Unusual square-wave around 30-40%B in an 1 - 40%B gradient over 180 minutes

<p><span>Dear all,</span></p><p></p><p><span>I had a good one with my UPLC for about 15 months. The complex peptide maps we developed has a run time of 240 min on a BEH C18 column 2.1x150mm with Guard column. The Flow rate is 200ul/min, Column temp: 30oC; Buffer A: 0.05% TFA, Buffer B: 0.04% TFA/ACN (all LCMS grade solvent from B&J). The maps were very reproducible with <0.4% RSD. However, after the PM performed a few weeks ago, the unusual square-wave noise appeared. Two Waters engineers are here trying to rectify these. </span></p><p></p><p><span>I would like to tap into the UPLC community for suggestions as well. I attach some screen shots of "good time" and "bad time" traces for your reference.</span></p><p></p><p></p><p><span>Thanks.</span></p><p></p><p><span>Albert</span></p>


  • I will forward this to the engineers too, for ideas.

    Can I add a little Product Management recommendation. As here, it is always tricky to know what has changed. If you make it a practice to regularly run bandspread test you can tell for example pretty fast if the system efficiency has changed from where it was. It's a simple test and very informative.

    This looks like a mixing issue, could be a bad connection somewhere or soemthing mechanical that needs tracking down. It may or may not be associated with eth PM.


  • Hi

    The baseline disturbance is most likely related to flow or mixing. The first symptom that I look for is a change in the frequency of the pulsation as the gradient runs. If the frequency increases as the gradient proceeds, it sugests a pulsation from the B pump. Conversely a decreasing frequncy suggests a pulse from the A pump. The pdf shows the pulse pattern very clearly, but there is not a long enough stretch of baseline to see a change in frequency. I woul compare the baseline from 20-30min with a similar segment much later, as 160-170min.

    If the frequency doesn't change, consider very small leaks after the mixing filter as well as malfunction of the pressure restrictor on the detector outlet.

    I am curious if the 425uL peptide mixer is installed on the system. Was it re-installed after the maintenance?

    Tom Wheat

  • Hi Tom,

    Thanks for your insight. I'll alert the engineers when they come today. I am more inclined to suspect the mixing rather than the flow with our regular UPLC since the retention times were spot on with traces that were hours and days apart in the same long run sequence over the weekend. Another observation of a low frequency square-wave noise (from the beginning to end of the whole gradient) on a recent upgrade of a nanoUPLC to handle 1mm ID column was fixed with a firmware upgrade without having to change any hardware components.

    I appreciate your assistance and thoughtful insight based on your wealth of chromatographic experience.

    Best regards,