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

Solution:

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.

Solution:

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

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

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