Gas Chromatography-Mass Spectroscopy Standard Operating Procedures

(GC Clarus 680 and MS Clarus SQ 8T)

In this analysis, we use BTEX (Benzene, Toluene, Ethyl Benzene and Xylene) as standard sample, Perkin
Elmer Elite-5MS chromatography column with 30 meter length and 0.25mm diameter and TurboMass as
GCMS operating software.

Part A. Hardware and column installation

Connecting new column to the Split/Split less injector
1. Unwind 20 cm (8 inches) from one end of the column

Elite-5MS Column

2. Insert a septum, 1/16-inch column nut, and 1/16-inch graphite ferrule (0.8-mm i.d., or 0.5-mm i.d.) over
one end of the column
3. Cut about 1 cm (3/8 inch) from the column end using a wafer scribe. Break off the tubing at the score
mark so that the break is clean and square.

4. Locate the capillary injector fitting inside the oven

Injector A

5. Position the septum on the column as shown in the following table

Injector A

Injector B

6. Insert the column into the capillary injector fitting. Hand-tighten the column nut ¼ turn past finger tight.
Using two ¼-inch wrenches tighten the column nut only until the column cannot be pulled out of the
fitting. Do not over tighten column nuts. Over tightening can cause damage to the ferrule and/or
column.
 Condition the Column: At the PPC panel, set the carrier gas flow at 1 mL/ min, oven temperature at 250
o
C (isothermal) for 2 hours.

Connecting a new column to Clarus SQ8T MS

1. Uncoil 50 cm (20 inches) from the new column. Place the column on the column hanger in the oven.

2. Slide the septum, 1/16-inch column nut and 1/16-inch graphite/Vesper ferrule over the end of the
column.
3. Slide the septum, column nut, and ferrule along the column to the position shown below. Score
and break 1.0 cm off the end of the column. Wipe the column with a methanol dampened lab
tissue to remove fingerprint on the column or wear glove during the installation of the column.

4. Carefully insert the column end to the transfer line into mass spectrometer until it is visible inside the
Plug Handle and Sight.

Plug Handle and Sight insertion

The correct position of the column end should be aligned with the edge of the circle
5. Hold the column and slide the column nut and ferrule to the transfer line and tighten the nut until hand
tight. Slide the septum until flush against the rear of column nut and this mark as the position of the
column end.

6. Pull back the column until the distance between nut and septum is 10mm. Then use the 10 mm
positioning tool (Gauge) to set the column nut at this position by placing the slot in the 10 mm Gauge
over the column between the nut and the septum. At this point be careful to avoid moving the septum
from its position on the column.
7. Remove the Plug Handle and Sight tool and reinsert back the inner source. Close the SQ8T MS access
door.

8. Using the 5 mm and 1/4 inch crescent wrenches, tighten the column nut until the ferrule is crimped
onto the column, making leak-free seal and making sure that the 10 mm spacing is retained between the
end of the column nut and the septum. Remove the 100m Gauge and column is now completely
installed into the MS.

9. Setup all the connection cable from GC-MS to the computer and carrier gas flow piping into GC-MS.
Part B: Preparing MS for Calibration
Starting up the GC-MS system and TurboMass Program

1. Turn on carrier gas (default Helium gas) and check inlet pressure (70-100 psi).
2. Turn on GC-MS and at the MS check for red light  yellowish green light blinking.
3. Power on PC and wait for Windows loading into stable.
4. Make sure connection cable from GC-MS is connected into PC Ethernet port and check for connection
status in the Control Panel.

5. Turn on TurboMass.

Create a new Project

1. Click ‘File” then ‘Project Wizard’ and enter ‘Project Name’ and ‘Description’ in the pop-up window.

2. Create project using ‘Default’ or existing project as template or Create new project
Air/Water check (Leak Test)

1. Open TunePage and load the default.ipr file.

TunePage icon

2. Before doing anything, check the ‘Inlet Line Temperature’ and ‘Source Temperature’ and make sure
both temperatures value is at 50c. This is to ensure that the inlet and source does not heat up
immediately as system try to read the last saved memory. When turning on the system as it may oxidize
and become black when heated up with high oxygen content inside.

Set to 50c and press ‘Enter’

Previous temperature memory,

reduce back to 50c to prevent
blackening of the inlet and inner
source
3. Click ‘Option’ then ‘Pump/Vacuum system ‘On’ to introduce vacuum into the system. Turbo molecular
pump sucks the air into the Roughing Pump, vacuum gauge goes down slowly.

4. Wait for status showing ‘Vacuum OK’

5. Key in the masses, span and gain as the table below into TunePage.

Mass (amu) Identity Span Gain

4 Helium 4 1
18 Water 4 1
28 Nitrogen 4 1
32 Oxygen 4 1

6. Click ‘Press for Operate’ to check for the leaking in the system
7. In normal cases, water percentage is high initially as introduce moisture to the system when changing
the column, heat the oven to 250c for 30 minutes and then cool down to 50c to remove all the
water/moisture in the system. Repeat the process if the water mass percentage is still high.

8. Tighten the 1/16’ nut at the transfer line and injector at every heat-up and cool-down time.

- In a leak free system, the percentage of helium mass must be 100%, since is a carrier gas; percentage
of water mass is 10%; mass of nitrogen should be less than 10% and oxygen mass should be lower
than 25% of nitrogen percentage and 5% of helium mass. Leaking is presence if percentile is more
than stated above and turns off filament immediately to prevent it from oxidizing.

9. Increase the temperature of ‘Inlet Line Temperature’ and ‘Source Temperature’ to 250c after 30
minutes pump down time, as in this period of time, water content is low enough to prevent damage to
the inner source and inlet line. Now you have a leak free system and safe condition to run the GC-MS.

Increase temperature to 250c

and press ‘Enter’
 Tuning

1. Before starting the tuning of the system, click ‘Save As’ to save a new tune file as tunexxxxxx.ipr,
example: tune15052013.ipr. This file will be located in C:\TurboMass\ProjectName\ACQUDB.

2. By using PFTBA (perfluorotributylamine) as reference gas, input the masses, span and gain as below in
the TunePage.

Mass (amu) Span Gain

69 4 1
131 4 1
219 4 3
502 4 30

3. Click to open Reference Gas Valve and allow reference gas flow in the system.

4. Press for Operate’ and print screen the TunePage as record for easy viewing.
5. Click ‘option’, open ‘UltraTune’ and ‘Custom/Auto Tune’.

6. Click ‘Setup’ to custom tuning.

 - AutoTune initiated

7. Monitor the peak and percentage of the graphs, picture below show desired graph shapes and
percentile.

8. Click ‘Press for Standby' and no need to pump out reference gas if need to continue carry out the mass
calibration process.
Mass Calibration

1. Before starting the calibration of the system, click ‘Save As’ to save a new calibration file as
calxxxxxx.cal, example: cal15052013.cal. This file will be located in
C:\TurboMass\ProjectName\ACQUDB.

2. Click ‘Calibration’, ‘Calibrate Instrument’

Reference file icon, which displays

mass spectrum fragment that
scanned for system calibration.

Reference file list

3. Click ‘Calibrate’, ‘Start Acquisition’.

Tick this option to generate report

for each three type of calibration
method, default printed out in
digital PDF format
- Scanning Calibration Report.
- Scan Speed Calibration Report.
4. Click ‘Process’, ‘Verification from File’ to verify the calibration parameters. Save the calibration file again.

5. Save the Tune file as .ipr again.

6. Pump out reference gas and ‘Press for Standby’

Part C: Data Aquisition
Create GC method
Method is based on analysis of BTEX standard in methanol as an example and injector is installed at injector
B, which is a PSSI injector site.
1. Click on the GC method icon on the TurboMass program.

- GC method main screen with method display (default setting).

2. Key in type of documentation here.

3. Data channel ‘A’ is for FID, choose ‘None’ if using MS as detector.

- Default setting for oven.
4. Temperature setting for oven. Set the temperature of the oven to 10c below the maximum of the
installed column.

5. Injector A is CAP injector, set it to ‘0’ when not used, while injector B is PSSI, temperature
programmable, set it ‘On’ and insert the temperature programming into the table above.
 - Carrier gas default value for injector B.

6. Carrier gas setting according to chromatography method with flow value of 1ml/min.
7. Set Detector A temperature to ‘0’ to turn off the detector, as it’s a FID, while we are using a MS
detector.

8. Save the GC method as xxxx.mth into the ACQUDB folder in TurboMass folder.
Create MS method

1. Click on the ‘MS Method Editor’ icon.

2. Click ‘Solvent Delay’ icon.

Choose either full scan or selected ion recording

3. Key in the solvent delay, taking BTEX in methanol as example, solvent peak will be shown in the
spectrum if solvent delay is not set, causing the pe4aks for the BTEX sample to be very low.
 - Default setting.

4. Method setting.

5. Save the MS method as xxxx.exp into the ACQUDB folder in TurboMass folder.
Create a sample list

1. Right click then click ‘Customize Display’ then ‘OK’, to display the selected information in the TurboMass
interface.

2. Key in the data as below in the Sample List

• Vials - key in vials position by sequences.

• File names - Key in sample name (BLANK, STD 1 and etc).
• MS method - use the dropdown and select the method accordingly. .exp Key in any details here for
• GC method - use the dropdown and select the method accordingly. .mth extra information purposes
• Injector - select injector A as we are using GCMS.
• Sample ID - key in sample ID for each vial.
• File text - this is not mandatory to key in. Can leave it blank if this not required.
• Condition - this is not mandatory to key in. Can leave it blank if this not required.
• Sample type - key in sample type. Example = analyte. Key in ‘calibration’ if need to run
• MS tune file - use the dropdown and select tune file accordingly. standard calibration for the first time
3. Highlight the row of sample(s) which need to run the analysis

4. Press start to run the sample according to the GC and MS method.

5. Press ‘Ok’ in the pop-up window and GC-MS will run automatically.
Part D: Data Processing (Method Qualification)
1. Check the chromatogram after the run has finished, click ‘View Chromatogram’.

2. Click on the clock icon to view real-time chromatogram during the progress of analysis.

3. Right click on any of the peak of the chromatogram window; it will pop up a ‘Spectrum’ window.
4. Press ‘F1’ on the Spectrum window it will search automatically using NIST library search.

7. We uses BTEX as sample and the spectrum from the chromatogram show that this peak is ortho-xylene.

8. Another peak at RT 2.25 shows a compound of benzene with probability of 79%.

9. Select another peak nearby to the selected peak to further identify the compound with greater
probability
10. Click ‘Process’ then ‘Integrate’ to view the difference of one peak to another

11. Click ‘Process’ then ‘Purity’ to check the purity of each peak. If the purity of each peak if not high enough
or in a desired value, choose a chromatography data from a high sample concentration.

12. From the chromatogram, major peaks has 100% purity show is formed by 100% of a single compound,
run the MS another for confirmation if desired.
Part E: MS Method for Acquire Data (Full Scan + SIR for BTEX)
By using Full Scan method in the mass spectrometer, we can expect to see 5 distinct peaks of retention time
from BTEX compound. By analyzing the compound using NIST search through the retention time peaks, we can
roughly identify the 5 peaks are given by benzene, toluene, ethyl benzene, meta & para-xylene and ortho-
xylene. To further increase confidence of the identification, we use the BTEX sample with 100ppm. Since we
have 5 different retention time peaks, we can conduct a full scan together with SIR scan to further identify the
compound presence in BTEX. SIR stands for selective ion recording in MS, scans a selective part of mass
spectrum according to the different elution time of compound out from the chromatography column. Each SIR
scan will generate a specific mass spectrum of individual scanning period, which the spectrum data is used in the
data processing and quantifying method.

Compound Retention Retention Mother ion (Base Qualifier ion/amu

time/min window/min Peak)/amu
Benzene 2.119 2.1-2.3 77 51,50
Toluene 2.962 2.8-3.1 92 91,65
Ethyl Benzene 4.122 4.0-4.3 106 91,51
Meta & Para-Xylene 4.250 4.1-4.4 106 91,77
Ortho-Xylene 4.571 4.4-4.7 106 91,77
Data from BTEX 100ppm spectrum.

1. Open the MS Method Editor. It will show the full scan method that being set during the initial MS scan.
2. Click on the SIR icon to manipulate the SIR scan.
3. It will show a ‘Function: 2 SIR’ pop-up window and key in the mass of mother ion, qualifier ion, dwell and
retention window. Click ‘Add’ when finish a set of molecular mass. Example of benzene SIR according to
the table prepared above:

4. Click ‘Ok’ when finish adding the data and the Scan Function window will show SIR of benzene from time
2.1 to 2.3 minutes.
5. Continue adding the data of other compound

6. Click ‘Save’ to save the SIR scan as BTEX_SIR.exp.

7. Initiate the GCMS run another time.
8. When the GCMS run has completed, click on the ‘View Chromatogram’ icon using BTEX 100ppm sample.
9. Click ‘Display’ then ‘Tic…’ to display the TIC Chromatogram

10. Highlight function number 2 to 6 by holding down ‘ALT’ button and select the ‘Add Trace’ option to stack
up the chromatogram of every SIR over the full scan chromatogram. Then, click ‘Ok’.
11. It will display a chromatogram with 6 functions. The small square beside each chromatogram indicates
the highlighted chromatogram.
 Part F: Data Processing (Method Quantification)
By generating the every chromatogram for each compound in BTEX, we can use the chromatogram to
quantify our sample by creating a calibration curve. By using 100ppm BTEX as standard sample, create a
calibration curve using individual SIR chromatogram and quantify for every other BTEX’s ppm analyte.

1. Select ‘Method’ then ‘Edit Method’.

Insert compound name

Function number
according to the
chromatogram. Eg.
Benzene spectrum is
function number 2

Insert retention time

Click ‘Append’ to add to EG

bottom of the list; ‘Insert’ to
add into middle of the list

Paste individual SIR spectrum

2. In the spectrum window, click ‘Edit’ then ‘Copy Spectrum List’, then paste the spectrum into the Method
Editor window by clicking ‘Edit’ then ‘Paste Spectrum’.
3. Fill in all the data for each of the five compounds in the BTEX compound.
4. Save the file as BTEX.mdb by clicking ‘File’ then ‘Save As’ into the MethDB folder.
5. After editing the quantify method, highlight the samples that wanted to be quantify, click ‘Quantify’
then ‘Process Samples’

Integrate sample Integrates all the sample data files named in the peak list.
Calibrate standard Uses Integration results to form Quantify calibration curves
from all the data files in the Sample List.
Quantify samples Uses Integration results and Quantify calibration curves to
calculate compound concentrations from all the data files in
the Sample List
Print quality report Produces hard copies of the results of integration and
quantification.
Quantify from sample to sample Sets the range of samples in the sample list that will be
quantified.

6. Check the quantify sample number is matching with the selected compound in the Sample List, then
click ‘Ok’. In the example we uses sample number 8 to 11 for quantify.
7. A pop-up window ‘Quantify-Summary’ showing the summary of response and ppm of each BTEX
concentration quantified according to the BTEX 100ppm data.
 Show current peak

8. Click on the ‘Show Current Peak’ icon to display the calibration curve of individual BTEX concentration.
9. Click ‘Display’, then ‘View’. Tick on ‘Curve’, ‘Residuals’ ‘Show summary window’ then click ‘Ok’ to display
the information. In this example, uses BTEX 5ppm as display example.
- In this window it displays the data of calibration curve, chromatogram, review spectra and ion ratio
data. Double click on #8 to display all the information of BTEX at 5ppm.

Double click here