GCMS Quick Start Manual: LC Mixture Analysis
This document is intended as a quick start manual for doing a basic liquid crystal
mixture analysis. It will describe the most common hardware configuration and the
most common parameter settings chosen by most users. If you require assistance
for other hardware configurations or additional parameter selections, please request
assistance from IPP staff.
Required Hardware:
1. Computer with Xcalibur Home Page software ver 1.4 SR1 running on a
Windows-based system.
2. Focus GC gas chromatography instrument by Thermo Electron Corporation, ver
1.4 SR1
3. DSQ mass spectrometer instrument by Thermo Electron Corporation, ver 1.4.1
SP4
4. Sample Insertion syringe, glass barrel, steel needle, 10 L capacity
5. Rotary Vane vacuum pump, Edwards, Model RV3
6. Ultra High Purity He (99.999) carrier gas, and gas regulator
Turning on the System:
(NOTE: It is recommended by the manufacturer that the system is left running in
standby mode with the carrier gas flowing at all times for stability of the
equipment. However, during period of extremely low usage, the instrument will
be shut down. The correct start up and shut down procedures are important as
improper starts and stops will damage the high temperature components of the
system. The start up and shut down procedures should only be performed by
qualified LCI Staff. If you need to use the GCMS system, call LCI Staff a day
ahead of time and the machine will be started up properly and stabilized before
the day you arrive to use it, and will by shut down by LCI Staff after you are
finished. Any damage to the equipment caused by unauthorized startup or
shutdown will be the financial responsibility of the person who performed the
unauthorized procedures. The Start Up and Shut Down procedures are only
included in this document for completeness in the in-house operational manual for
this system.)
1. Turn on He carrier gas source, main tank valve. Set output pressure to 20 PSI.
a. NOTE: DAMAGE occurs if Mass Spec turned on without column flow.
This forces air through the column and will damage it and this large air
leakage into the Mass Spec will cause the ion source to require cleaning.
2. Turn on the Mass Spectrometer (MS) by flipping the white power toggle UP
located at the rear, right, top of the instrument.
a. NOTE: This will turn on the Rotary Vane Pump, allow the Fore pressure
to reach proper operating pressure and then turn on the Turbomolecular
pump.
3. Turn on Gas Chromatograph (GC) with the white power toggle located at the rear,
right, bottom of the instrument.
4. Start the Xcalibur software by double clicking the Xcalibur icon on the computer
desktop to establish communication and initialize each instrument for use.
a. NOTE: Wait a minute or so for the Mass Spec to initialize, then notice a
message that will say ‘Insufficient Vacuum’. Leave this message
displayed. It will take about 2 minutes for sufficient vacuum to be reached
and the message will change to: “Vacuum OK. System will start in 10
min” and the 10 min timer will run backwards as the instrument counts
down to system start.
5. In the ‘Status’ list on the left side of the Xcalibur screen, the two instruments
(DSQ-MS, Focus GC) are listed at the bottom of the tree. Click once on the
Focus GC. A second ‘Status’ tab window will now appear below. Scroll the
window all the way to the bottom. Click once on the button labeled ‘Keypad’.
The Keypad is the direct parameter control for the GC instrument hardware
conditions. Earlier versions of the Focus GC product line had the keypad
physically located and accessible on the front of the actual GC unit so parameters
could be adjusted without accessing the software. Our instrument does not have
this direct physical keypad, but the manual that came with the system still
describes parameter setting as if this is possible. Whenever the manual says to
use the keypad to set a parameter, the software must be launched and the keypad
accessed as described above.
6. The Keypad has a hierarchy of parmeters and variables. If you click on the down
arrow with the mouse, you will step through the main parameters in this order:
Oven, Inlet, Carrier, Detector, Signal, Autosampler, Others. Each of these has its
own subvariables. When the desired parameter is selected, hit the blue ‘Enter’
button and the menu will drop into the subvariables for that parameter.
7. For now, hit the down arrow until Detector is displayed and then hit the blue enter
button. MS X-Line (MS Transfer Line) is displayed. Type in 2,5,0 on the blue
number pad and now ‘250’ will be flashing in the set point location when you are
typing and this will stop flashing when you hit enter. Now you will notice the
‘Actual’ temp begin to climb toward your new value, from its standby temp of
50C. It should take about 3 min to climb frlom 50 C to 250 C. The instrument
will overshoot a little and then stabilize.
8. Close Keypad window.
9. Minimize Xcalibur software.
10. Double click DSQ Tune icon on computer desktop. Select menu item
‘Instrument’ and pull down to ‘Set Temperature’. Type in Ion Source to 200 C
(standy by mode will have it set to 25C. Close DSQ Tune
11. Maximize Xcalibur software again. In DSQ Parameters, Heaters, Ion Source will
now show set point 200 C, and Actual will begin to climb toward that value. This
will take about 30 minutes or more to stabilize.
12. Also in DSQ Parameters, Vacuum will show about 200 mtorr close to startup.
The system must be run overnight with all parameters and heaters set, allowing
the vacuum and temperatures to stabilize before the instrument can be run.
Overnight, the vacuum will pump down and stabilize to about 30 mtorr.
13. In Focus GC, Keypad, parameter Oven, set oven to 150C and hit enter.
14. Come back the next day for use of the instruments.
Sample Run Procedure:
NOTE: This instrument is capable of detecting and analyzing materials in the
pictogram range since it is new. If the instrument becomes dirty through misuse, the
sensitivity and accuracy of results will suffer.
1. Using a pre-weighed, sterile glass vial, place a very small amount of your test
material into the vial, the smallest amount you can weigh. Make sure the
instruments you use for transferring the material to the vial and perfectly clean.
Use an ultrahigh purity solvent to dilute and dissolve your material, one suitable
for GCMS work such as MeOH, Acetonitrile, Dichloromethane. It is best to use
solvents that are very low boiling points and that have very few carbons, and do
not engage in strong hydrogen bonding or at least, do not have more than one –
OH group. Higher boiling solvents will not flash off inside the machine and
cause the machine to shove a large amount of solvent into the ion source and
column which will cause things to burn out. The instrument is only designed to
handle extremely small quantities of material and a large dose of solvent is very
bad for it.
2. Stir the contents lightly with the transfer object (clean spatula, glass eye dropper).
Do not put on a cap and shake up the contents to mix. All of the caps are either
plastic or have liners that will release some of their formulation to the solvent if
touched. Only very clean glass can be used. Once the material is mixed, a cap
may be placed on to prevent contamination and evaporation, but the contents of
the vial must not come in contact with the capo at any time.
3. Weigh the vial with material and solvent in and subtract the preweigh. Calculate
the concentration of your material. A good starting place for concentration is
about 100 pg/ml. If yours is higher, you could figure out what dilution to make to
get there from what you have, preweigh a new vial, load the required dilution of
GC solvent into the vial and then dispense a small amount of your first vial
contents into the second vial. If you choose to go with a concentration that is too
high, you may find that your GC elution plot is topped out and linearity will
suffer. If a material is present is such high amounts as to top out over a range of
elution times, other material will similar elution times will be masked from view.
The MS will still find them but their MS plots will be complicated by having
several materials get fragmented at overlapping elution times. But, if your
concentration is too low, you will get GC and MS data that looks very
complicated and this will be due only to impurities inherent in the instrument such
as from the injection needle septum, the solvent, the vials/caps, etc. To be sure
that you can recognize a false plot of a material that is too weak to be detected,
you should run a blank injection using only your solvent to see what the baseline
GC and MS data for the instrument look like. Then if your sample plots look like
that, your sample concentration is too weak.
4. The machine must have a ‘Method’ or a program sequence to operate. First, one
must be created for your intended purpose. A general Method has been created
which will suffice for all the most common needs. This method is called
USR_runfile.
5. To load the method, open up the Xcalibur software on the desktop.
a. In the instrument icon map on the right side of the screen, click on
Instrument Setup.
b. Go to menu heading ‘File’ and pull down to the bottom where
USR_runfile should be listed as a recent past opened file. Silect this file.
(If the file is not listed, choose ‘File’, ‘Open’ and you will be directed to
the folder C:\Xcalibur\Methods where the file USR_runfile is located and
load it)
c. There are two large square buttons on the left side once the method is
loaded, ‘DSQ’ and ‘Focus GC’. Click on DSQ (the top one). The little
green triangle in the corner of the button will be lit if it is the one currently
selected.
d. Many parameters are listed here. In general nothing will need to be
changed. The exception would be if a high boiling solvent HAD to be
used for solubility reasons and in this case, a longer start delay would have
to be built in. Currently it is set to 5.00 min. This delay allows the solvent
to flash off and purge away before the filament is turned on so that the
majority of the solvent is gone and doesn’t burn the filament when it turns
on.
e. Click on the large ‘Focus GC’ button next. Many tab windows are found
here with many run parameters listed in each tab window. Again, these
parameters are set up to cover most expected uses and should not need to
be changed. The exception might be to raise the Oven, SSL inlet, and
Transfer line temps in the case of needing higher temps for a troublesome
material. This will NOT normally be necessary!
f. Once all parameters have been set (hopefully unchanged), while still in the
Focus GC area, select the main file header ‘Focus’ and pull down and
select ‘Send Method to GC’.
g. IF the method was changed in any way, you must now use File, Save As
to give the method a new name so that the default method everyone uses is
not overwritten. Store a renamed Method file in the default folder
C:\Xcalibur\Methods so that it can be easy to find later. Write down the
name of your method file in your notebook so you will know what you
used, although this information is stored in the datafile header parameters
if you forget.
h. Close Instrument Setup.
6. Back on the main Xcalibur software window now, showing the icon map on the
right side, select Sequence Setup. This is where you will name your current
sample run, tell the machine which method to use to run it, and where to store the
data.
a. Click once in the ‘Filename’ text box and type a new filename.
b. Double click in the ‘Inst Meth’ box and select your method file
(USER_runfile), and then hit ‘Open’.
c. Double click in the ‘Path’ text box and select the folder where you want to
dump your data. (A subfolder \User and several subfolders |User_1, etc.
have already been created for this purpose since ‘Create new folder’ in this
window is not an option. You can direct all of your data to one of these
and then find the folder and give it your name later, or you can do this
ahead of time.
7. Now that the method is set and sent to the GC, and the sequence is set and ready
to run, you need to load a syringe, inject the sample and run the instrument and let
it do its thing.
8. There is a microliter syringe with a very narrow metal plunger located at the
instrument. The previous user has already cleaned it thoroughly…
a. Have an empty vial next to it.
b. Draw in some of your pure solvent to about halfway up the syringe.
c. Eject the solvent into the empty vial.
d. Repeat this 4-5 time to make sure the syringe is cleaned out and only has
your solvent in it.
e. Next, draw up about 0.1 uL of your pure solvent.
f. Remove the syringe from the solvent and draw up about 0.2-0.3 uL of air.
g. Wipe the tip off gently with a clean wipe and gloved hand.
h. Your syringe is now ready to inject, but the machine is not. Set it down on
its side without letting the needle tip touch anything.
9. The next series of steps will happen quickly, one right after the other, and you
must be ready to inject the syringe efficiently. On the left side of the window, the
drop down tree listing DSQ and Focus GC are listed at the bottom as before.
Focus GC should be the one selected so you can get to the Keypad during this
next series of steps.
a. In the main window’s series of many buttons along the top, click on the
‘Run Sample’ button (7th button from the right, a half filled test tube with
a right-pointing green arrow through it).
b. Click the ‘Start Analysis’ button (looks like a VCR Play button, a dark
green triangle pointing to the right, 4th button from the right)
c. Scroll down all the way in the Focus GC parameter window on the lower
left side and click on the ‘Keypad’ button again. There are a series of
indicator lights along the left side of the keypad frame. A yellow light
alongside Standby will be lit initially. When you hit the ‘Start Analysis’
button, the machine will fire up and then the light next to the line ‘Ready
to Inject’ will turn on. When this happens it is time to quickly inject your
sample.
d. Shove (carefully) the syringe needle all the way until it bottoms out in the
instrument, inject the sample by discharging the contents of the syringe in
one quick push, and then remove the syringe.
e. Immediately after removing the syringe, hit the round, blue ‘Start’ button
on the keypad panel to initiate the run.
f. Set down the syringe and close the keypad window.
Now it is time to watch the data as it comes through:
10. Click on the button for Real Time Plot View (third button from the left).
11. The GC data and MS data will begin to come across once the 5 min delay time
has elapsed. The run time of the typical method file is about 20 minutes.
Once the run is completed, it is time to look at your data:
12. From the main icon map window, select ‘Qual Browser’.
13. Select ‘File’, ‘Open’, and select the name of the file that you just ran.
14. The top window will display the GC data which is a series of peaks that are
different materials coming out of the GC column at different elution times based
on their affinity for the column. Each discreet peak usually represents a different,
unique material. If you are running a pure material, and your concentration is
high enough to get above the baseline impurities of the machine, you should have
only one major peak.
15. Select the pushpin icon in the upper right corner of the top window to activate the
GC window (if not already active)
16. Use the mouse to click drag along the width of your peak of interest and allow the
computer to zoom in on it.
17. Now click on the pushpin icon in the upper right corner of the lower window
(MS) to activate that window, and click on the center of your peak in the top
window. By doing this you will get MS data for just the peak you selected.
Now you have the ability to set up the instrument, prepare samples, run samples, and look
at data. The majority of the work researchers using this instrument wish to do is to
identify purity (how many compounds in my supposedly pure sample), to determine the
molecular ion (and thus the molecular weight of the compound), and sometimes to
fragmentation pattern of major fragment ions to help determine structure. There are
many ways to analyze GCMS data based on the intended purpose, and data analysis is
beyond the scope of this tutorial. Please see LCI staff trained in the use of this equipment
for any help regarding the use of this instrument, training, and analysis of data.
REV 1.0 9/6/2007 Bentley Wall