Chromatography Forum

Hey everyone,

I'm a grad student working on an LC/MS method, where I have several internal standards. My mobile phase A is Water 6 mM Ammonium Bicarbonate. B is Methanol/Water 95:5, same modifier and concentration. My gradient runs to 98% Acetonitrile and the column is reequilibrated for 4 minutes with the initial conditions. I am running on a standard C18 column. I am having a problem where the RTs for certain nonpolar compounds drift and become lower as the run goes on in negative MS. I am trying to design an experiment around determining the cause of this. So far I have not been able to figure it out. I was turning to the experience of this board for help! I wasn't sure whether or not to post this in the MS section as it's an LC issue. Thanks all!

Retention time drift is an LC problem. More information is needed to help you with your problem. Please provide details on the size and type of column and the gradient conditions including flow rate. You list a buffer as mobile phase A and methanol/water as mobile phase B yet you state that your gradient goes to 98% acetonitrile? The 4 minute equilibration may not be sufficient. Please list your analytes if possible.

Hello!

I totally apologize for the error! I was playing with my method and switching mobile phase B!

To clarify, mobile phase A is Water with 6 mM Ammonium Bicarbonate and mobile phase B is 95/5 MeOH:H20 with 6 mM Ammonium Bicarbonate. The gradient goes to 98% B after 8 minutes and I'm starting at 99% A.

My flow rate is 0.5 mL/min and the column I am using is a Waters C18 2.1 mm x 100 mm UPLC column.

The retention time shift is only occurring in the later eluting compounds that are very non polar. For example, over the course of a long run, the retention time will decrease! Also the next day when I try to rerun the same samples sometimes the later eluting compounds retention times will change!

Let me know if you need any more information

pH, pH????????????? Temperature changes also pH etc. and charged compounds will change RT.
Keep pH constant.