Significant Decrease in retention time

[skunked_once]

I'm surprised no one has mentioned column re-equilibration at the end of the run. In some cases, up to ten column volumes of the initial mobile phase composition need to run through the column to re-equilibrate to initial conditions. Using a 150 mm column with a column volume of about 1.5 mL would require 15 mL of initial solvent conditions which would be 15 minutes at 1 mL/min. Your re-equilibration time is only about 6 minutes. Try increasing the equilibration time at the end of the run by a factor of two or more. Good luck!

[letsdoit]

But then my gradient stays the same at the beginning of the run for 5 minutes.... So i guess effectively the column re-equilibration runs for 11 minutes
althought therun has started and the separation takes place simultaneously

So should I just start the gradient earlier and run the requeilibriation at the end for a longer time

[skunked_once]

You are correct that the column is equilibrated for 11 minutes but you have already injected your sample and it is already on the column during the last five minutes of the equilibration. You can test whether column reequilibration is the problem by making several runs with a much longer equilibration time at the end of the run. If you retention times are now stable, you can decrease the equilibration time incrementally until the retention time begin to shift. This way you can determine the minimum equilibration necessary. I know it takes time but as one or our retired custodians used to say "The pay is the same!" Good luck.