coeluting peaks


I am currently encountering coeluted peaks in my chromatographic analysis for two specific analytes following a recent maintenance session. To provide a comprehensive overview, here is a detailed timeline of the actions taken:

- Prep MPA (0.1 % formic acid aq) and MPB (0.05% formic acid aq)

 --> ran a blank (MeOH) [Figure 1A] and system stability check of a Continuing Calibration Verification Standard [Figure 1B].The chromatography of this run mirrors our normal results

- Same day: Following the calibration run, the chromatogram reveals RT shifts and coeluted peaks

--> blank (MeOH) [figure 2A] and a calibration level [figure 2B]

- Replace the guard column, ran a blank (MeOH) [figure 3A] and a calibration level [figure 3B]

- Replace guard column and analytical column [Figure 4A]


Any ideas on how I can fix this issue?

    • The issue of coeluted peaks and retention time (RT) shifts in chromatographic analysis can be quite challenging, especially following maintenance activities. Based on the detailed timeline you provided, it seems like the system was initially performing as expected, but issues arose after some maintenance actions. Here are a few steps and considerations that might help resolve the problem, keeping in mind best practices and safety guidelines from typical chromatography instrument manuals:

    Check System Installation and Column Alignment: Ensure that the guard column and analytical column were properly installed. Misalignment or improper seating can cause unexpected changes in flow path and peak profiles.

    Reevaluate Mobile Phase Preparation: Double-check the preparation of your mobile phases (MPA and MPB) to ensure they are correct and consistent with previous preparations. Even slight variations in the concentration of formic acid can affect the chromatography, especially for sensitive analyses.

    System Equilibration: After replacing any column, ensure the system is adequately equilibrated with the new column before running any samples. This might require more time than initially anticipated.

    Flow Rate and Pressure Check: Verify that the flow rate and system pressure are within expected ranges after the maintenance. An unexpected change in these parameters can indicate issues with the system setup or column packing.

    Temperature Control: If your system uses temperature control, ensure that the column oven is set to the correct temperature and is stable. Temperature fluctuations can cause retention time variability and affect peak separation.

    Mobile Phase Degassing: Ensure that the mobile phases are adequately degassed. Dissolved gases can lead to baseline noise and instability, potentially affecting peak resolution.

    Sample Preparation and Injection Volume: Reassess the sample preparation and injection volume. Overloading the column or changes in the sample matrix can lead to coelution.

    Gradient Elution Profile: If you are using a gradient elution, review the gradient profile to ensure it matches the intended method. A slight deviation in the gradient can lead to significant changes in retention times and peak separation.

    Method Development and Optimization: If the problem persists, consider revisiting the method development stage. Adjusting the mobile phase composition, gradient slope, column temperature, or even trying a column with a different stationary phase might be necessary to resolve the coelution.

    Instrument Diagnostics and Leak Check: Perform a thorough instrument diagnostic to check for any leaks or issues in the detector, pump, or autosampler that might have been overlooked during maintenance.

    Consult Manufacturer’s Guidelines: Refer to the specific instrument and column manufacturer's guidelines for troubleshooting and maintenance. They often provide detailed steps for resolving such issues based on the equipment's design and common challenges.

    Record Keeping: Document all changes made during troubleshooting. This documentation can be invaluable for future reference and in identifying what adjustments lead to improvements.

    Given the complexity of chromatographic systems and the multitude of factors that can affect performance, a systematic approach to troubleshooting is essential. It may require several iterations of these steps to identify and resolve the underlying issue. Always prioritize safety and adhere to the manufacturer's guidelines for equipment use and maintenance.

  • Wow, your peaks are super close to each other! Anything minor will affect your chromatogram. Maybe you should search for a better method? Anyways, what impacts the most are flow (are you running a gradient method?), organic concentration (again, gradient method?), temperature and pH. Maybe you could check if your MCGV (gradient valve) is working fine and maybe replace that? it is unlikely, but those are the starting points

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