Buffer vs. water = no signal vs. signal?

Hi everybody!Has any of you experienced a similar problem or have a comment regarding the one outlined below: 1) When I dissolve my substance of interest (little and polar) in waterand use my UPLC-MS/MS (Xevo TQ MS) machinery I can measure my substance at nM scale. 2) When I dissolve same substance inphosphate buffer (0.1 mM, pH adjusted to 7.4) and run upon the same machinery and with same eluent as above I get no signal at all! Both 1 and 2 was run at same time. 3) When I dissolve same substance in water and run it upon my HPLC-MS/MS (Quatro Micro) machinery (same eluent) I can measure my substance at nM scale. 4) When I dissolve same substance in phosphate bufferand run it upon my HPLC-MS/MS (Quatro Micro) machinery I can measure my substance at nM scale. I am really puzzled, as I have checked for matrix effect (which seems much the same for buffer and water injection at retention time). Any answer will do.Best regards, Kasper

Answers

  • Hello Kasper.

    What I didn't get is what kind of mobile phase are you using and at what retention time is your peak eluating in HPLC-MS/MS and in UPLC-MS/MS.

    Please keep in mind that one great difference with uplc and hplc is the time all substances needs to pass the column.

    While with hplc the injection has pleanty of time to get solved in mobile phase this time is drastically reduced with uplc and that means that every uplc seperation may run into great problems when the time needed for diluting the injection in mobile phase isn't long enough.

    It is very likely that your substance of interest loves to be surrounded by phosphoric ions and with high speed uplc there is no time to get this "hull" diluted by mobile phase,so this "quasi ion pair molecule" enters the ms and won't be seen an expected m/z.

    Some questions:

    - your substance of interest is likely of basic nature?

    - retention time is quite low?

    - your mobile phase isn't buffered?

    Andreas

  • Dear Kasper!

    I think phosphate buffer is not a good choice, it can make salt and polluted your system. You should try with some organic buffer such as acetic acid/ammonium acetate, formic acid/formiate.

    Probably pH also can cause problem. Is the water pH and buffer pH is equal?

    I have no other idea. I hope that I can help you.

    Best regards, Bobszlee

  • Kasper

    Can we have some details more details about the inlet/column configurations so we can troubleshoot further.

    Thank you

    Liz

  • Hey and thank you for the quick respons!

    Additionel information is provided below:

    I use a UPLC HSS T3 column, where my neutral substance of interest, have a retention time of 1.96 min in water.

    The following gradient is used with water (0.2 % HCOOH) and MeOH (0.1 % HCOOH) with a flow-rate of 0.4 mL/min:

    0-0.30 min:15 % MeOH, isocratic.

    1.30-1 min: linear gradient to 50% MeOH.

    1.0-1.5 min: linear gradinet to 90% MeOH.

    1.5-2.0 min:90 % MeOH

    For the HPLC I use a T3 Atlantis column, where the retention time is approx 6 min, composition the same and with a flow-rate of 0.150 mL/min:

    0-8 min: 15%, isocratic.

    8-12 min: wash 90%.

    P.S. The reason for using 0.1 M phosphate buffer in my experiments is to mimic in vivo (cell) conditions.

    Best regards, Kasper

  • Hello Kasper,

    guess I asked the wrong question:

    does your compount has basic groups which can interact with the phosphoric acid?

    To test if my assumption of the phosphoric-cluster is right, programm a method where the flow is stopped for 5 min shortly after injection.

    Do you get a signal for both the sample with water and the sample with phosphoric ions?

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