Basic SOP for Agilent 6890/5973 system
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This document provides standard operating procedures for using a gas chromatography mass spectrometry (GCMS) instrument. Key steps include: 1. Creating GC and MS data acquisition methods to control instrument parameters and acquire data from both the GC and MS separately. 2. Tuning the mass spectrometer by injecting perfluorotributylamine and adjusting parameters like entrance lens, EM volts, and AMU gain/offset to satisfy targets for peak numbers, mass accuracy, relative abundances, and peak widths. 3. Creating a sample log table to input sample information and run a sequence of samples using the optimized GC-MS method. 4. Editing data reports and setting up quantitation databases to identify
Basic SOP for Agilent 6890/5973 system
- 1. SOP FOR GCMS L-201 x-2452 L-205 x-2356 G.C. HP 6890 (fid,tcd) M.S.5973 Chemstation Mod. G1540A G 1098A G 1701CA Ver. C.00.00 S.N. US00023315 US82311190 lic. BN26CE1DCB ● PLEASE READ THE COMPLETE SENTENCE , STEPS, BEFORE selecting INPUTS ●First the GC parameters must be satisfied, then the MS parameters and Auto Tuning MS. ● FID and MS have separate injections, therefore 2 DATA Acquisition Methods will be created. . . LOSS OF GC COLUMN FLOW could degrade the GC Column. 1.) Check column(s) flow & MS vacuum; On GC key pad select Col. 1or Col.2 display window will indicate column 1 flow, should be ~1 ml/min. On top of the MS, the Triode Gauge will indicate the MS vacuum, desired vac. 2.5 – 5 . 2.) Check utilities such as; gases for GC He, Air, Hydrogen and vacuum oil level on MS diffusion pump ( rough pump on the floor). 3.) Check AUX. 2,which is the GC/MS interface temperature; set point is 280 ° C. . 4.) Being an FID, the detector will not ignite until the detector temperature is at 150 ° C, also at this temperature both Air & Hydrogen flows will automatically be turned on to preset flow rates for detector ignition. Air flow: Hydrogen flow, usually ~10:1, 400/40 ml/min . I.) Both the GC & MS are being controlled by the computer. II.) Turn the computer on. On Desk Top , select Instrument #1 icon . The opening screen will either be TOP menu or Instrument Control Menu There are 3 main menus; TOP menu , INSTRUMENT CONTROL menu & DATA ANALYSIS ( offline). ●These 3 MAIN menus are available under VIEW on either Top menu & in INSTRUMENT CONTROL menu. ●Also when the computer is powered-up, it will load the parameters of the last method used or maybe the Default method parameters. 5.) Next is creating a Method operating parameters for the GCMS. ● From either TOP menu or INSTRUMENT CONTROL menu, select METHOD. i.) Then select Edit Entire Method .. When finished, name the method, by selecting SAVE AS ii.)The Edit Method window will appear with 3 Edit options. Method Info Instr/Acquire Data Anal Select all 3, then click O.K. iii.)The next screen will be as figure 170 below with a smaller window overlapping the right bottom corner of larger window. The smaller window is info for sample injection source . Then click O.K. iiii.)Then click on the Injector (syringe) and follow the instruction on the screen. Then select the Inlets and fill-in the parameters. When finished editing parameters , SAVE AS with new name.
- 2. NEXT THE MASS SPECTROMETER MUST BE TUNED
- 3. GC/MS Standard Operations Procedure Before samples can be analyzed using the GC/MS, the mass spectrometer must be tuned. The MS is tuned by adjusting several parameters while the Perfluorotributylamine (PFTBA,aka FC-43) is injected into the MS chamber. Options are MANUAL tune or AUTOTUNE. ● AUTO TUNING is preferred. In INSTRUMENT CONTROL ,select Instrument, then select PERFORM MS AUTOTUNE. After tune is finished name the results and save the results. Below is the MANUAL procedure for tuning the GC/MS. However. 1) Click on the “Instrument #1” icon to start the Chemstation software. 2) From the “View” menu select “Manual Tune”. 3) When prompted “be sure changes are saved. Switch View Now?”, select “Yes” 4) In the “Manual Tune” view select the “AdjParam” menu to adjust the tune parameters. 5) From The “AdjParam” menu select “Edit MS Params” menu 6) From the ”edit MS Params” menu select “Scan” and you should see a similar PTFBA spectra a s shown in Figure 2-1. Wait 30 seconds for the PFTBA levels to stabilize before changing any tune parameters. Figure 2-1. - Tuning with the PTFBA spectra The scan from 10-700 amu should have less than 200 peaks. If there are more than 200 peaks then some sort of contamination is entering the MS chamber and further trouble shooting should be done.
- 4. The mass spectrum of PFTBA has a base peak of 69 amu. The relative abundance of the mass peak of 219 to the base peak in the PTFBA spectra should be between 40 and 85%. The relative abundance of the mass peak of 502 to the base peak in the PTFBA spectra should be between 2.5 and 5%. In the “mass” column in Figure 2-1, if the masses of 69, 219, and 502 are off by more than 0.2amu then the mass axis should be adjusted. This is done by selecting “Mass Axis” from the calibrate menu. By doing this the mass axis is automatically adjusted by the software. The “Rel Abund” column shows the relative abundance of the 69, 219, and 502 masses. To adjust the relative abundance, the EN lens and the EntLens offset parameters should be adjusted. The Entrance lens parameters are inversely proportional to the relative abundance, which means that if you wish to increase the relative abundance you should lower the entrance lens setting and vise versa. The abundance of mass 69 should be between 200,000 and 400,000. To adjust the abundance the EM volts parameter should be adjusted. The EM volts are directly proportional to the abundance. An increase in the EM volts parameter will increase the abundance. Click on the “Prof” button to get a profile of the 69, 219, and 502 peaks. The profiles are shown in Figure 2-2. The peak with at 50% of the peak height (Pw50) should be between 0.57- 0.63 → Desired TARGETS Peak #, should be < than 200 Adjust effect Em volts Base 69 , Abundance should between 200,000 400,000 Entr lens Mass values should be 0.2 amu Mass % (ratio to base peak ) 69 = 100 219 = 40 85 502 = 2.5 5 after targets are satisfied then select PROFILE A chromatogram of 3 Masses will be on screen amu Peaks widths ( pw ) should be between 0.57 0.63
- 5. PROFILE OF MASSES 69,219 & 502 Figure 2-2. Peak widths To adjust the peak widths the amu gain and the amu off set parameters should be adjusted. The relationship between the peak widths and the amu gain and the AMU off set are shown in Figure 2-3. In order to increase the peak width the amu gain and the amu off set parameters should be decreased, and vice versa to decrease the peak width.
- 6. Figure 2-3 Relationship between peak width and AMU gain and AMU offset 7) After changing the tune parameters print the parameter settings by selecting “print” from the file menu. 8) Click on “OK” to leave the edit Parameters view. 9) Save the tune values by selecting “save tune values” from the file menu. Name the file & save . 10) Select “Top” from the view menu, to switch to the top view. When Prompted “be sure changes are saved. Switch View Now?”, select “Yes” Note, if changes have not been saved go ahead and do so now other wise the changes will be lost once the view has been switched. After the mass spectrometer has been tuned then a sequence table will be prepared to run the samples using the EXAMPLE in figure 2-4 below. A STANDARD SHOULD BE THE FIRST INJECTION. PREPARE a STANDARD, the system requires an injection, a chromatogram to activate the system’s DATA ACQUISITION software. After first Analysis Report the system will permit editing the data report. Edit items that relate to the components’ id on both the GC & MS .
- 7. ●Next From TOP Menu select SEQUENCE, then select Edit Sample Log Table, to create a SAMPLE LOG TABLE. Fig.2-4 will be on screen Figure 2-4: Sample Log Table Select line 1, which then inputs in boxes Type, vial relate to line 1. The vial number will always be 1 The data file name is limited to eight characters The method for GC/MS analysis in fig.2-4 is AAVOC1 The sample name can be more descriptive and can be longer than eight characters. More information about the sample can be entered in the Miscellaneous Information Box. Normally such information and the sample volume and internal standard volume injected . Repeat for other lines The Expected Barcode Box is left blank. Click on “OK” to close the sample log table. Using the AS up to 8 samples can be analyzed. * For a Single run; from INSTRUMENT CONTROL select Method, then RUN **Another option is togo to INSTRUMENT CONTROL,then click on the upright sample vial. *** A third option is running a SEQUENCE. Ω Regardless of run option, figure 209 Acquisition- Sample Information will appear on screen, just after deciding on injection option . Next page was scanned, unable to edit it, jr
- 8. EXAMPLE; Figure 2-5 will appear for running a SEQUENCE
- 9. Fig. 2-5 Start sequence option * Select “Run” from the “Sequence” menu to run the sequence. Figure 2-5 shows the start sequence options * Select “Full Method”, select “Inject anyway on Barcode Mismatch”. Do not select “Overwrite existing data files”. * Entering a sequence comment is optional. * Enter your name under the operator name. * Enter C:msdchem1DATAMeasurements Class for the data file directory * Click on the “Run Sequence” button to run the sequence After first Analysis Report the system will permit editing of the data report , parameters that relate to the components’ id on both the GC & MS . ●Next are the following items , included are Agilent reference pages #s which are in more detail. * Creating a GC Quantitation Data Base . * Creating a MS Quantitation Data, (targent ion, qualifying ions, etc..) * Calibrations Curves (levels) * Library Search. Quantification, Peak purity ●●●●Included are Agilent’s procedures for some items, which would probably be better communicated on actual instruments , sort of hand-on . See pages 8-33 ** The items are: Pftba MSDS included
- 10. CREATING A QUANTITATION DATABASE Procedure is below is for the MS data.For the GC there will only be the chromatogram, no ion spectrum. Note; “ The order of compounds in the data base is VERY important. Internal standards must precede all compounds that will be quantitated relative to it. Retention times order is NOT very important.” “Any compound that will be treated as external standards must be entered before the first internal standard.” ●From the Desk Top select DATA ANALYSIS From Data Analysis, select CALIBRATE. Under Calibrate select SET-UP QUANTITATION. ●Opening screen is named, Quantitation DatabaseGlobals 1.)Enter a name (id) for title, in measure select %, in Curve Fit select Avg,.Response Factors, then click O.K. 2.)The next screen is Edit Compound, which is empty. Click on Insert Above. 3.)The next screen will have the first injection’s chromatogram on top, and a smaller window on the bottom right corner, labeled Quant Setup. 4.)To get the retention times. On the chromatogram move the mouse curser to the apex (top) of first component,then double right click and the retention time will be appear Quant Setup window. Also the spectrum of the compound will appear below the chromatogram. Name the Compound. 5.)Next the targent ion and qualifying ions must be selected from the spectrum. “ A target ion - - is an ion characteristic of the target compound, preferably one that distinguishes this compound from any others with similar retention times. The extracted ion chromatogram for this ion will be used for the quantitation. “ “ Qualifier ions—are selected from the mass spectrum of the target compound . The presence of these ions in the correct amounts relative to the target ion gives evidence of correct target compound identification. Depending on your choice of peak selection parameters, the qualifiers may influence how the software chooses between multiple hits. “ For example isomers. Selecting the taget ion.. On the compound spectrum place the curser which now is shaped like cross-hair, over the selected target ion then click both mouse button at the same time. This will place that ion in the quant setup box and will also calculation the relative ratio. REPEAT this step for the rest of the qualifying ions. ***Then click the SAVE button 6.) Repeat all of step 4 above for the rest of the compounds. 7.) When finished click SAVE, then exit, then exit again. Next is Calibration Curves
- 11. CREATING A CALIBRATION CURVE FOR A COMPOUND * The procedure is bit similar to the SET-UP QUANTITATION from previous page ● Load the file that was just saved above on SET-QUANTITATION ●From DATA Analysis, select Generate Report, Save & name report. ●From DATA ANALYSIS , select Calibrate, then select Update,update level one, then click O.K. i.)Move Mouse curser to desired component. The edit compound screen will appear . Select insert Above and a screen will appear to add the concentration of the standard. SAVE. ii.) Repeat this for each component, Saving each time. iii.) It will ask if there are more levels (Std % concentrations). ●To add another level ( Std concentration) Repeat steps i,ii,iii. When finished name id and save Calibrations Curves. PFTBA MSDS will be included in Desk Copy.