Exactive GC Tune

Use Exactive GC Tune to operate the Exactive GC mass spectrometer via the data system computer. It has the
following components:

 The menu bar provides drop-down menus with commands for operating the program. On its right side it
shows the active user role.
 The toolbar provides symbol shortcuts for frequently used commands.
 The tasks panel provides six windows: Instrument Control, Tune, Mass Traces, Calibrate, Evaluate, and
Vacuum/Bakeout. The instrument control window is divided into three additional windows for defining
scan parameters, setting EI/CI source parameters, and acquiring and storing measurement data.
 The display panel provides various windows for showing real-time information about the current
measurement and the instrument status.

❖ To display the Exactive GC Tune program

Chose Start > Programs > Thermo Exactive Series > Tune.

Preparation
Before going on, check that the following preconditions are fulfilled:

 The main power circuit breaker switch is in the On (|) position and the electronics service switch is in the
Operating Mode position, the gas pressure is within the operational limit, and the vacuum levels are
sufficient for operating the instrument.

Refer to the Q Exactive GC and Exactive GC Operating Manual for instructions about turning on the GC
with vacuum compensation.

 The data system computer with the Exactive GC Tune software is switched on and the mass spectrometer is

placed in Standby mode.

NOTICE Unless the instrument is undergoing maintenance or service, it should be left in Standby mode when not
in use. Power supplies and trap gas are stable in Standby mode. If the instrument is in the Off mode, wait 90 minutes
before calibrating the system.▲

For information about performing these tasks, refer to the Q Exactive GC and Exactive GC Operating Manual, the
Exactive GC Software Manual or the Exactive GC Tune Help.

Getting Ions from Experiments

This topic describes how to test the instrument before you can calibrate your Exactive GC mass spectrometer
automatically.

Setting up Exactive GC Tune

❖ To set up the mass spectrometer in the Exactive GC Tune software for tuning and calibration

1. If you have not already done so, choose Start > Programs > Thermo Exactive Series > Tune to start the
Exactive GC Tune program.
 2. In the Exactive GC Tune window, on the toolbar, click the On/Off/Standby button to take the
mass spectrometer out of the Standby mode or Off mode and turn it On. When you turn the mass
spectrometer to On, you initiate the following events:
 The mass spectrometer begins scanning.
 Nitrogen flows into the C-Trap and HCD cell.
 The spectrum window of Exactive GC Tune shows a real-time display of the mass spectrum.
3. Open an existing tune file:
a. Chose File > Load Tune File to display the Open dialog box.
b. Browse for the folder C:\Xcalibur\methods\. Select a tune file.
c. Click Open to open the file. Exactive GC Tune downloads the Tune Method parameters to the
mass spectrometer.

A tune file is specific to your particular analyte. You can load the tune file again and use it as a starting
point for optimization of the mass spectrometer for a different analyte of interest.

4. Examine the pre-tune EI/CI source settings:

a. From the Instrument Control panel, open the
EI/CI source window. See the figure on the
right.
b. The default MS transfer line temperature is 250
°C.
NOTICE The MS transfer line temperature does not need to be
set to the maximum oven program temperature because the outlet
of the column in the MS transfer line is at reduced pressure. ▲

c. The default ion source temperature is 200 °C.

NOTICE The default settings for the ions source and MS transfer line temperature are 200 °C and 250 °C. These
are the minimum recommended temperatures for a typical analysis with the installed test column. For best robust
operation when running samples in matrix, it is recommended to set the ion source temperature higher than the MS
transfer line temperature. For example, maximum settings recommended for of high boiling analytes in matrix are:
ion source temperature set to 350 °C and MS Transfer line set to 280 °C. ▲

d. Turn the filament to On.

e. Set the calibration gas to EI.
f. Set the ionization mode to EI or CI depending on the installed ion volume.
g. If using CI mode, set the CI gas flow to 1.5 mL/min.
h. Set the CI gas port to A or B as installed.
i. See the Q Exactive GC and Exactive GC Operating Manual for instructions to configure the CI
gas type installed at Ports A and B.
j. If you have made any changes in the window, click Apply.

NOTICE Select the Hot link check box to apply all changes in real time. A green frame around the parameter box
indicates an active hot link. ▲

5. Set the scan parameters for tuning and calibration:

a. From the Instrument Control panel, open the Scan parameters window. See the figure on the right.
b. Set Scan type to Full MS.
c. Enter a scan range between m/z 50 and 550.
d. Set Fragmentation to None to specify that the HCD fragmentation option is turned off.
e. Select 60000 for Resolution.
 f. Set the ion polarity mode to Positive.
g. Set the total number of microscans to 1.
h. Set Lock masses to Off.
i. Select 1e6 for AGC target.
j. Specify a maximum injection time of 50 ms.
k. If you have made any changes in the
window, click Apply or select the Hot link
check box.

The mass spectrometer is now properly set up for tuning and

calibration in EI mode.
Testing the Mass Spectrometer

In this section, you can test whether the mass spectrometer is operating properly. Admit a low flow of calibration
gas for EI mode – containing perfluorotribuylamine. Monitor the real-time display of the mass spectrum to ensure
that the signal is stable.

1. At the top of the spectrum window, notice the values for the inject time (IT) in milliseconds and the
normalized target level (NL). Reasonable performance on a clean, tuned system in EI modes shows the
Inject Time is typically less than 5 ms and the height of the tallest peak is typically greater than 1 –e8. See
figure below.
2. Observe the values for NL and IT in the spectrum window. Check the ion current and readback values
while scanning calibration gas:
 Is the signal present?
 Is the signal stable? Check in
the instrument status window
that the TIC variation has
stabilized at 10% or less from
scan to scan (green check
mark). See the figure on the
right.

If you answered “yes” to the questions in step 2, then your mass spectrometer is operating properly.

If you answered “no” to either of these questions, try the following troubleshooting measures:

 Ensure that the EI ion volume is installed in the ion source cartridge.
 Ensure that the ion source cartridge is properly inserted into the ion source block.
 Ensure that the transfer line nut is not leaking air. It will leak if cooled from high to low temperature
quickly.
Your mass spectrometer is operating properly in EI mode. You are now (nearly) ready to calibrate the mass
spectrometer.

Leak Check the MS system

The Exactive GC mass spectrometer needs to be “leak free” to ensure that background noise remain as low as
possible. To conduct a leak check be sure to leave the perfluorotributylamine spectra scanning as directed above.

❖ To conduct a leak check on the Exactive GC mass spectrometer for positive ion mode

1. Click the On/Off/Standby button to take the mass spectrometer out of the Standby mode
and turn it On.
2. Turn the Filament on and set the Calibration Gas to EI.
3. Click the title bar of the Evaluate window to display it.
4. In the Positive Ion Evaluation sub-window, select the Leak Check check box.
5. In the Evaluate window, click Evaluate to start the automatic calibration procedure.
6. An icon appears in the toolbar to indicate an active procedure.
7. A message box appears when the leak check procedure is completed.
 The leak check will automatically pass when less than 10%, however, you should have a strong
preference to it being less than 5% for the best data quality.
 If the leak check fails, you will need to tighten the nut/ferrule adapters at either the column inlet of
column exit/mass spectrometer inlet with the ¼ inch wrench (or both).

NOTICE You should not OVERTIGHTEN the ferrules as this will warp the fitting and create a worse leak in the
system. If you do not feel comfortable preforming this step, PLEASE SEEK IMMEDIATE ASSISTANCE before
proceeding. ▲

8. Record the leak rate in the log book for the Exactive GC system before moving on to the next step.
9. Click OK to close the message box.

Mass Calibration
The Exactive GC mass spectrometer should be calibrated to ensure the mass accuracy of the results. Usually, it is
sufficient to perform a mass calibration only. Before starting the calibration, check again that you have a stable ion
signal: Focus on the injection time (IT) and on the normalized level (NL) in the header information on the spectrum
window.

NOTICE If the system was in Off mode before, it is necessary to put the instrument into On mode for at least 90
minutes before a mass calibration is performed. The filament and calibration gas do not need to be on during this
time. ▲

Positive Ion Mode Calibration

❖ To calibrate the Exactive GC mass spectrometer for positive ion mode

1. Click the On/Off/Standby button to take the mass spectrometer out of the Standby mode or Off mode and
turn it On.

NOTICE Any time the system is placed in standby or off, the calibration gas and filament are turned off. The
Calibration gas will show the last level set (EI or CI) when the system was on, when in standby or off mode and the
displayed level will be set on again when the system is set On. The filament will show off when placed in Standby
or Off mode and it must be set to on in order for the filament to turn on when setting the system to On. The filament
and calibration gas may be turned on when in standby mode by making the appropriate selection and selecting apply
or using the Hot link. ▲

2. Turn the Filament on and set the Calibration Gas to EI.

3. Click the title bar of the Calibrate window to display it.
4. In the Calmix Calibration sub-window, select the MS Mass Calibration (pos) check box to specify a mass
calibration for the positive ion mode. See the figure on the right.
5. In the Calibrate window, click Calibrate to start the automatic calibration procedure:
a. The Calibrate window
indicates the completed
percentage of the calibration
process by a progress bar.
b. Exactive GC Tune displays a
message box with information
about the active calibration
procedure and the currently
calibrated parameter.
c. The analysis graphs window
displays a graphic representation of the calibration process for the currently tuned parameter.
d. The messages window shows a record of the calibration.
e. A link to a report of the calibration is available in the Reports menu.
6. An icon appears on the toolbar to indicate an active procedure.
7. A message box appears when the calibration procedure is completed. The message box also
informs you that Exactive GC Tune has automatically saved the calibration results. Please
record the rms error values in the Exactive GC logbook.
8. Click OK to close the message box.

Negative Ion Mode Calibration

Before performing measurements in the negative ion mode, the mass spectrometer must be set up for and calibrated
in NCI.

❖ To calibrate the Exactive GC mass spectrometer for negative ion mode

 1. Click the On/Off/Standby button to take the mass spectrometer out of the Standby mode or Off mode and
turn it On.
2. Insert the CI ion volume into the ion source cartridge and reinsert the ion source cartridge.
3. Change the CI Gas Type to methane. Use methane gas for all built-in calibrations.
4. Set the CI Gas Flow to 1.25 mL/min.
5. Turn on the Filament and CI level calibration gas.
6. In the spectrum window of
Exactive GC Tune, observe the
mass spectra of the singly-charged
ions of the negative ion mode
calibration gas.
7. Click the title bar of the Calibrate
window to display it.
8. In the Calmix Calibration sub-
window, select the MS Mass
Calibration (neg) check box to
specify a mass calibration for the
negative ion mode.
9. In the Calibrate window, click Calibrate to start the automatic calibration procedure.
10. The Calibrate window indicates the completed percentage of the calibration process by a progress bar. See
the figure on the right.
11. A message box informs you when the calibration procedure is completed. Please record the rms error
values in the Exactive GC logbook.
12. Click OK to close the message box.

Manual Injection/Data Acquisition

NOTICE This section is for those who plan to manually inject their sample into the GC system. If you intend to
utilize the autosampler for your injections, please skip this section and proceed to the “After Operation” section
below. ▲

After you have entered the scan parameters of your experiment, you can finally start to acquire data.

❖ To perform data acquisition from Tune

1. Specify the acquisition parameters in the Acquisition window:

a. In the Destination file field, type the full path (Drive:\path\file name) of the raw file that is used to
store the acquired data. Alternatively, click
the 💾 button to the right of the text box to
open a dialog box to browse your
directories. See the figure on the right.
b. Click into the Acquisition time field to
open the dialog box. Select the
continuously option button to specify that
the acquisition will continue until you click
Stop. The Acquisition time field shows
continuously.
2. Click the On/Off/Standby button to take the mass
spectrometer out of the Standby mode or Off mode
and turn it On.
3. Turn on the filament.
4. In the Acquisition window, click Start to initiate the data acquisition. The spectrum window shows a real-
time display of the mass spectrum. The data are saved in the selected raw file.
 5. Click Stop to stop the acquisition.

After Operation
When the measurement is finished, Thermo Fisher Scientific recommends leaving the Exactive GC mass
spectrometer in Standby mode and not in Off mode to provide the best mass accuracy for the next analysis.

❖ To place the Exactive GC mass spectrometer in Standby mode

1. Turn the Filament off and set the Calibration Gas to Off.
2. Click the On/Off/Standby button to take the mass spectrometer out of the On mode and turn it Standby.
Setting up an Automated Sequence
The following example describes how to create an instrument method in the Instrument Setup view of the
Thermo Scientific™ Xcalibur™ software application. You can then use the instrument method to acquire data
with the Exactive GC mass spectrometer. The tune file used in the instrument method is used for data
acquisition. Make sure in the Exactive GC Tune software that your tune file has appropriate settings and save it
accordingly.

Creating the Instrument Method

❖ To set up an instrument method
1. Display the Instrument Setup page in Xcalibur:
a. Choose Start > Programs > Thermo Xcalibur > Xcalibur to display the Xcalibur Home Page
window.
b. Choose GoTo > Instrument Setup to display the Instrument Setup window.
c. If necessary, click Exactive GC - Orbitrap MS in the View bar to display the Method Editor.
2. In the Method Editor, drag an experiment symbol (for example, Full MS - SIM) from the Experiments pane
(lower left) to the gray bar in the Graph pane. A corresponding time bar is displayed in the Scan Groups
pane.
3. Edit the fields of the Properties pane (right) to set up the experiment parameters. Refer to the Q Exactive
GC Software Manual or the Exactive GC Tune Help for a description of the available parameters.
4. Assign a tune file:
a. On the External Hardware pane, click the icon to expand the Tune Files pane.
b. In the Properties of Tunefiles pane, click the Base Tunefile field.
c. Click the button to open a dialog box.
d. Browse for the tune file (for example, ab.mstune) and assign it to the experiment.
5. Click on the toolbar to save the new instrument method:
a. In the Save As dialog box, enter the name (Full_MS.meth, for example) and the location for your
instrument method. Then click Save.
b. In the File Summary Information Dialog Box, enter a comment as header information for your
instrument method. Then click OK to close the dialog box.

The instrument method can now be used to acquire data with your Exactive GC mass spectrometer, either from
the Xcalibur Sequence Setup, TraceFinder, or from the Exactive GC Tune window. The next figure shows the
example of an instrument method that was set up as described above.
Creating an automated sequence for data acquisition
❖ Configuring a sequence
1. Open TraceFinder
a. Choose Start > Programs > Thermo TraceFinder 4.1 > TraceFinder 4.1 General Quan
2. Launch the Quick Acquire Sample menu
a. Choose Tools > Quick Acquire Sample
3. Complete the Sequence Table with the following fields:
a. Filename: the name of your sample
b. File Path: where you want to save the data on the computer hard drive
c. Vial Position: the position of the vial in the tray/sample holder; the number is located in the
UPPER LEFT corner of the hole
d. Injection volume: 1 uL for nearly all injection in both splitless and split mode
e. Instrument method: your preferred instrument method for analyzing the samples

4. To add more samples to the sequence, click the “Samples: # Add button.
5. Complete all relevant fields in Step 3 above for each sample you inject. We recommend that you
ALWAYS begin your analysis with a “blank” of the solvent that your samples are solvated in.
6. Once you have added the appropriate number of injections/samples to your queue, you are ready to begin
the sequence.

a. Click the Acquire: Sample to inject a single sample form the queue after
highlighting that sample of interest OR

b. Click the Acquire: Sequence to inject all sample in the listed spreadsheet.
7. Your start menu should look
something like this:
8. Please VERIFY that the sample rows
that you intend to run are referenced
in the “Samples” field of this menu.
9. Also VERIFY that the “Post-run
system state” is set to Standby.
10. You will receive a confirmation
prompt that your samples have been
submitted to the queue. You can
“OK” this message.
11. Data will present itself in the
TraceFinder window in real-time;
however, only the chromatogram will
be viewed.
12. You can review your data in
Xcalibur > Qual Browser post
acquisition.