Missing qTOF Reference Ions



I am not seeing all of the reference ions (negative or positive) while my qTOF instrument is in idle or acquisition.  Spectrum and Work List icons remain yellow whereas Vial icon remains red. A fresh reference solution was prepared, flow and function of nebulizer confirmed. Some additional details as follows:


  • In negative mode, seeing only 112 and 1033 m/z.  Not seeing 119, 301, 966, 980, 1633 m/z.  Similar scenario in positive mode; seeing only 121 and 922 m/z.
  • Played with MP flow rate, reference nebulizer pressure and other parameters to no avail.
  • I have a dual ESI source.  Set up for small molecule analysis; 1700 m/z, 2 GHz.
  • Instrument recently passed calibration and tune for TOF and Quad.
  • Acquisition software recently upgraded to Version 10.  Methinks this is the culprit..?


Best regards,


  • Hi,


    Do you have the other reference ions in your reference ion solution?   The default ingredients only include the reference ions that you have already found.




  • No other reference ions beyond the TFA, Purine and HP-0921 that were used to prep the ref mix.  I see TFA (112 m/z) and HP-0921+TFA (1034 m/z) but not purine (119 m/z).  Also, I do have HOAc and HCOOH in my MP, so I should see 980 and 966 m/z.  

  • Hi Mark,


    Are you running in Pos or Neg mode or both?


    If you are running in Pos mode you only need to see the ions at 922 for HP-0921 and 121 (Purine).        As long as these reference ions bracket your ions of interest within your analytical run,   the reference ion correction will work as expected.    Please also keep an eye on abundances of each of these ions across your gradient and you may need to fine tune these abundances.  The stock recipe for making up the reference ion solutions typically works well.

  • I'm really only concerned with negative ion mode.

  • Hi Mark,

    Just something quick, are the neg ions of interest checked in ref tab in Method?


  • Check.


    Agilent service rep addressed the issue by building the method from scratch in the recently updated software.  This has appeared to solve the problem however, I still need to sit down with the instrument.

  • I spent a couple days with the instrument.  I was initially seeing the ions for TFA and purine in negative ion mode.  Now they are no longer observable.  Rather bizarre.  Do see the HP-0921 but not + acetate; and yes, I have 0.1% HOAc in the MP.

  • Agilent service rep came out a second time and was able to clear up issue (cleaning of ref nebulizer and associated lines).  After running my method (gradient of 0.1%HOAc MeOH into H2O), ions (112 and 119) lost; indicators changing from green to yellow (same as observed on Dec 27).  Methinks the fixed 10 psi used to deliver the ref ion solution is to low.  From what I gather, others may have similar issues.

  • Conclusion:  The nebulizer is ineffective for delivery of reference ion solution to the source.  Apparently this is the case for at least one other user of the 6350 instrument and I suspect there are more out there.  The work around includes configuring an isocratic pump and a splitter to deliver ref ion solution via a T post column.  The pressure of the isocratic must slightly exceed (~2-3 bar) pressure of HPLC pump; this gets fun when you're running a gradient!  Not perfect and will require more futzing.


    Addendum:  TFA should be eliminated from the tuning mix since it functions as an ionization suppressor.  

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