Poor Resolution with High Concentrations

Hello,

We notice that when analyzing our calibration standards, the resolution between peaks decreases as the concentrations increase. We are using a Microsorb-MV 100 C18 250 x 4.6 mm 5um column @ 1.000mL/min and 5uL sample volume. How can we improve resolution for samples with higher concentration?

Parents
  • Are you able to attach any chromatograms? It may simply be a case that the method is not suitable and requires further development if the peaks are too close together.

    Other things to consider are the condition of the column? An old/degraded column may cause peak shape to worsen and tail/front causing them to co-elute, or mobile phase preparation, especially with buffers, can cause a shift in retention time if they are prepared even slightly different.

Reply
  • Are you able to attach any chromatograms? It may simply be a case that the method is not suitable and requires further development if the peaks are too close together.

    Other things to consider are the condition of the column? An old/degraded column may cause peak shape to worsen and tail/front causing them to co-elute, or mobile phase preparation, especially with buffers, can cause a shift in retention time if they are prepared even slightly different.

Children
  • Marcus.jones,

     

    We do not use buffers in our mobile phase, and we prepare it using HPLC grade reagents. We have also replaced our column and guard column without any improvement on the resolution. I have even replaced our HPLC tubing just to see if it had any effect (it did not). We really want to avoid reducing the concentration of our calibration standard, while still achieving good separation. However, reducing the concentration certainly improves the separation…

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