AB液质联用使用说明tune mass calibration and CEM

1.1 IntroductionThis chapter is intended for the user to become familiar with manually tuning and calibrating the instrument.In this section, users will:•Open a PPG acquisition method•Acquire 10 MCA scans of PPG spectrum•Adjust Peak Widths to achieve Unit Resolution•Manually tune and calibrate the instrument using the Calibrate from Spectrum icon•Replace Mass Calibration•Review Calibration Results with instructor•Optimize the CEM1.2 Materials•Syringe Pump•Syringe•PEEK tubing and union adapter•Appropriate PPG calibrant solution1.3 Procedure•Insert the syringe into the PEEK tubing and union adapter•Tighten fittings to ensure there will be no leaks•Place syringe on pump and lock in place•Slide push plate up against plunger of syringe•Set flow rate and diameter of syringe•Plumb the loose end of the PEEK tubing to the inlet of the source•Press start to begin infusion1.4 Manual Tuning and Mass Calibration1.4.1 Choosing Hardware Configuration1.Choose the API Instrument Project folder from the drop down menu nextto the yellow folder icon on the task bar.2.Double-click the Hardware Configuration and activate the Mass SpecOnly Profile. After a green check appears for the selected profile, closethe window.Manual Tune, Mass Calibration and CEM1.4.2 Manual Tuning Set-up1.Click on the Tune and Calibrate Mode on the Navigation Bar. You shouldhear the gases turn on as the instrument becomes active for use. A greenmass spec status icon also appears in the bottom right corner of thewindow.2.Under the View menu, activate the Sample Queue or click on the ViewQueue icon to open the Queue Manager window. Minimize the QueueManager window.3.Double-click on Manual Tuning. Under File, select Open and choose anacquisition method (*.dam) for calibration. Start with Q1 positivecalibration. For positive Q1, select Q1PosPPG.dam, for negative Q3,select Q3NegPPG.dam and so on. The method then comes up in theTune Method Editor window, shown in Figure1-1.Figure 1-1Manual Tune Window with “Q1 PosPPG.dam” Method Open4.The settings on the Source/Gas, Compound, Resolution and Detectorsettings found on the tabs on the left side of the window as well as the MSand Advanced MS settings on the right side are values established by theinstallation engineer. Consult the help files if you have any questionsregarding the settings in this window.5.The API 2000™ and API 3200™ Systems have integrated syringe pumps,be sure to create the hardware profile with it activated on the Mass SpecConfiguration tab. Click on the Mass Spec and click Set Up.Manual Tune, Mass Calibration and CEMFigure 1-2Integrated Syringe Pump Set Up1.4.3 PPG Dilution and Syringe Pump Set UpDifferent calibration solutions must be used, depending on the specific instrument and the polarity being calibrated.1.Load a syringe with the appropriate PPG solution.Table 1-1PPG Solutions and Dilutions By Instrument InstrumentTypeNegative ModePositive ModeHigh MassAPI 2000™ System PPG 3000 Standard PPG Standard (10-4M)NA API 3000™ System PPG 3000 Standard PPG Standard, Diluted 1:10 (10-5M)PPG 3000 Standard API 3200™ System PPG 3000 Standard PPG Standard, Diluted 1:10 (10-5M)PPG 3000 StandardAPI 4000™ System PPG 3000 Standard PPG Standard, Diluted 1:50(2x10-6M)PPG 3000Standard API 5000™ SystemPPG 3000 Standard, Diluted 1:10PPG Standard, Diluted 1:500(2x10-7M)PPG 3000 Standard Note:The PPG Standard should be diluted with the appropriate diluent. DONOT use the vial marked “Diluent” in the Mass Spec Standards Kit. The appropriate dilution solution for the PPG Standard is: dissolve 15.4 mg of ammonium acetate in 49.9mL of water, then add 49.9 mL of methanol, 0.1 mL acetonitrile, and 0.1 mL formic acid. The appropriate dilution solution for the PPG 3000 standard is: dissolve 385 mg ammonium acetate in 49.9 mL of water, then add, 49.9 mL of methanol, 0.1 mL acetonitrile, and 0.1 mL formic acid.Manual Tune, Mass Calibration and CEM2.API 3000™, API 4000™, API 5000™ Systems: Start the infusion pump at aflow rate of 10 μL/min. Ensure that the appropriate syringe diameter is being used.3.API 2000™ and API 3200™ Systems: Use the drop down menu to theright of Edit Ramp to access the Syringe Pump Method dialog box. Input a rate of 10 μL/min and an appropriate diameter for the syringe being used. Click Set Flow Rate or Start Syringe Pump to start the infusion. Then go back to the MS Method Editor by selecting MS Method from the drop down menu to the right of Edit Ramp.Figure 1-3API 2000™ and API 3200™ Systems Syringe Pump Method Set-up4.Allow enough time for the infusion rate to equilibrate.5.Verify that the sprayer is positioned properly.API 2000™, API 3000™ Systems: The edge of the spray should be at least 2 mm from the curtain plate hole. Momentarily increase flow rate to 100 μL/min to visibly see the spray for proper spray adjustment relative to the orifice.API 3200™, API 4000™, & API 5000™ Systems: Settings of 10 vertical and 4 to 6 horizontal are recommended.1.4.4 Verify PPG Ion Intensities and Peak Shape1.On the MS tab, enter 10 cycles and verify that MCA box is checked. ClickStart to acquire 10 scans in MCA mode.Table 1-2Syringe DiametersSyringe Volume (μL)Diameter (mm)1000 4.61500 3.262502.30Manual Tune, Mass Calibration and CEM2.Right click in the bottom-right pane and select Open File.3.Right click in one of the spectral panes and select List Data.A new pane will open below the spectra with three tabs: Data List, Calibration Peak List, and Peak List.Figure 1-4Calibration Peak List Tabpare the PPG intensities displayed to a previous calibration of theinstrument. Guidelines for intensities at Unit resolution can be found at the end of this chapter.5.If the instrument is operating correctly, all intensity values should bewithin 15% of the previous calibration values. 6.Observe the isotopic pattern of each panel. For all PPG ions >1700 amu,the second isotope is the ion of interest for calibration purposes. Also, forNote:MCA is Multi-Channel Acquisition and when it is activated the successive scans will be summed in a running total of intensities starting from the first scan up to the current scan. This is most useful when acquiring data from low intensity peaks, because adding several scans can produce better signal-to-noise ratios. This mode of operation is used for discrete sample analysis by infusion. For PPG tuning and calibration, 10 MCA scans is the standard approach.Tip!If the Calibration Peak List tab is not visible, navigate to the Appearance Options to activate it by following Figure1-5.Manual Tune, Mass Calibration and CEMall PPG ions >1700 amu, a change in the isotope pattern might beobserved if solution is old.Figure 1-5Activating Mass Calibration Peak List Tab1.4.5 Adjusting Peak Widths to Achieve Unit ResolutionOn the Calibration Peak List tab, examine the peak width column. Peak widths orresolution should be consistent across the entire mass calibration range. Toachieve “Unit” resolution, the DC offsets of the PPG ions whose peak widthvalues are outside 0.6 - 0.8 amu should be adjusted.To adjust Peak Widths:1.Return to the Tune Method Editor window. Click on the Resolution tab.The Ion Energy (IE1) value was established at installation and is seldomchanged. This parameter affects peak shape, sensitivity and resolution.Click the Advanced button. The Resolution Table (see Figure1-6) thatappears contains a list of all the PPG ions being calibrated on with theircorresponding DC Offset values.2.Capture the offset values prior to making any changes by usingCtrl+PrntScrn and paste the capture into NotePad (that way you have astarting point to return to). Make small changes (+/- 0.005) in the offsetvalues of the PPG ions whose peak widths are out of range. Click Apply,then re-acquire another 10 MCA scans (click YES when prompted to“Save changes made in manual tune to instrument data file”). Go back tothe spectral panes, right click and List Data again and observe if therewere any changes to the peak width.Try making big changes (+/- 0.05),click Apply, re-acquire 10 MCA scans, and note how peak width changes.Increasing the offset will increase the resolution, which in turndecreases the Peak WidthsManual Tune, Mass Calibration and CEMDecreasing the offset will decrease the resolution, which in turn increases the peak widths3.After changes are made, re-acquire 10 scans in MCA mode, right click inbottom right pane and select Open File, then use the List Data tab again, and examine the peak widths for Unit Resolution.Figure 1-6Resolution Table for Q1 Positive4.Determine if any changes need to be made and adjust the offsets asnecessary, re-acquiring 10 MCA scans to observe the changes in peak widths.5.Continue to adjust Offsets to achieve Unit resolution for the peak widthsacross the entire mass range.Table 1-3Peak Resolution Specifications ResolutionPeak Width (amu)FWHHLow ~1.1 amu Unit 0.6 - 0.8 amu High0.4 - 0.6 amuNote:You MUST Tune and Calibrate High Resolution if you are going to use it. Each has its own resolution and calibration tables. Low Resolution is a specified drop of all the Offsets from Unit Resolution Offsets and cannot be calibrated.Manual Tune, Mass Calibration and CEM1.4.6 Replacing the Mass Calibration1.Once Unit Resolution is achieved across the mass range, use the Toolsmenu to select Calibrate from Spectrum, or click the Calibrate from Spectrum icon on the toolbar to begin manual mass calibration.2.The Mass Calibration Options dialogue box appears (Figure 1-7). Selectthe appropriate standard from the drop down list. Click on Edit to update the Reference Table if needed. Be sure to enter an appropriate Search Range and Threshold for the calibration.Figure 1-7Mass Calibration Options3.Click Start.The Mass Calibration Report window opens (Figure 1-8) whichcontains three graphs.Tip!There is a tendency for resolution to decrease as ion mass increases;therefore, it is necessary to adjust the ratio of the RF to DC voltages applied to the quadrupole. Increasing the DC voltage as a function of mass allows you to achieve a constant peak width for each ion over a large mass range. The rate at which the DC voltage is varied with mass is set using the Mass and Offset settings in the Resolution Table window. In general, as the resolution is increased (i.e narrow peak widths), the ion intensity decreases (i.e less sensitive). The normal trade-off between high ion transmission (increasedsensitivity) and narrow peak widths (increased resolution) should be optimized for each application. Most applications will require “unit” mass resolution (i.e., isotope peaks one amu apart are clearly defined) that corresponds to peak widths of approximately 0.7 +/- 0.1 amu at 50% intensity (Full Width at Half Height, FWHH). It is usually desirable to have a consistent peak width over theentire mass range.Manual Tune, Mass Calibration and CEMFigure 1-8Mass Calibration Report GraphsThe top graph is a plot of the mass shift from the previous calibration, where the solid line represents a 0 amu shift and the dotted lines arethe limits of + and - 0.1 amu shift.The middle graph is a plot of the full peak widths at half height for all of the PPG ions used in the calibration. The solid line represents 0.7amu peak width, and the dotted lines are the limits of + and - 0.1 amu.The bottom graph is a plot of the % change in intensity of the PPG ions from the previous calibration.4.If there are any outliers in the top two graphs, you must not attemptrecalibration, instead you must determine the cause of the outliers. If the peak widths are outside the specifications of Unit Resolution, then you must adjust the peak widths appropriately. If the peak widths are meet Unit Resolution and there are still outliers in the top graph, thenrecalibration is necessary.5.To replace the existing calibration, click on the bottom red arrow iconlocated at the top left corner of the results window (See Figure1-8). Click Yes when prompted to accept the new calibration.The top red arrow is to update the calibration, for example, if there were masses added to the method and Reference Table to extend the calibration.6.Print (Ctrl+P) the Mass Calibration Report.7.There is also a Mass Calibration Results text window (Figure1-9) thatopens behind the graphs. Minimize the graph window to access the text report.This text report contains the same information in tabular form and also has the updated DAC values which are the RF/DC values for massManual Tune, Mass Calibration and CEMcalibration on the quad, as well as another parameter called SlopeVariation.If you plot the masses vs. DAC values, you should get a straight lineand that is where the Slope Variation is derived. This value should bebetween 0.998 and 1.002. If it is not, then the instrument may havebeen calibrated on the wrong isotopic peak for that ion.Figure 1-9Partial Mass Calibration Results Text WindowTune and Calibration Review•You now have successfully calibrated the instrument in the specifiedpolarity and quadrupole. Remember that for each resolution setting (unitand high), there are 4 instrument calibrations: Q1 positive, Q3 positive,Q1 negative, Q3 negative.•The DC offsets and DAC values are stored in the Instrument Data Filefolder which is located in the API Instrument Project folder. You can accessthese values in any project in Analyst® software. Click Configure in thenavigation bar. Click on Tools in the taskbar and select Settings thenInstrument Options. Click Quad 1, Positive, Unit. Then click CalibrationTable. The DAC values are displayed. Click Resolution Table. The DCoffsets and Ion Energy are displayed.•Each combination of quad, polarity and resolution will have its owncalibration and resolution settings. These settings will automatically getupdated whenever the instrument is calibrated in that polarity andresolution.•Click Configure in the navigation bar. Click on Tools in the taskbar andselect Settings then Tuning Options. The Resolution tab allows the user toset the Resolution specifications for Unit, High and Low. The Lowresolution offsets are automatically generated by taking the UnitResolution DC offsets and lowering each offset by the specified OffsetDrop.1.4.7 Automatic Tuning and CalibrationThe instrument can be tuned and calibrated automatically using the InstrumentOptimization function under the Tune and Calibrate mode. Please refer to theGetting Started Guide for detailed instructions for using the automatic feature.1.4.8 CEM OptimizationThe Channel Electron Multiplier (CEM) degrades with time and continued use and must therefore be re-optimized periodically. Manual CEM Optimization can be done using the standard positive PPG calibration solution. PPG mass 1254 amu is monitored for the optimization (mass 906 amu for API 5000™ System).1.Infuse the standard positive PPG calibration solution at 10μL/min.2.Open the PPG Q1 Pos method in Manual Tune. Modify the method asfollows:Check the “Center/Width” (if it isn’t already) to alter how the masses are displayed (toggle the check mark to familiarize yourself with thedifferent displays). Uncheck Parameter Range if it is checked.Highlight and delete all rows except the one corresponding to mass 1254. You can save the method with a different method name, such as“CEM Opt.dam”.Set the duration to 10 minutes and uncheck MCA.3.In the Detector tab, drop the CEM voltage by 200 V. Click Start and beginacquiring data.When a stable baseline has been established, increase CEM voltage in increments of 100 V, allowing enough time between changes todetermine intensity at a given setting. When the increase in signalintensity is 20 – 40% (optimal is 30%) with a 200 V increase, theoptimum CEM voltage has been determined. Remember – increasingthe CEM voltage not only increases the signal, but also the noise.Figure 1-10CEM Gain Check TICNote:API 2000™ and API 3200™ Systems have a different kind of detector, so the procedure for optimization is slightly different. Instead of a 200 V decrease, use a 100 V decrease and 50 V increments to gather the data points. Theoptimal setting will be determined with a 100 V difference producing the 20% - 40% gain in signal.4.In this example, 2150 V would be the optimal CEM setting, with 36%gain.The percentage signal gain will vary depending on the mass of the ion. In general, the gain is higher at higher mass. Therefore, it is very important to use the proper criteria for determining the CEM setting to be used.1.4.9 Intensity Guidelines For Positive ModeThe intensity values in these tables serve as guidelines only. The calibration intensities from installation are the best reference. All values are based on MCA of 10 scans.WARNING!Setting the bias voltage above the optimized voltage shortens the CEM life.Table 1-4Intensity Guidelines for API 5000™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)59NA NA 0.6 - 0.8175NA NA 0.6 - 0.8500 1.1e7 1.1e70.6 - 0.8906 1.7e7 1.4e70.6 - 0.811964.3e63.4e60.6 - 0.8Table 1-5Intensity Guidelines for API 4000™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)59 3.0e7 2.0e70.6 - 0.8175 1.0e7 1.0e70.6 - 0.8616 1.5e7 1.0e70.6 - 0.8906 2.0e7 2.0e70.6 - 0.81254 3.0e6 3.0e60.6 - 0.81545 1.0e6 5.0e50.6 - 0.82010 1.5e6 1.0E60.6 - 0.8522421.0E68.0E50.6 - 0.85Table 1-6Intensity Guidelines for API 3200™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)599.0E69.0E60.6 - 0.81759.0E67.7E60.6 - 0.8616 4.5E6 3.8E60.6 - 0.8906 1.1E78.1E60.6 - 0.81254 1.3E68.6E50.6 - 0.815452.7E51.4E50.6 - 0.81.4.10 Intensity Guidelines for Negative ModeThe intensity values in these tables serve as guidelines only. The calibrationintensities from installation is the best reference. All values are based on MCA of 10 scans.Table 1-7Intensity Guidelines for API 3000™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)59 1.2E7 1.2E70.6 - 0.8175 1.2E7 1.2E70.6 - 0.8616 1.0E79.1E60.6 - 0.8906 1.9E7 1.5E70.6 - 0.81254 1.3E6 1.2E60.6 - 0.81545 4.5E5 3.7E50.6 - 0.82010 1.5E6 1.0E60.6 - 0.8522421.0e66.1e50.6 - 0.85Table 1-8Intensity Guidelines for API 2000™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)59 2.0e61.0E6 1.2E70.6 - 0.8175 2.0E6 1.0E60.6 - 0.8906 2.0E78.0E60.6 - 0.81254 2.0E6 1.0E50.6 - 0.81545 1.0E6 1.0E50.6 - 0.817781.0E61.0E50.6 - 0.85Table 1-9Intensity Guidelines for API 5000™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)9341.1e79.0e60.6 - 0.8Table 1-10Intensity Guidelines for API 4000™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)934 2.0e7 1.0e70.6 - 0.820363.0e6-0.6 - 0.85Table 1-11Intensity Guidelines for API 3200™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)934 1.5e67.2e50.6 - 0.815721.4e5-0.6 - 0.81.5 Discuss and Answer the following Questions:1.In most labs, the service engineer performs the calibration every 6 monthsduring PM service. Why do I need to know how to calibrate the instrument?2.How often should the instrument be calibrated?3.If you would like to add another PPG ion to the acquisition method, wherecan you find a list of PPG ions to choose from? How can you calculate the DAC value for that ion and estimate the DC offset?4.What should you do so that in the event of a major computer crash, youcan easily recover your mass calibration?5.Under the Resolution tab, what is the function of the parameter IonEnergy (IE1)? See lecture presentation.6.I opened a Q1Pos PPG method that was set at Unit Resolution and wassuccessful at manual calibration. I used the same method but changed Resolution to High --- the peaks disappeared. What should I do? I used the same method but changed Resolution to Low ---- what should I expect?7.What is the difference in the mass displayed when the Center/Widthcheck box is activated or not?8.How often should I optimize CEM?9.What are some factors that can lead to a short CEM life?Table 1-12Intensity Guidelines for API 3000™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)45 2.0e5 1.2e50.6 - 0.8585 2.0e5 1.2e50.6 - 0.89347.1e6 5.5e60.6 - 0.81224 2.0e5 1.2e50.6 - 0.81572 2.0e5 2.0e50.6 - 0.82036 1.5e67.5e50.6 - 0.822115.0e54.0e40.6 - 0.85Table 1-13Intensity Guidelines for API 2000™ System Nominal m/zQ1 (cps)Q3 (cps)FWHH (amu)45 2.0e4 2.0e30.6 - 0.8585 2.0e4 2.0e30.6 - 0.8934 1.0e5 2.0e40.6 - 0.81224 4.0e4 1.0e30.6 - 0.81572 4.0e4 1.0e30.6 - 0.817471.0e41.0e30.6 - 0.810.What is the maximum CEM voltage before replacement is needed? At the end of this chapter, you should have the following printouts:•Mass Calibration Results text file.•Mass Calibration Report Graphs.。