Christie Hunter


Troubleshooting low flow LC – Spread of peptide elution is compressed or delayed

Symptom: The observed retention times of the peptides eluting are compressed, are not as spread out as expected, suggesting the elution gradient is compressed

Test: Use PepCalMix LC-MS test to check the peptide elution time and spread


  • Make sure you are performing the recommended regular maintenance tests on your low flow LC system
  • Ensure there are no leaks in your flow path
  • For the SCIEX low flow LC systems:
    • Re-initialize pressure transducers regularly (at a minimum, biweekly is recommended for best performance)
    • Perform a leak test on your LC pump. If this fails, contact your service representative
    • If the leak test passes, check flow module calibration on your LC pump, recalibrate if necessary. If this does not help or the calibration is off by more than 20%, contact your service representative


Symptom: The elution of peptides is delayed, appearing later than expected.

Test: Use PepCalMix LC-MS test to check the peptide elution time and spread, typical chromatograms are shown in the protocol.


  • Check for leaks in your gradient flow path, both pre- and post-column. Check the pressure in the gradient pump to see if it is lower than expected.
  • Dead volume in pre-column flow path will create a gradient delay, check tubing lengths and connections for excess dead volume



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