Poor Resolution with High Concentrations


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?

  • Just some thoughts without having the full details of the method and hardware setup.....

    Do you have enough sensitivity with your lowest standard to decrease the injection volume? If your lowest standard is at or close to the LOQ then that probably isn't an option. What is the data rate you are using on the detector and is there scope to increase this? This would depend on the model of detector, but an increase in data rate normally gives an increase in resolution but will increase the noise (assuming the detector is UV).

    Another approach would be to decrease the particle size of the packing material in the column. This will increase the number of plates (N) and will increase resolution. However, decreasing the particle size increases the back pressure generated and, depending on the hardware (pump) you have installed, this may not be possible. Also, I don't see a smaller particle size in the Microsorb material.

  • 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.

  • Dannymobbs,


    Unfortunately, our lowest standard is close to our LOQ. Our FL detector settings are: Response Time = 2.0 s, Sampling Period = 200 ms (5Hz).

  • 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…

  • This question has been marked as assumed answered.

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