Agilent LC/QTOF 6546- Calibration/ System Tune

Hello, 

The instrument went down due to its pump failing and sitting without use for a couple months. It vented itself once the pump was no longer functioning. I was out of the office on leave during this time. I was able to bring it back up once a new pump arrived. I followed the start up process (pumped down etc.) and everything seemed to be in order. All three vacuums appear to be within normal range for this instrument. However upon further investigation the TOF vac is E^-06 vs E^-07. I was advised to try and let the instrument sit for another day to see if the TOF vac value changes. Just in case it doesn't I want to put this information out there to see if anyone has experienced the problem listed below. 

 I tried running a mass calibration check but it failed. I made fresh positive tuning mix since the one in place was over a year old before I attempted to tune it. I have added a photo of my tune reports below. I tried to do a system tune and it aborted itself during the post tune calibration. I have included those results as well. 

Does anyone have any experience with this issue?

Thanks,

Kelsey

Backup current tune file before autotune
Running QTOF System Tune in Positive Polarity
Begin Running System Tune
Backup All Parameters
Spraying Calibrant Solution
Disable sureMass optimization
Setup Dual AJS ESI Source to Achieve Stable Signals for Auto Tune
   Restore Collision Cell Pressure to its default value 22 psi
Reset All Parameters to Their Default Values
Baseline Adjustment for 10 GHz Instrument
Binary search the linear range of high gain base line
Sucessfully found the linear starting point of Amp Offset at 2666.0 DAC
Finding High Gain Baseline Using Linear Regression
Adjust High Gain Base Line
         Estimate Hi Gain Amp Offset Found at 2353
Binary search the linear range of low gain base line
Sucessfully found the linear starting point of Low Gain Preamp Offset at 1877.9 DAC
Finding Low Gain Baseline Using Linear Regression
Estimate Low Gain Base Line
         Optimum Low Gain Amp Offset Found at 1814
            Final Optimum Low Gain Pre Amp Offset Found at 1814 DAC
            Final Optimum High Gain Pre Amp Offset Found at 2353 DAC
   Waiting for Calibrant to Elute...
   Monitoring 118.06 m/z Peak for Intensity and Stability
      Signal Intensity and Stability Satisfied Auto Tune Requirements
   Performing Coarse TOF Mass Calibration Using 6/10 Calibrant Masses 
      Successfully Perform Mass Calibration with RMS Deviation (ppm): 0.1
   Reset Optics 2 and TOF Parameters to Their Default Values
   Coarse TOF Resolution - Mirror Mid Optimization
      Change Mirror Mid from -2,270.0 to Optimum Value at -2,265.1
   Coarse TOF Resolution - Puller Offset Optimization
      Change Puller Offset from 40.0 to Optimum Value at 30.8
   Performing Coarse TOF Mass Calibration Using 6/10 Calibrant Masses 
      Successfully Perform Mass Calibration with RMS Deviation (ppm): 0.35
   Adjusting VCap to reduce signals to reasonable height 100000
   Reduce high mass signals by reduce VCap to 1844 V
   Adjusting Quad AMU to Produce Single Ion Event at MCP of 700
      Coarse optimum Quad AMU Found at 9.4 with Peak Height of 4174
      Optimum Quad AMU Found at 9.2 with Arrival Frequency of 4.3 %
   Searching MCP to Produce 20 Counts per Single Ion
           Counts         MCP     Frequency               Status
           30.7              700        4.7 %                in Progress
           16.5              668        3.9 %                in Linear Range
           17.7              671        3.9 %                in Linear Range
           18.4              674        3.6 %                in Linear Range
           19.0              675        4.4 %                in Linear Range
           20.6              676        4.0 %                in Linear Range
           21.2              676        3.9 %                in Linear Range
           19.7              675        4.2 %                in Linear Range
           19.4              675        4.3 %                in Linear Range
Optimum of MCP Found at 675 V
Running Transmission Tune for 3200 m/z Range
Initializing Parameters for 3200 m/z Range Standard Tune
         Fragmentor:   165.00
         Skimmer:   45.00
         Oct 1 DC:   28.50
         Lens 1:   27.00
         Quad DC:   25.00
         PostFilter DC:   25.00
         Cell Entrance:   24.00
         Hex DC:   23.00
         Hex Delta:   -7.00
         Hex2 DC:   16.00
         Hex2 DV:   -1.00
         Hex3 DC:   14.80
         Ion Focus:   10.00
         Lens 2:   18.00
         Quad AMU:   140.00
      Optimizing Optics Rail From Ion Source to Detector
   Adjusting Optics Rail from Oct 1 DC to TOF for 3200 m/z Range
      Change Optics Rail from Oct 1 DC to TOF from 0.0 to Optimum Value at 0.1
         Optimum of Oct 1 DC Found at 28.6
   Adjusting Optics Rail from Lens 1 to TOF for 3200 m/z Range
      Change Optics Rail from Lens 1 to TOF from 0.0 to Optimum Value at -0.2
         Optimum of Lens 1 Found at 26.9
   Adjusting Optics Rail from Quad DC to TOF for 3200 m/z Range
      Change Optics Rail from Quad DC to TOF from 0.0 to Optimum Value at -0.1
         Optimum of Quad DC Found at 24.8
   Adjusting Optics Rail from PostFilter DC to TOF for 3200 m/z Range
      Change Optics Rail from PostFilter DC to TOF from 0.0 to Optimum Value at -0.2
         Optimum of PostFilter DC Found at 24.6
   Adjusting Optics Rail from Cell Entrance to TOF for 3200 m/z Range
      Change Optics Rail from Cell Entrance to TOF from 0.0 to Optimum Value at -0.3
         Optimum of Cell Entrance Found at 23.3
   Adjusting Optics Rail from Hex DC to TOF for 3200 m/z Range
      Change Optics Rail from Hex DC to TOF from 0.0 to Optimum Value at 0.3
         Optimum of Hex DC Found at 22.7
         Skimmer:   45.00
         Oct 1 DC:   28.59
         Lens 1:   26.92
         Quad DC:   24.79
         PostFilter DC:   24.63
         Cell Entrance:   23.33
         Hex DC:   22.66
         Hex Delta:   -7.00
         Hex2 DC:   15.66
         Hex2 DV:   -1.00
         Hex3 DC:   14.46
         Ion Focus:   9.66
   Performing Coarse TOF Mass Calibration Using 6/10 Calibrant Masses 
      Successfully Perform Mass Calibration with RMS Deviation (ppm): 0.39
   Quad Transmission Optimization
      Change Quad AMU from 140.0 to Optimum Value at 128.6
   Balancing TOF Mirrors and Slits at Puller Offset of 35.0
   Mirror Mid         Bot Slit            Resolution        Abundance           Score
   -2,265.6            -46.0                31,519             146,204                  128
   -2,265.6            -45.0                33,316             226,379                  143
   -2,265.6            -44.0                34,577             243,817                  148
   -2,265.6            -44.5                32,843             237,540                  145
   -2,265.6            -43.5                37,170             214,610                  155
   -2,265.8            -43.0                40,666             162,751                  158
   -2,265.8            -42.5                38,936             115,312                  146
   Local maxima found Bot Slit: -43.0
   Refine Puller Ofsset - Puller Offset Optimization
      Change Puller Offset from 35.0 to Optimum Value at 33.4
      Mapping Area 1:  Resolution: 39,555 Abundance: 160,858 Score: 158
   Balancing TOF Mirrors and Slits at Puller Offset of 45.0
   Mirror Mid         Bot Slit            Resolution        Abundance           Score
   -2,265.8            -46.0                22,177             141,482                  94
   -2,265.3            -45.0                25,307             200,638                  111
   -2,265.3            -44.0                27,388             191,482                  118
   -2,265.3            -44.5                26,424             206,379                  116
   -2,266.3            -43.5                27,937             164,228                  118
   Local maxima found Bot Slit: -44.0
   Refine Puller Ofsset - Puller Offset Optimization
      Change Puller Offset from 45.0 to Optimum Value at 36.8
      Mapping Area 2:  Resolution: 35,098 Abundance: 244,304 Score: 153
      Best Multidimensional Optimization Solution Found:
         Bot Slit:   -43.00
         Mirror Mid:   -2,265.84
         Puller Offset:   33.43
   Refine TOF Resolution - Pusher Offset Optimization
      Change Pusher Offset from 0.0 to Optimum Value at -39.8
   Reoptimizer delta slit with newly found optimum Pusher Offset of -39.8
   Balancing TOF Mirrors and Slits at Puller Offset of 33.4
   Mirror Mid         Bot Slit            Resolution        Abundance           Score
   -2,265.8            -46.0                31,164             153,047                  129
   -2,265.4            -45.0                35,755             232,517                  152
   -2,265.4            -44.0                37,931             240,122                  160
   -2,265.4            -44.5                35,808             248,296                  155
   -2,265.4            -43.5                38,605             204,571                  161
   -2,265.4            -43.0                39,281             159,479                  154
   Local maxima found Bot Slit: -43.5
   Final Multidimensional Optimization Mirror Mid, Bottom Slit, and Puller Offset
      Optimized Score Improved 2.5 %  to 128
      Best Solution Found:
         Bot Slit:   -43.64
         Mirror Mid:   -2,265.35
         Puller Offset:   33.32
   Refine TOF Resolution - Puller Offset Optimization
      Change Puller Offset from 33.3 to Optimum Value at 34.5
   Refine TOF Resolution - Pusher Offset Optimization
      Change Pusher Offset from -39.8 to Optimum Value at 30.2
   Refine TOF Resolution - Slit Offset Optimization
      Change Slit Offset from 0.0 to Optimum Value at 3.3
   Refine TOF Resolution - Puller Offset Optimization
      Change Puller Offset from 34.5 to Optimum Value at 32.6
   Refine TOF Resolution - Pusher Offset Optimization
      Change Pusher Offset from 30.2 to Optimum Value at -14.8
   Standard TOF Resolution - Lens 3 Optimization
      Change Lens 3 from -60.0 to Optimum Value at -61.4
   Fine TOF Resolution - Extractor DC Optimization
      Change Extractor DC from -10.0 to Optimum Value at -14.0
   Fine TOF Resolution - Ion Focus Optimization
      Change Ion Focus from 9.7 to Optimum Value at 5.4
   Reduce signals before optimization Acc Focus in high resolution mode
   All Calibrant Peaks Passed Saturation Check - No Abundance Adjustment Needed
      Do not need to reduce signals
   Fine TOF Resolution - Acc Focus Optimization
      Optimum for Acc Focus Found at -2,146.0
   Refine TOF Resolution - Puller Offset Optimization
      Change Puller Offset from 32.6 to Optimum Value at 31.4
   Fine TOF Resolution - Mirror Mid Optimization
      Change Mirror Mid from -2,265.4 to Optimum Value at -2,265.1
   Performing Coarse TOF Mass Calibration Using 6/10 Calibrant Masses 
      Successfully Perform Mass Calibration with RMS Deviation (ppm): 0.31
   TOF Energy Offset Optimization
      Change TOF Energy Offset from 0.0 to Optimum Value at -0.2
Enable sureMass optimization
   Performing Coarse TOF Mass Calibration Using 6/10 Calibrant Masses 
      Successfully Perform Mass Calibration with RMS Deviation (ppm): 0.36
Set Quad State to Initial Calibration
 Mass Calibrants: 322.0m/z and 2121.9m/z

Coarse Quad Calibration for Wide Peak Width
Wide - Mass Tolerance = 1. amu 
Wide - PeakWidth Target = 9 +/- 1

 Iteration        Delta m/z          Delta PW            Score            Result
                   (322./2122.)       (322./2122.)
     1           -21.70/-52.26      11.48/59.88            0             in Progress
     2           01.98/-04.85      00.08/13.95            0             in Progress
     3           -01.40/-01.46      01.55/06.87            0             in Progress
     4           00.64/01.86      00.38/01.45            4             in Progress
     5           00.24/05.74      00.12/-03.43            0             in Progress
     6           01.44/02.82      -01.21/-02.25            0             in Progress
     7           -00.38/00.10      00.23/-00.18            78             Passed
Results for Wide Peak Width: Score of 78

Coarse Quad Calibration for Medium Peak Width
Medium - Mass Tolerance = .5 amu 
Medium - PeakWidth Target = 4 +/- 0.5

 Iteration        Delta m/z          Delta PW            Score            Result
                   (322./2122.)       (322./2122.)
     1           02.51/01.84      -01.05/-00.81            0             in Progress
     2           -00.65/-00.72      00.00/00.04            9             in Progress
     3           00.02/00.04      00.10/-00.02            96             Passed
Results for Medium Peak Width: Score of 96

Coarse Quad Calibration for Narrow Peak Width
Narrow - Mass Tolerance = .2 amu 
Narrow - PeakWidth Target = 1.3 +/- 0.15

 Iteration        Delta m/z          Delta PW            Score            Result
                   (322./2122.)       (322./2122.)
     1           01.30/00.78      -00.23/00.20            0             in Progress
     2           -00.22/-00.37      00.02/00.30            0             in Progress
     3           00.03/-00.02      00.00/00.11            64             Passed
Results for Narrow Peak Width: Score of 64
      Finish Quad Tune
Optimize Lens 2 RF Phase 
   Lens 2 RF Ph Optimization
      Change Lens 2 RF Ph from 0.0 to Optimum Value at 97.0
Optimize Lens 2 RF as a Function of m/z
                    m/z               Lens 2 RF
                    0118.1                00.0
                    0322.0                79.5
                    0922.0                147.8
                    1522.0                212.8
                    2121.9                271.2
                    2721.9                316.1
Optimize Lens 2 DC as a Function of m/z
                    m/z               Lens 2 DC
                    0118.1                00.1
                    0322.0                20.3
                    0922.0                15.0
                    1522.0                07.3
                    2121.9                03.3
                    2721.9                03.4

Fine Quad Calibration for Wide Peak Width
Wide - Mass Tolerance = 1. amu 
Wide - PeakWidth Target = 9 +/- 1

 Iteration        Delta m/z          Delta PW            Score            Result
                   (322./2122.)       (322./2122.)
     1           -00.93/-05.24      01.39/04.61            0             in Progress
     2           -00.58/-03.37      00.91/02.90            0             in Progress
     3           -00.06/-02.25      00.50/01.94            0             in Progress
     4           -00.30/-01.41      00.08/01.24            9             in Progress
     5           -00.21/-01.19      00.65/00.90            9             in Progress
     6           -00.18/-01.00      -00.26/00.66            31             Passed - Refine
     7           -00.12/-00.78      -00.40/00.88            25             Passed - Refine
     8           -00.12/-00.65      -00.18/00.49            58             Passed - Refine
     9           -00.12/-00.68      00.36/00.68            57             Passed - Refine
     10           00.03/-00.41      00.39/00.46            76             Passed - Refine
     11           00.06/-00.37      00.11/00.29            84             Passed - Refine
     12           -00.18/-00.32      -00.03/00.30            87             Passed - Refine
     13           -00.13/-00.21      -00.03/00.34            88             Passed - Refine
     14           -00.06/00.04      00.32/00.01            92             Passed - Refine
     15           00.14/-00.13      -00.02/-00.15            94             Passed
Final Results for Wide Peak Width: Isolation Efficiency: 83, Score: 94

Fine Quad Calibration for Medium Peak Width
Medium - Mass Tolerance = .5 amu 
Medium - PeakWidth Target = 4 +/- 0.5

 Iteration        Delta m/z          Delta PW            Score            Result
                   (322./2122.)       (322./2122.)
     1           -00.73/-04.39      01.32/03.24            0             in Progress
     2           -00.19/-03.46      00.54/03.25            0             in Progress
     3           -00.23/-02.40      00.32/02.03            0             in Progress
     4           -00.18/-01.50      00.15/01.34            0             in Progress
     5           -00.18/-01.08      00.07/01.12            0             in Progress
     6           -00.06/-00.70      00.19/00.80            5             in Progress
     7           -00.07/-00.38      00.03/00.47            37             Passed - Refine
     8           -00.10/-00.43      00.17/00.52            9             in Progress
     9           00.00/-00.28      00.11/00.45            48             Passed - Refine
     10           -00.06/-00.26      -00.13/00.29            55             Passed - Refine
     11           -00.01/-00.26      00.01/00.29            63             Passed - Refine
     12           -00.05/-00.22      00.00/00.29            68             Passed - Refine
     13           00.01/-00.07      00.01/00.28            77             Passed - Refine
     14           -00.05/-00.08      00.05/00.21            88             Passed - Refine
     15           -00.01/-00.10      00.08/00.16            92             Passed - Refine
     16           00.04/-00.10      00.14/00.12            91             Passed - Refine
     17           -00.03/-00.07      -00.01/00.08            97             Passed
Final Results for Medium Peak Width: Isolation Efficiency: 74, Score: 97

Fine Quad Calibration for Narrow Peak Width
Narrow - Mass Tolerance = .2 amu 
Narrow - PeakWidth Target = 1.3 +/- 0.15

 Iteration        Delta m/z          Delta PW            Score            Result
                   (322./2122.)       (322./2122.)
     1           -00.78/01.94      00.74/-00.38            0             in Progress
     2           -00.07/-02.29      00.28/-00.27            0             in Progress
     3           -00.09/-03.53      00.05/01.64            0             in Progress
     4           -00.19/-03.45      00.13/03.20            0             in Progress
     5           -00.32/-02.46      00.23/02.40            0             in Progress
     6           -00.24/-01.61      00.20/01.66            0             in Progress
     7           -00.16/-01.10      00.09/01.22            0             in Progress
     8           -00.12/-00.71      00.08/00.98            0             in Progress
     9           -00.07/-00.42      00.12/00.73            0             in Progress
     10           -00.02/-00.26      00.07/00.53            0             in Progress
     11           -00.04/-00.19      00.08/00.43            0             in Progress
     12           -00.02/-00.09      00.07/00.30            6             in Progress
     13           00.00/-00.08      00.04/00.29            7             in Progress
     14           -00.04/-00.07      00.03/00.23            9             in Progress
     15           00.00/-00.03      -00.01/00.23            9             in Progress
     16           -00.03/00.00      00.02/00.21            9             in Progress
     17           00.02/00.00      00.02/00.18            9             in Progress
     18           -00.02/00.00      00.06/00.10            75             Passed - Refine
     19           00.02/00.00      00.02/00.09            78             Passed - Refine
     20           00.01/00.04      00.01/00.12            59             Passed - Refine
Final Results for Narrow Peak Width: Isolation Efficiency: 50, Score: 78
      Finish Quad Tune
Start Post Tune Calibration(may take a few min).....
Progress  0%......
Progress  3%......
Progress  4%......
Post Tune Calibration failed, abort!
Abort called, terminate the calibration
Stop Spraying Calibrant Solution
System Tune did not complete due to the following reason(s): 

Post Tune Calibration failed
Completed QTOF System Tune in Positive Polarity

  • It looks like you may have some interference at the low m/z calibration ions.  Can you turn on the calibrant and zoom in on those ions and check the zoomed spectra?  Maybe a new bottle of calibrant carefully prepared or the old calibrant if it looks cleaner and a new system tune.

  • I made a new calibrant solution and did a full system tune. The system tune passed. However when I switched back to my low 1700 m/z range and did a mass cal/check tune my positive failed but my negative did not. 

  • Running a Mass Calibration \ Check after switching to 1700 m/z range will always Fail Quad results in a Mass Calibration \ Check if a Quad Transmission Tune has not been performed in the new Acquisition Board mass range.


    If you switch to Low Mass range you should always run a Quadrupole (Only) Transmission Tune because:

    -The number of Transients per spectrum has increased which means more abundance/intensity.

    -The Lens 2 RF table needs to be re-written for the Low (1700 m/z) because dynamics have changed.

    I need to check to the there is a service note, but I'm pretty certain that there is. If not there will be one published ASAP

    Quad Transmission tune when moving to 1700 mode (after System Tune), save the tune file as "Low Mass.tun" and you're good to run Mass Calibration \ Check in that mode for a pass report.

    Transients per spectrum are increased in 1700 m/z mode and the Lens 2 Table for isolation/transmission is different from System Tune (3200 m/z)

  • With the release of the new QTOF AQ software we published a PDF document that reviews the best procedures and protocols for doing Tuning and calibration on all the Agilent QTOF/TOF systems.  As WMORE mentioned if starting fresh after a system tune (with both Quad and TOF and Both Polarities Initially Selected) when going to 1700 mass range one should do a transmission tune with both TOF and Quad selected.  Once this has been completed the Quad voltages will be set.  Always Check the Quad AMU voltage when changing mass ranges also.

  • Only execute a Transmission tune for the Quadrupole unless by choosing "Both" you desire sensitivity at the TOF as opposed to resolution.

    When switching to 1700 m/z range, at least a Quadrupole Transmission tune must be performed because the transients per spectrum have changed and the Lens 2 RF table/ (compensation during Quad isolation) has also changed.

    If you choose a TOF Transmission tune as well (or "Both") after changing the mass range selection you will receive greater sensitivity from the Tune but decreased resolution because the TOF will be tuned for Transmission

  • Do you mind sharing such protocols with forum members, please. Thank you!

  • Hi Raphasanz,

    See if this works for you.

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