Peak tailing and drift throughout a run

We've had this problem on and off for a while but every so often two of the analytes we look for start drifting and tailing throughout the course of a run, none of the other compounds are affected - we look for a total of nine steroids in animal urine. At the start of each run the peaks look normal but they gradually become broader and more retained as the run goes on. On average each run is ~120 injections.I have attached two pictures of how the peaks look at the start of the run and how they look at the end.We use a UPLC Acquity system coupled to a Premier mass spectrometer, the mobile phases are 0.1% ammonia in water and 0.1% ammonia in methanol (gradient) and we use an Acquity C18 BEH column (the column we are using now is quite new, only about 500 injections). After each run the column is cleaned by flushing with 50:50 water:methanol.Any thoughts about why this happens and how it can be prevented would be greatly appreciated.

Answers

  • Quickly looking at this it feels like chemistry, but will forward to smarter chemists and MS'ists than I.

    The first thing is why chromatography and MS sensitivity is changing as progresses.

    The first thing I would like to see is more detail on the gradient and washes, but I would try and injection with the run time extendied - extending the final hold and see what that looks like.

    In the eman time I will collect answers from some smarter people.

    Liz

  • It could be a solubility issue.

    You might want to replace your NH4OH mobile phase with AmBicarb buffer at pH 10.0 to do a test run.

  • Gradient, Weak/Strong Needle wash solvents, seal wash time, injection volume/type (Full/PLUNO), and column length would be helpful before making a suggestion. If you cannot change the MP (validated method, etc) then consider making them in smaller volumes and protect from light. .

    Also I have to agree to try another mobile phase additive than NH4OH. Needle wash solvent has burned me in the past so choose compatible solvents that are miscible with sample and MP's. Also what is the column temp you are using?

  • What conc is your 0,1% ammonia? Are you using Ammonia 33% or 0,1% of 0,1% Ammonia? What is the pH of your mobile phase (at start and at end)?

    Ammonia is gasing out of the mobile phase so this could be one possibility of your problem. As Jane mentioned this could be solved by using a buffer.

    When flushing the column with water/methanol regenerated the column you may consider flushing the column after every 10 or 20 injections and recondition back to analytical conditions. This will increase total run time but maybe solves your problem?

  • One other thing to try is to clean the column with a higher level of organic during your wash step.

    Another thing to consider is changes in samples. Maybe a cleanup step through an SPE cartridge might be in order.

    Also you state that this happens once in a while. Any ideas what happens when the problem goes away? Do you let the column set, new mobile phases, longer cleaning before shutdown?

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