What cause deterioration of separation from instability with pressure curves?

<p> Can anyone help to solve this puzzle?</p><p></p><p>I found an usual situation when I set up some UPLC petpide separation runs on 20-Dec-10 just before Christmas. The separation and pressure curves seemed to be normal for the first two days (typical profile shown in slide 2 in the attached file). However, on 22-Dec the peptides seemed to be shifting, which co-incided with progressive deterioration of subsequent pressure curves (Slide 1). At first, I suspected that it could be a column problem (Column 3, which was good when last used in Nov), and switched to another BEH C18 column to trouble-shoot with Waters Peptide Mixture (Column 2, slide 3). I included a typical normal trace of the Waters Pepitde Mixture separated with Column 3 on 20-Dec when the system was still behaving.</p><p></p><p>I know the issues were not sample related, because they ran fine (checked today) when I transferred all the samples to the newly installed UPLC (early December).</p><p></p><p>This particular UPLC system was just PM back in October. The Column Manager plumbing were re-built because of leak found at Column 2 outlet valve position in Dec. I have never seen this type of issuse before. Based on slide 3 & 4, I suspect Pump B might be the culprit (at least one of them).</p><p></p><p>Thank you.</p><p></p><p>Albert</p>


  • KWB

    Hi Albert,

    Based on the pressure traces and chromatograms I think you may be correct in suspecting the B pump. It may be something simple such as a connection on one of the B pump check valves being loose.  A very small leak can cause havok. You can perform a Leak Test on the BSM from the Acquity Console. If you have a failure, snug all of the compression screws on the pumps just to make sure there are no leaks. Try the test again.

    Ken Blakeslee

  • atus

    Dear Kenneth,

    Thank you for your suggestion. You are spot on! I have called in Waters Engineer to fix the problem. He found pump B failed the static decay test. He replaced the check valve. The decay test still failed even the delta ripple improved. When the tubing between the transducer to the pump head of B accumulator, the decay tests passed.

    I ran some Peptide mixture again after that. The chromatograms and the pressure cureves are almost super-imposable.

    Thanks for taking your time and expertise to help out. Very much appreciated.