1.1 Introduction

This 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 instructorOptimize the CEM

1.2 Materials

Syringe PumpSyringe

PEEK tubing and union adapterAppropriate PPG calibrant solution

1.3 Procedure

Insert the syringe into the PEEK tubing and union adapterTighten fittings to ensure there will be no leaksPlace syringe on pump and lock in place

Slide push plate up against plunger of syringeSet flow rate and diameter of syringe

Plumb the loose end of the PEEK tubing to the inlet of the sourcePress start to begin infusion

1.4 Manual Tuning and Mass Calibration

1.4.1 Choosing Hardware Configuration

1.Choose the API Instrument Project folder from the drop down menu next

to the yellow folder icon on the task bar.2.Double-click the Hardware Configuration and activate the Mass Spec

Only Profile. After a green check appears for the selected profile, close the window.

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1.4.2 Manual Tuning Set-up

1.Click on the Tune and Calibrate Mode on the Navigation Bar. You should

hear the gases turn on as the instrument becomes active for use. A green mass spec status icon also appears in the bottom right corner of the window. 2.Under the View menu, activate the Sample Queue or click on the View

Queue icon to open the Queue Manager window. Minimize the Queue Manager window.3.Double-click on Manual Tuning. Under File, select Open and choose an

acquisition method (*.dam) for calibration. Start with Q1 positive calibration. For positive Q1, select Q1PosPPG.dam, for negative Q3, select Q3NegPPG.dam and so on. The method then comes up in the Tune 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 Detector

settings found on the tabs on the left side of the window as well as the MS and Advanced MS settings on the right side are values established by the installation engineer. Consult the help files if you have any questions regarding 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 Spec

Configuration tab. Click on the Mass Spec and click Set Up.

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Figure 1-2Integrated Syringe Pump Set Up

1.4.3 PPG Dilution and Syringe Pump Set Up

Different calibration solutions must be used, depending on the specific instrument and the polarity being calibrated.

Table1-1PPG Solutions and Dilutions By InstrumentInstrument Type

API 2000 SystemAPI 3000 SystemAPI 3200 SystemAPI 4000 SystemAPI 5000 System

Negative Mode

PPG 3000 StandardPPG 3000 StandardPPG 3000 StandardPPG 3000 StandardPPG 3000 Standard, Diluted 1:10

Positive Mode

PPG Standard (10-4M)PPG Standard, Diluted 1:10 (10-5M)

PPG Standard, Diluted 1:10 (10-5M)

High Mass

NAPPG 3000 StandardPPG 3000 Standard

PPG Standard, Diluted PPG 3000

Standard1:50(2x10-6M)

PPG Standard, Diluted PPG 3000 Standard1:500(2x10-7M)

Note:The PPG Standard should be diluted with the appropriate diluent. DO

NOT 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.

1.Load a syringe with the appropriate PPG solution.

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2.API 3000 , API 4000 , API 5000 Systems: Start the infusion pump at a

flow rate of 10 μL/min. Ensure that the appropriate syringe diameter is being used.

Table1-2Syringe Diameters

Syringe Volume (μ

L)

1000500250

Diameter (mm)

4.613.262.30

3.API 2000 and API 3200 Systems: Use the drop down menu to the

right 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-up

4.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 Shape

1.On the MS tab, enter 10 cycles and verify that MCA box is checked. Click

Start to acquire 10 scans in MCA mode.

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Note: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.

2.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 Tab

Tip!If the Calibration Peak List tab is not visible, navigate to the Appearance Options to activate it by following Figure

1-5.

http://www.77cn.com.cnpare the PPG intensities displayed to a previous calibration of the

instrument. 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 be

within 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, for

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all PPG ions >1700 amu, a change in the isotope pattern might be observed if solution is old.

Figure 1-5Activating Mass Calibration Peak List Tab

1.4.5 Adjusting Peak Widths to Achieve Unit Resolution

On the Calibration Peak List tab, examine the peak width column. Peak widths or resolution should be consistent across the entire mass calibration range. To achieve “Unit” resolution, the DC offsets of the PPG ions whose peak width values 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 seldom changed. This parameter affects peak shape, sensitivity and resolution. Click the Advanced button. The Resolution Table (see Figure1-6) that appears contains a list of all the PPG ions being calibrated on with their corresponding DC Offset values.2.Capture the offset values prior to making any changes by using

Ctrl+PrntScrn and paste the capture into NotePad (that way you have a starting point to return to). Make small changes (+/- 0.005) in the offset values 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 to the spectral panes, right click and List Data again and observe if there were 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 turn

decreases the Peak Widths

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Decreasing the offset will decrease the resolution, which in turn increases the peak widths

3.After changes are made, re-acquire 10 scans in MCA mode, right click in

bottom right pane and select Open File, then use the List Data tab again, and examine the peak widths for Unit Resolution.

Table1-3Peak Resolution SpecificationsResolution

LowUnitHigh

Peak Width (amu) FWHH

~1.1 amu0.6 - 0.8 amu0.4 - 0.6 amu

Note: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.

Figure 1-6Resolution Table for Q1 Positive

4.Determine if any changes need to be made and adjust the offsets as

necessary, re-acquiring 10 MCA scans to observe the changes in peak widths.5.Continue to adjust Offsets to achieve Unit resolution for the peak widths

across the entire mass range.

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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 (increased

sensitivity) 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 the

entire mass range.

1.4.6 Replacing the Mass Calibration

1.Once Unit Resolution is achieved across the mass range, use the Tools

menu 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 (Figure1-7). Select

the 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 Options

3.Click Start.The Mass Calibration Report window opens (Figure1-8) which

contains three graphs.

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Figure 1-8Mass Calibration Report Graphs

The 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 are the 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 0.7 amu 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 attempt

recalibration, 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, then recalibration is necessary.5.To replace the existing calibration, click on the bottom red arrow icon

located 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) that

opens 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 mass

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calibration on the quad, as well as another parameter called Slope Variation.

If you plot the masses vs. DAC values, you should get a straight line and that is where the Slope Variation is derived. This value should be between 0.998 and 1.002. If it is not, then the instrument may have been calibrated on the wrong isotopic peak for that ion.

Figure 1-9Partial Mass Calibration Results Text Window

Tune and Calibration Review

You now have successfully calibrated the instrument in the specified

polarity and quadrupole. Remember that for each resolution setting (unit and 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 File folder which is located in the API Instrument Project folder. You can access these values in any project in Analyst® software. Click Configure in the navigation bar. Click on Tools in the taskbar and select Settings then Instrument Options. Click Quad 1, Positive, Unit. Then click Calibration Table. The DAC values are displayed. Click Resolution Table. The DC offsets and Ion Energy are displayed.

Each combination of quad, polarity and resolution will have its own calibration and resolution settings. These settings will automatically get updated whenever the instrument is calibrated in that polarity and resolution.

Click Configure in the navigation bar. Click on Tools in the taskbar and select Settings then Tuning Options. The Resolution tab allows the user to set the Resolution specifications for Unit, High and Low. The Low resolution offsets are automatically generated by taking the Unit

Resolution DC offsets and lowering each offset by the specified Offset Drop.

1.4.7 Automatic Tuning and Calibration

The instrument can be tuned and calibrated automatically using the Instrument Optimization function under the Tune and Calibrate mode. Please refer to the

Getting Started Guide for detailed instructions for using the automatic feature.

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1.4.8 CEM Optimization

The 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 as

follows:

Check the “Center/Width” (if it isn’t already) to alter how the masses are displayed (toggle the check mark to familiarize yourself with the different 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 begin

acquiring data.

When a stable baseline has been established, increase CEM voltage in increments of 100 V, allowing enough time between changes to determine intensity at a given setting. When the increase in signal intensity is 20 – 40% (optimal is 30%) with a 200 V increase, the

optimum CEM voltage has been determined. Remember – increasing the CEM voltage not only increases the signal, but also the noise.

Figure 1-10CEM Gain Check TIC

Note: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. The

optimal setting will be determined with a 100 V difference producing the 20% -

40% gain in signal.

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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.

WARNING!Setting the bias voltage above the optimized voltage shortens the CEM life.

1.4.9 Intensity Guidelines For Positive Mode

The 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.

Table1-4Intensity Guidelines for API 5000 SystemNominal m/z

591755009061196

Q1 (cps)

NANA1.1e71.7e74.3e6

Q3 (cps)

NANA1.1e71.4e73.4e6

0.6 - 0.8

0.6-0.80.6 - 0.80.6 - 0.80.6 - 0.8

FWHH (amu)

Table1-5Intensity Guidelines for API 4000 SystemNominal m/z

591756169061254154520102242

Q1 (cps)

3.0e71.0e71.5e72.0e73.0e61.0e61.5e61.0E6

Q3 (cps)

2.0e71.0e71.0e72.0e73.0e65.0e51.0E68.0E5

FWHH (amu)

0.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.850.6 - 0.85

Table1-6Intensity Guidelines for API 3200 SystemNominal m/z

5917561690612541545

Q1 (cps)

9.0E69.0E64.5E61.1E71.3E62.7E5

Q3 (cps)

9.0E67.7E63.8E68.1E68.6E51.4E5

FWHH (amu)

0.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.8

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Table1-7Intensity Guidelines for API 3000 SystemNominal m/z

591756169061254154520102242

Q1 (cps)

1.2E71.2E71.0E71.9E71.3E64.5E51.5E61.0e6

Q3 (cps)

1.2E71.2E79.1E61.5E71.2E63.7E51.0E66.1e5

FWHH (amu)

0.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.850.6 - 0.85

Table1-8Intensity Guidelines for API 2000 SystemNominal m/z

59175906125415451778

Q1 (cps)

2.0e61.0E62.0E62.0E72.0E61.0E61.0E6

Q3 (cps)

1.2E71.0E68.0E61.0E51.0E51.0E5

FWHH (amu)

0.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.85

1.4.10 Intensity Guidelines for Negative Mode

The intensity values in these tables serve as guidelines only. The calibration

intensities from installation is the best reference. All values are based on MCA of 10 scans.

Table1-9Intensity Guidelines for API 5000 SystemNominal m/z

934

Q1 (cps)

1.1e7

Q3 (cps)

9.0e6

FWHH (amu)

0.6 - 0.8

Table1-10Intensity Guidelines for API 4000 SystemNominal m/z

9342036

Q1 (cps)

2.0e73.0e6

Q3 (cps)

1.0e7-

FWHH (amu)

0.6 - 0.80.6 - 0.85

Table1-11Intensity Guidelines for API 3200 SystemNominal m/z

9341572

Q1 (cps)

1.5e61.4e5

Q3 (cps)

7.2e5-

FWHH (amu)

0.6 - 0.80.6 - 0.8

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Table1-12Intensity Guidelines for API 3000 SystemNominal m/z

455859341224157220362211

Q1 (cps)

2.0e52.0e57.1e62.0e52.0e51.5e65.0e5

Q3 (cps)

1.2e51.2e55.5e61.2e52.0e57.5e54.0e4

FWHH (amu)

0.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.85

Table1-13Intensity Guidelines for API 2000 SystemNominal m/z

45585934122415721747

Q1 (cps)

2.0e42.0e41.0e54.0e44.0e41.0e4

Q3 (cps)

2.0e32.0e32.0e41.0e31.0e31.0e3

FWHH (amu)

0.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.80.6 - 0.8

1.5 Discuss and Answer the following Questions:

1.In most labs, the service engineer performs the calibration every 6 months

during 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, where

can 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, you

can easily recover your mass calibration?5.Under the Resolution tab, what is the function of the parameter Ion

Energy (IE1)? See lecture presentation.6.I opened a Q1Pos PPG method that was set at Unit Resolution and was

successful 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/Width

check 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?

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10.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.

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