 months old by now and used twice a week so I wonder is it just deteriorating. I always do a very long start up on my UPLC before use to purge out air and the system was only serviced a month back too. The only thing I can think of was I changed the check valves 2 weeks ago because the pressure was going up and down and when I changed it the pressure was stable but the check valve seemed to be hard to close before I screwed it in place, I wonder is it sitting slightly off kilter in the system.

    Any other suggestions?