Hello My Agilent HPLC has problem with retention time change every day. We changed MCV and plungers and all. But still same. How to fix this

Regarding HPLC retention time variation.

  • There might be numerous reasons. Analysing pressure profiles between runs can give some hints. Use Agilent - HPLC Advisor as a starting point for your troubleshooting.

  • Hi  

    Maybe a bit more detail would help to get you an answer.  Here's a few questions:

    • Is the method gradient or isocratic
    • Do you prepare the mobile phase freshly each day
    • Is the column maintained at a set temperature using a column oven/thermostat
    • Is there just variability day-to-day, or is there the same variability across the day
    • Provide us more details about the HPLC - what modules does the system contain; I can tell that you have a quaternary pump, since you mention changing the MCGV
    • Have you carried out any tests on the pump, either leak rate test or gradient test?

    Probably enough questions for now.


    1. Check Mobile Phase and Solvent Strength: Ensure the mobile phase composition is consistent and properly mixed. Small changes in solvent composition can significantly affect retention times. Also, use HPLC grade solvents to minimize impurities.

    2. Column Conditioning: If the column is new or has not been used for a while, it might require conditioning. Follow the manufacturer's instructions for column conditioning to ensure it is equilibrated properly.

    3. Column Temperature: Fluctuations in column temperature can cause retention time variability. Ensure the column oven is set to a constant temperature and is working correctly.

    4. Sample Preparation: Inconsistencies in sample preparation can lead to variability. Ensure that the samples are prepared in the same way each time, including the same diluent and concentration.

    5. Systematic Pump Issues: Despite changing plungers, there might still be issues with the pump such as inconsistent flow rate or pressure fluctuations. Check for air bubbles in the system and ensure the pump is properly primed.

    6. Injection Volume: Ensure that the injection volume is consistent for each run. Variability in injection volume can cause changes in retention time.

    7. Check for Leaks or Blockages: Any leaks in the system or blockages in the flow path can cause pressure fluctuations and affect retention times. Inspect all fittings and connections for tightness and check for blockages in the flow path, including the column, filters, and frits.

    8. Mobile Phase Degassing: Inadequately degassed mobile phases can lead to bubble formation in the system, affecting the retention time. Ensure the mobile phase is properly degassed before use.

    9. Method Development: If the retention time variability is specific to certain compounds, it might be necessary to adjust the method parameters, such as the mobile phase composition, flow rate, or column selection.

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