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					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
   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 80-90 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.
         b. For leak detection, use a digital leak detection device. Do not use
             anything that uses liquids such as ‘Snoop’ as these materials will get
             drawn into the system and contaminate it.
         c. Also remember to check and replace the triple filter which is in line with
             the carrier gas flow at the back of the machine. The filter takes Oxygen,
             water and hydrocarbons out of the carrier gas stream. The three section
             sof the filter will change color as they become expired. The one that
             usually goes first is Oxygen. There is a pamphlet in the drawer under
             GCMS that describes this filter and how to reorder and change it out. It is
             not necessary to buy the whole fixture to replace, just the filter tube and
             contents. This filter should be changed roughly once a year through
             normal GCMS use.
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
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.
           a. Under 40 mtorr is good for flow of 0.5
           b. Higher flow rate of carrier gas will result in higher pressure
           c. The lower this number is the better
           d. Up to 50 mtoor is OK, but at 55 mtoor with flow of 1.0 or less and there is
               cause for concern that there is a leak.
   13. 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
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
       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
       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
       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 10 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 1 uL of your pure solvent. (no more than 2 uL)
       f. Remove the syringe from the solvent and draw up about 2-3 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
         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

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