Quadrupole Calibration

This tutorial will cover the calibration of Q1 and Q3 using the PPG Standard solution and the PPG
3000 Standard solution for the Q TRAPTM System and the 4000 Q TRAPTM System.
A method for manual calibration will be described as well as an automatic method for PPG
optimization and calibration. The method described here uses direct infusion via the syringe pump
in positive ion mode using the TurboIonSpray source or the TurboVTM Source. The PPG
solutions are available in the Mass Spectrometer Test Kit P/N 401936.

Quadrupole Calibration

2.1

Creating a Hardware Profile

Open up the Hardware Configuration Editor by clicking the Hardware Configuration item on the
Navigation bar (left hand side of the screen).
For the Q TRAPTM System with the integrated syringe pump: Click the New Profile button to
create a new hardware profile and name it MS + syringe. Select Add Device and choose the
Mass Spectrometer from the list of peripherals. Click OK to make it appear in the Edit
Hardware Profile window. Click Setup Device and ensure the integrated syringe pump is
activated by checking the box Use integrated syringe pump at the bottom of the pane. Return
to the Hardware Configuration Editor by clicking OK, twice.
For the 4000 Q TRAPTM System: The syringe pump is external and no specific modification
is required in the hardware profile. Click the New Profile button to create a new hardware
profile and name it MS only. Select Add Device and choose the Mass Spectrometer from the
list of peripherals. Click OK to make it appear in the Edit Hardware Profile window. Return
to the Hardware Configuration Editor by clicking OK, twice.

When you are back to the Hardware Configuration Editor, click Activate Profile. The computer
will establish communication with the devices in the profile and checkmark will turn green. Close
the editor.

Quadrupole Calibration

2.2

Infusing the PPG Calibration Solution

Create a new project named Training by selecting Projects>Create Project from the Tools menu.
Once the hardware profile has been activated, click Tune in the navigation bar (the left-hand panel
in the Analyst Software main window), then click the T icon to put the instrument into Ready
mode. You will hear the gases come on as the instrument becomes active.

Double-click Manual Tuning in the navigation bar to open a blank template.

For the Q TRAPTM System: Fill the 1 mL syringe with the PPG solution and place it in the
syringe pump on the Q TRAPTM System. For calibration of the Q1 and Q3 quadrupoles in
positive mode, use the PPG Standard solution. For calibration of Q1 and Q3 in negative
mode, use the PPG 3000 Standard solution. To start the syringe pump, select Syringe Pump
Method from the menu by MS Method.

In this window, define the syringe diameter and flow rate. For a 1 mL syringe, the syringe
diameter is 4.61 mm (see table in Table of Hamilton Syringe Diameters for other diameters). Click
Start Syringe Pump once the parameters have been adjusted. Use a flow rate of 5-10 uL/min for
infusion of the calibrant.

Note: If the flow rate or syringe diameter must be changed once the syringe has been started, enter
the new value and click Set Flow Rate. Changes will not take effect until you click Set Flow Rate.
For the 4000 Q TRAPTM System: Fill the 1 mL syringe with the appropriate PPG solution and
place it in the external syringe pump. For calibration of the Q1 and Q3 quadrupoles in positive
mode, use the diluted PPG Standard solution (see Dilution of Standard PPG Solution). For
calibration of Q1 and Q3 in negative mode, use the PPG 3000 Standard solution. Set the
diameter on the syringe pump to 4.61 and the flow rate to 5-10 L/min.

Quadrupole Calibration

2.3

Building an Acquisition Method

Note: Methods for calibrating the quadrupoles in positive and negative mode may already exist in
the API Instrument folder from installation. You can open those methods now instead of building
a new method. Example acquisition methods for the Q TRAPTM System and the 4000 Q TRAPTM
System can be found in the API Instrument folder.

Create a Method for Positive Mode Calibration

Choose the Q1 Scan from the Scan type menu. Click the Center/Width button and enter the PPG
masses.
For the Q TRAPTM System, enter the masses 59.1, 175.0, 616.5, 906.7, 1254.9 and 1545.1.
For the 4000 Q TRAPTM System, enter the masses 59.1, 175.0, 616.5, 906.7, 1254.9, 1545.1,
2010.5 and 2242.6.
Set all the values on the Source/Gas tab to the values shown below for the source. Click MCA on
under the MS tab. Set the number of Cycles to 10.

Set the Declustering Potential (DP) on the Compound tab; start with a value of 110. Alternatively,
individual settings for DP can be set for each PPG mass. Right-click the gray area beside the
masses and select Declustering Potential from the menu. Enter the values 110 respectively in the
rows, these values can now be separately adjusted for each PPG ion as each DP value is
optimized.

Quadrupole Calibration

Set Q1 resolution to Unit on the Resolution tab.

Save this method with the name Q1 Pos PPG.dam.

Create a similar method for calibrating Q3, using the same masses, the same declustering
potentials and setting Q3 resolution to Unit. Save this method with the name Q3 Pos PPG.dam.
Create a Method for Negative Mode Calibration
Use a similar procedure as described above to create a method for negative mode calibration.
Choose the Q1 Scan from the Scan type menu. Click the Negative polarity radio button to switch
the polarity of the method. Click the Center/Width button and enter the PPG masses. Enter a
declustering potential of -60 as a starting point.
For the Q TRAPTM System, enter the masses 45.0, 585.4 933.6, 1223.8 and 1572.1.
For the 4000 Q TRAPTM System, enter the masses 45.0, 585.4 933.6, 1223.8, 1572.1, 1863.3,
2037.4 and 2211.6.

Quadrupole Calibration

2.4

Creating a Reference File for Calibration

To calibrate the instrument based on a data spectrum, you must pre-define the reference table for
the mass calibrant being used. Once this has been set up for the calibrant, it does not need to be
repeated.
In the Tools pull-down menu, choose Settings, then Tuning Options. This opens the Tuning
Options pane on the Calibration tab.

Select the PPGs Pos from the Standard menu. Ensure the Positive polarity is checked, then select
the two methods that were created in the previous section for the Q1 Method and the Q3 Method.
A method must be selected for LIT Method, any LIT method can be chosen as it wont be used in
this tutorial (a method for LIT calibration using PPG will be created in Section 3.8 in the tutorial
on the Calibration of the Linear Ion Trap Scan Speeds).

Quadrupole Calibration

Click the Reference button to view the reference table for the PPG positive calibration.

You can select which PPG ions to use during calibration by checking/unchecking the Use boxes.
Click Update Ref to save any changes, then click Close.
Note: The 4000 Q TRAPTM System does not require mass dependent parameter optimization.
Click OK to close the Tuning Options window.

Quadrupole Calibration

2.5

Manual Acquisition of PPG Data

Now that the acquisition method has been created and the syringe pump is infusing, data
acquisition can begin.
Click the Start button and two new panes will appear in the Manual Tune window. The Total Ion
Chromatogram (TIC) for the acquisition, shown in theleft pane represents the total number of ions
reaching the detector in each scan. The right hand pane contains the spectrum that is currently
being acquired.

Quadrupole Calibration

The PPG masses will appear in the bottom right pane, right-click on the pane and select Open File
to view all of the PPGs in one window.

Ensure each PPG ions meets its intensity specification, as reported in the Appendix.

Quadrupole Calibration

2.6

Calibration of Quadrupoles from Manually

Acquired Data
Next, click the Calibrate icon in the tuning toolbar to open up the Mass Calibration Option
window.

Choose the reference file for this calibration from the Standard menu: PPGs Pos. Notice that the
quadrupole, the resolution and the polarity used in the data collection are automatically entered
into this window.

You can directly edit the Reference Table Editor by clicking Edit.

Quadrupole Calibration

10

Click Start to begin calibration.

The first pane that opens is the Mass Calibration Report pane. The top graph illustrates the mass
shift in the PPG ions since the last calibration. If the mass spectrometer is mass calibrated, the
typical mass shifts seen are 0.1 to 0.2 amu.
The middle pane illustrates the peak width at half height.
The bottom pane shows the changes in intensity compared to the last calibration.

Quadrupole Calibration

11

The second pane that opens is the Mass Calibration Results pane. This is a numerical
representation of the calibration results. Check the Peak Width column in the report and verify that
the peak widths are between 0.6 0.8 for all of the PPG ions.

If the data spectrum looks good and the mass shifts arent too large, click the Replace Calibration
button on the Mass Calibration Report pane. If all the mass shifts fall between the two dotted lines
shown on the graphical report, the instrument does not need to be recalibrated.

The following message is displayed, click Yes to replace the instrument calibration.

Quadrupole Calibration

12

The values for the new calibration are now shown in the bottom of the mass calibration report. The
report shown below is for the Q TRAPTM System

Note: If there is a drastic change in mass or intensity of one of the fragment ions, it is important to
determine why before this ion is used in calibration. Click No and refer to the mass calibration
report shown above. In the Found Mass column, determine the mass of the ion that is off. Next,
observe the quality of this ion in the raw data spectrum. If the incorrect ion was chosen, the Search
Range used in the LIT Mass Calibration may need to be widened or narrowed to allow the
software to find the correct ion. The software will use the centroid of the most intense peak in the
search range for calibration.

Quadrupole Calibration

13

2.7

Automatic Mass Calibration

The Resolution Optimization tool in Analyst Software can be used to automatically optimize and
calibrate the quadrupoles using the PPG solutions. While infusing the appropriate PPG solution,
this tool will optimize the resolution offsets until peak widths specifications are met, and then
calibrate the instrument on the resulting data.
Note: Before using the automatic resolution optimization tool, perform a quick manual data
acquisition to ensure the PPG signals are of reasonable intensity.
Click the Resolution Optimization button in the Tune section of the Navigation Bar.

This will open the Resolution Optimization Window.

Select the appropriate calibration standard from the Standard list. Select the quadrupole and
resolution to be optimized and calibrated. Check the Mass Calibrate on Success button to ensure
the calibration is updated after the resolution offsets have been adjusted.
For the Q TRAP System only, the Update Mass Dependent Parameters box can also be checked
to determine the optimal values for CEP and CXP at the low and high masses as part of the
resolution optimization process. Extrapolated values will be applied in experiments where the CEP
and CXP is not specified.
The Peak Search Parameters can be used to control where the automatic tool will look for the
peak to be calibrated on. Use Search range settings of 0.5 amu to ensure the program doesnt pick
another strong isotope in the window. Threshold is best left at the default value of 200 cps.

Quadrupole Calibration

14

Select the polarity to be calibrated. Select only one polarity at a time as the calibration solutions
are different for each polarity. A direct link to the Reference Table Editor is available from this
window by clicking Edit.
Click Start to begin automatic calibration.

Note: A text file is generated and displayed during the optimization process. This file records all
changes as it goes through the process. View the text file and the data file, as they are being
generated, to follow the progress of the optimization you may need to minimize windows. If an
ion is not found, abort the optimization. Expand the search range and re-start the process.
If the specifications are not reached within 20 iterations, the automated calibration will be aborted.
If it is able to reach the specifications, it will note successful completion in the text file and on the
screen.

Quadrupole Calibration

15

Before saving the Resolution Optimization results, review the generated text report carefully to
ensure that the desired sensitivity and resolution was obtained, and that the isotope peak used for
calibration was correct.
Click Yes to accept the Resolution Optimization results. Now the Q1 and Q3 are both calibrated in
positive mode.
Calibrate Q1 and Q3 in Negative Mode
Change solutions from PPG Standard to PPG 3000 and repeat the process described above to
calibrate Q1 and Q3 in negative mode.

Quadrupole Calibration

16

2.8

Appendix

Dilution of Standard PPG Solution

Dilution Solvent

Dissolve 154 mg of ammonium acetate in 300 mL water.

Combine 699 mL of acetonitrile and 1.0 mL of formic acid.
Mix the above two solutions together to make final dilution solvent. (70/30 acetonitrile/water
with 2mM ammonium acetate).
Dilution Protocol

Using the dilution solvent prepared above, perform a 1:50 dilution of the Standard PPG
solution.
For accuracy, it is recommended that a 1:10 dilution is prepared, followed by a 1:5 dilution.

Table of Hamilton Syringe Diameters

Volume
(
L)

Diameter
(mm)

1000

4.61

500

3.26

250

2.3

100

1.46

50

1.031

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17

Q TRAPTM System Specifications for Q1 and Q3 Quadrupoles

Positive mode PPG specifications
These intensity specifications are for the PPG Standard Solution.
Nominal m/z

Q1 (cps)

Q3 (cps)

FWHH (amu)

59.050

2.0 E6

1.0 E6

0.6 - 0.8

175.133

2.0 E6

1.0 E6

0.6 - 0.8

616.464

2.5 E6

1.0 E6

0.6 - 0.8

906.673

1.5 E7

8.0 E6

0.6 - 0.8

1254.925

2.0 E6

1.0 E5

0.6 - 0.8

1545.134

5.0 E5

N/A

0.6 - 0.85

Negative mode PPG specifications

These intensity specifications are for the PPG 3000 Solution.
Nominal m/z

Q1 (cps)

Q3 (cps)

FWHH (amu)

44.998

N/A

N/A

0.6 - 0.8

585.385

N/A

N/A

0.6 - 0.8

933.636

1.0 E6

2.0 E5

0.6 - 0.8

1223.845

N/A

N/A

0.6 - 0.8

1572.097

1.0 E5

N/A

0.6 - 0.8

Quadrupole Calibration

18

4000 Q TRAPTM System Specifications for Q1 and Q3 Quadrupoles

Positive mode PPG specifications
These intensity specifications are for the diluted PPG Standard Solution.
Nominal m/z

Q1 (cps)

Q3 (cps)

FWHH (amu)

559.050

3.0 E7

2.0 E7

0.6 - 0.8

175.133

1.0 E7

1.0 E7

0.6 - 0.8

616.464

1.5 E7

1.0 E7

0.6 - 0.8

906.673

2.0 E7

2.0 E7

0.6 - 0.8

1254.925

3.0 E6

3.0 E6

0.6 - 0.8

1545.134

1.0 E6

5.0 E5

0.6 - 0.8

2010.5

1.5 E6

1.0 E6

0.6 - 0.85

2242.6

1.0 E6

8.0 E5

0.6 - 0.85

Negative mode PPG specifications

These intensity specifications are for the PPG 3000 Solution.
Nominal m/z

Q1 (cps)

Q3 (cps)

FWHH (amu)

44.998

N/A

N/A

0.6 - 0.8

585.385

N/A

N/A

0.6 - 0.8

933.636

2.0 E7

1.0 E7

0.6 - 0.8

1223.845

N/A

N/A

0.6 - 0.8

1572.097

1.0 E5

N/A

0.6 - 0.8

1863.3

N/A

N/A

0.6 - 0.85

2037.4

3.0 E7

N/A

0.6 - 0.85

Quadrupole Calibration

19