Chromatography Forum

Hi everyone,

I'm developing a gradient HPLC method for pharmaceuticals. My last chromatographic peak appears at B ~70%. To shortn the method I'm considering to start the column equilibration directly after the last peak instead of waiting until B reaches 95%. Is this a good idea taking into account that I will run environmental samples with a high degree of impurities or is it better to implement a flushing window with B 95%?
I'm running an LC-MS/MS method in MRM mode so that there are usually no disturbing peaks orgining from impurities.

I think it's good practice to go to high % organic in RPLC for dirty samples and hold for a couple of minutes depending on your flow, since matrix can start eluting in random places in subsequent injections and cause matrix effects. After the last analyte eluted, you can use a steep gradient to high % organic to save some time. You could also take the risk to stop at 70% and perform a blank run at high % organic every 5 samples or so.

To shorten the flushing time at high organic for dirty samples I tend to increase the flow after the elution of analytes and flow state to waste. The LC has to be able to handle the pressure bump of course.

Proper scientific method development always includes ramping up the solvent to a higher concentration than needed to elute the last peak, then hold it for period of time. This is a critical part of the method that should not be skipped. Column washing after the analysis may also be needed to.

There are better ways of reducing the overall run-time of an HPLC method which do not include ignoring such key fundamental concepts (e.g. higher resolution method which elutes peaks earlier).

Thanks a lot for the helpful answers. Due to my dirty samples I set the final B[%] to 95 and added a flushing step.