Chromatography Forum

Hello,

I am running 3 differnt samples preps with the same compoent of interest, but as run progresses the retention time would jump from the usual 12 mins to 25min the next injection but the response/area is comparable. This happens to all 3 preps and standards as well.

I suspect it could be my mobile phase changing in composition over time (it's a 45 hour run and the jumping started about 6 hours into the run) as my MPA is 100% water with minute TFA and MPB is 100% ACN with minute TFA, or it could be the pH of my mobil phases having an effect on the column, ie. hydrolysis of the bonded phase. How should I troublshoot this problem?

p.s the column and sample heating are both on.

Does you retention time return to normal when doing a new series of runs after washing the column? Or is the change permanent?

Are you giving enough time for column re-equilibration with the initial mobile phase once each run is complete?

Are the changes related to the different sample diluents? How different are these diluents from the initial mobile phase?

How much TFA are you using? What is the pH of your mobile phase? What column are you using? The instruction sheet coming with your column should inform you on what conditions are acceptable for the column.