AB SCIEX TripleTOF® 5600/5600+ Instruments System User Guide Document Number: D5033331 A Release Date: May 2012
This document is provided to customers who have purchased AB Sciex equipment to use in the operation of such AB Sciex equipment. This document is copyright protected and any reproduction of this document or any part of this document is strictly prohibited, except as AB Sciex may authorize in writing. Software that may be described in this document is furnished under a license agreement.
Contents Safety and Regulatory Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 General Safety Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Symbols and Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Qualified Personnel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 Equipment Use and Modification . . . . . . . . . . . . .
Contents Chapter 3 Instrument Tuning and Calibrating. . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Required material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39 Prerequisites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39 Optimize the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39 Chapter 4 Basic Acquisition Methods . . . . . . . . . . . . . .
Contents Show a Spectrum from a TIC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 Generate XICs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 Generate BPCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72 Generate XWCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74 View DAD Data . . . . . . . . . . . . . . . . . . . . . . . . .
Contents Chapter 9 Basic System Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 Appendix A Recommended Calibration Ions . . . . . . . . . . . . . . . . . . . . . . . . . . .113 Appendix B Exact Masses and Chemical Formulas . . . . . . . . . . . . . . . . . . . . .115 PPG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .115 Reserpine (C33H40N2O9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Safety and Regulatory Information This section contains general safety-related information, describes the symbols and conventions used in the documentation, and provides regulatory compliance information. It also describes potential hazards and associated warnings for the system, and the precautions that should be taken to minimize the hazards. In addition to this section, refer to the Site Planning Guide.
Safety and Regulatory Information WARNING! Biohazard: This symbol indicates a warning of biohazardous materials. Read the warning and follow all precautions before performing any operation described in the guide. Failure to do so can result in serious injury. Caution: A caution indicates an operation that could cause damage to the instrument or loss of data if precautions are not followed.
Safety and Regulatory Information Shipping Crate Labels and Indicators Table 1 Labels and Indicators on the Crate External Labels and Indicators Definition Action TIP N TELL Blue beads in the arrow Write on the Bill of Lading and indicate that the container was check for damage. Any claims tipped or mishandled. for tipping require a notation. or Shock Indicator The indicator is broken if the container has suffered a shock greater than the level marked on the indicator.
Safety and Regulatory Information Regulatory Compliance This system complies with the standards and regulations listed in this section. Applicable labels have been affixed to the system. Australia and New Zealand • Electromagnetic Interference—AS/NZ CISPR 11 (Class A) • Safety—AS/NZ 61010-1 Canada • Electromagnetic Interference—CAN/CSA CISPR11-04. This ISM device complies with Canadian ICES-001. • Safety—CAN/CSA C22.2 No. 61010-1-04, CAN/CSA C22.2 No.
Safety and Regulatory Information International • Electromagnetic Compatibility—IEC 61326-1; CEI/IEC CISPR 11 • Safety—IEC 61010-1; IEC 61010-2-061 For more information, refer to the Declaration of Conformance included with the system. Symbols and Labels on the Mass Spectrometer Table 2 Labels on the Mass Spectrometer External Labels Definition High Voltage WARNING: NO USER SERVICEABLE PARTS INSIDE. REFER SERVICING TO QUALIFIED PERSONNEL.
Safety and Regulatory Information Table 2 Labels on the Mass Spectrometer (Continued) External Labels Definition Do not dispose of equipment as unsorted municipal waste (WEEE). FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. This mass spectrometer is for research use only. It is not intended for use in diagnostic procedures. This instrument contains the following technology: LINAC® Collision Cell Curtain Gas™ Interface WARNING: Hot Surface Hazard.
Safety and Regulatory Information Table 2 Labels on the Mass Spectrometer (Continued) External Labels Definition V-A Volts - Amperes (power) W Watts (power) Occupational Health and Safety Symbols This section describes some occupational health and safety symbols found in the laboratory environment.
Safety and Regulatory Information Table 4 Mechanical Hazard Symbols (Continued) Safety Symbol Description Crushing Hazard — From Side Fire Hazard Hot Surface Hazard Laser Radiation Hazard Lifting Hazard Magnetic Hazard Puncture Hazard Table 5 Pressurized Gas Hazard Warning Symbols Safety Symbol Description Pressurized Gas Hazard Mains Supply WARNING! Electrical Shock Hazard: Use only qualified personnel for the installation of all electrical supplies and fixtures, and make sure that all installati
Safety and Regulatory Information Protective Earth Conductor The mains supply should include a correctly installed protective earth conductor that must be installed or checked by a qualified electrician before connecting the mass spectrometer. WARNING! Electrical Shock Hazard: Do not intentionally interrupt the protective conductor. Any interruption of the protective earth conductor is likely to make the installation dangerous.
Safety and Regulatory Information WARNING! Biohazard: For biohazardous material use, always apply local regulations for hazard assessment, control, and handling. This instrument or any part is not intended to act as a biological containment safety cabinet. Instrument Disposal (Waste Electrical and Electronic Equipment) Do not dispose of system components or subassemblies, including computer parts, as unsorted municipal waste.
1 System Information The AB SCIEX TripleTOF® 5600/5600+ system is designed for the qualitative and quantitative analysis of chemical species. The system includes a mass spectrometer, a DuoSpray™ ion source, the optional calibrant delivery system (CDS), and a computer running the Analyst® TF software.
System Information System Overview Figure 1-1 to Figure 1-2 show the mass spectrometer components and connections. 1 2 4 3 7 6 8 5 Figure 1-1 Front and right side Item Description For more information... 1 Optional CDS Refer to the CDS Operator Guide. 2 DuoSpray™ ion source Refer to DuoSpray™ Ion Source User Reference on page 83 3 Syringe pump Refer to Adjust the Integrated Syringe Pump Position on page 23. 4 Dress panel LEDs Refer to Dress Panel LEDs on page 20.
System Information Figure 1-1 Front and right side (Continued) Item Description 8 For more information... Serial (RS-232) cable connection for the syringe pump 1 Contact an AB SCIEX FSE. 11 10 9 2 8 3 4 7 5 12 6 13 14 15 Figure 1-2 Left side view Item Description For more information... 1 Gas and vacuum bulkhead Contact an AB SCIEX FSE. 2 Calibrant control connection See the CDS Operator Guide. 3 AUX IO connection. The optional LC system start signal connects to this port.
System Information Figure 1-2 Left side view (Continued) Item Description For more information... 7 Gas 1 and Gas 2 (zero) supply connection Contact an AB SCIEX FSE. 8 Source exhaust gas (zero air or nitrogen) supply connection Contact an AB SCIEX FSE. 9 CAD gas (nitrogen) supply connection Contact an AB SCIEX FSE. 10 Source exhaust waste connection Contact an AB SCIEX FSE. 11 Roughing pump vacuum connection Contact an AB SCIEX FSE. 12 AC distribution panel Contact an AB SCIEX FSE.
System Information Use the following procedures if you need to turn on or shut down the system. You may need to shut down the system to perform maintenance. 1. Make sure there is clear access to the mass spectrometer AC mains power cord. The cord must be accessible in order to disconnect the instrument from the AC mains power supply. 2. Make sure the 4 L drain bottle is connected to the Exhaust Waste connection on the rear of the instrument and to the laboratory ventilation system. 3.
System Information Use the Integrated Syringe Pump Edit the Hardware Profile for the Integrated Syringe Pump Make sure that the serial (RS-232) cable is connected between the computer and mass spectrometer. Make sure the syringe pump is seated properly to avoid damaging the syringe. For more information about creating and editing hardware profiles, refer to Create a Hardware Profile on page 29. 1. In the Navigation bar, under Configure, double-click Hardware Configuration. 2.
System Information 5. Click Configure Pump. Figure 1-4 Harvard Syringe dialog 6. Select the COM port for the connection to the syringe pump. 7. Click OK until the Hardware Configuration Editor dialog box appears. 8. Activate the hardware profile. Adjust the Integrated Syringe Pump Position 1. Press the Release button on the right side of the syringe pump to lower the base and then insert the syringe as shown in Figure 1-5.
System Information 1 2 Figure 1-5 Lowering the syringe Item Description 1 Syringe plunger. 2 Release button. Press to raise or lower the base. 2. Adjust the post, shown in Figure 1-6, so that it triggers the automatic syringe stop before the syringe plunger hits the glass syringe. 1 2 3 Figure 1-6 Safety stop Item Description 1 Automatic syringe stop. After the post hits the automatic syringe stop, the syringe pump stops.
System Information Figure 1-6 Safety stop (Continued) Item Description 2 Post. Adjust the height to prevent the syringe plunger from hitting the syringe during sample infusion. 3 Post lock screw. Tighten the screw after you have adjusted the height of the post. 3. Turn the side screws as shown in Figure 1-7 to secure the syringe. Figure 1-7 Syringe pump 4. In the Analyst TF software, on the Navigation bar, double-click Manual Tuning. 5. Click Start Syringe. 6.
System Information Reset the Syringe Pump If the Analyst TF software stops communicating with the syringe pump, you can reset the syringe pump. • Use a paper clip or similar tool to press the reset button, shown in Figure 1-8.
2 Hardware Profiles and Projects Sample Workflows • Instrument Setup on page 27 • Sample Acquisition Workflow on page 27 • Experienced User Workflow on page 28 Table 2-1 Instrument Setup Step To do this... Find the information in... What does it do? 1 Create a hardware profile. Create a Hardware Profile on page 29 Each hardware profile must include a mass spectrometer. Only devices included in the active hardware profile can be used when creating acquisition methods.
Hardware Profiles and Projects Table 2-2 Sample Acquisition Workflow (Continued) Step To do this... Find the information What does it do? in... 2 Create an acquisition Basic Acquisition method. Methods on page 41 To analyze samples, create an acquisition method for the mass spectrometer and any LC devices. An acquisition method indicates which peripheral devices to use, when to use them to acquire data, and the associated parameters. 4 Create and submit a batch.
Hardware Profiles and Projects Hardware Profiles A hardware profile tells the software what instrument and peripheral devices to use, and how the instrument and the devices are configured and connected to the computer. Each hardware profile must include a mass spectrometer and only peripheral devices included in the active hardware profile can be used when creating acquisition methods. Before creating an acquisition method, make sure that all devices used in the method are included in the hardware profile.
Hardware Profiles and Projects 3. In the Profile Name field (Figure 2-2), type a name for the profile. For example, TripleTOF5600+Shimadzu.
Hardware Profiles and Projects 4. Click Add Device. In the Available Devices dialog, in the Device Type field, Mass Spectrometer is the preset value (Figure 2-3). Figure 2-3 Available Devices dialog 5. In the Devices list, select the Mass Spectrometer TripleTOF 5600 instrument and then click OK. 6. In the Devices in current profile list, select the instrument. 7. Click Setup Device. 8.
Hardware Profiles and Projects 9. (Optional) If using an integrated syringe pump, select Use integrated syringe pump. Figure 2-4 Configuration tab 10. Click OK to return to the Create New Hardware Profile dialog. 11. Click Add Device and then add and configure each peripheral device that is used with the instrument. Refer to Add Peripheral Devices to Hardware Profiles on page 33. 12. Ensure all changes are accepted and then click OK. 13.
Hardware Profiles and Projects Add Peripheral Devices to Hardware Profiles Peripheral devices must be configured to enable the software to communicate with them. Configuring the peripheral devices requires two procedures: setting up the physical connections and configuring the software to communicate with the peripheral devices.
Hardware Profiles and Projects • If the device uses Ethernet communication, type the IP address assigned to the device or use the corresponding host name for the address. • If the device uses a GPIB board as a communication interface, do not change the settings for the GPIB board. The rest of the preset values for the device are likely appropriate; do not change them. For information about the Configuration and Communication tabs, refer to the Help. 10.
Hardware Profiles and Projects Create Projects and Subprojects To use a subproject structure within a project, create the subproject structure when the project is created. 1. Click Tools > Project > Create Project. 2. In the Project name field, type a project name. 3. To use subprojects in this project, select the required folders in the Projects folders list and then use the arrow buttons to move them to the Subproject folders list (Figure 2-6).
Hardware Profiles and Projects Create a New Subproject Subprojects can only be created in a project that has an existing subproject structure. 1. On the Project toolbar, from the Project list, select the project. 2. Click Tools > Project > Create Subproject. 3. In the Subproject name box, type a name for the subproject. 4. Click OK. Copy a Subproject Note: The user can copy a subproject from another project that has existing subprojects.
Hardware Profiles and Projects Switch Between Projects and Subprojects • On the software toolbar (Figure 2-7), from the project list, click the required project or subproject. 1 Figure 2-7 Project List Item Description 1 Project list showing a folder, Tutorial, and the Tutorial folders subfolders. Installed Project Folders Three project folders are installed with the software: API Instrument, Default, and Example.
Hardware Profiles and Projects Back up the API Instrument Project Back up the API Instrument folder regularly and after routine maintenance has been performed. 1. To create the backup, copy the API Instrument project, paste it to a different location, preferably to another computer, and then rename the folder. Use the date and an instrument reference if there is more than one instrument when the folder is named. 2.
3 Instrument Tuning and Calibrating Run the Verify Performance Only option at any time; however, tune the instrument only if a loss of sensitivity or resolution is noticed. For more information about tuning and calibration, refer to the Advanced User Guide. For tuning the system, use the following solutions that come with the installation kit: For positive mode: • For optimizing TOF MS - MSMS high resolution or MSMS High Sensitivity, use the Tuning Solution.
Instrument Tuning and Calibrating 4. Select a tuning solution. Make sure that the tuning solution matches the reference table. 5. The Verify Performance Only check box is preselected. Click Next. For this example, leave this option selected. If the report indicates that the instrument needs tuning, then run Instrument Optimization again and select one or more scan modes to optimize. 6. Make sure that the ion source and syringe parameters are suitable. Note: Users can also use the CDS to inject the solution.
4 Basic Acquisition Methods An acquisition method consists of the method for the mass spectrometer and for liquid chromatography (LC) devices. Users can easily create an acquisition method using the Method Wizard. The Acquisition Method Editor can also be used to create acquisition methods and to add a sequence of periods and experiments for the instrument and devices.
Basic Acquisition Methods Tip! If required, users can further edit the acquisition method using the Acquisition Method Editor. In Acquire mode, click File > Open and then open the method that was created using the Method Wizard. 10. Next steps: The newly created acquisition method can now be used to acquire data for preliminary analysis.
Basic Acquisition Methods Add an Experiment 1. Right-click the period where an experiment needs to be added and then click Add experiment. An experiment is added below the last experiment in the period. Note: An experiment or a period cannot be inserted between experiments or periods. Users can only add an experiment at the end of the period. 2. In the Acquisition Method Editor pane, select the appropriate device or instrument parameters.
Basic Acquisition Methods Scan Techniques The system is a versatile and reliable system for performing liquid chromatography mass spectrometry analysis on liquid sample streams to identify, quantify, and examine polar compounds.
Basic Acquisition Methods Product Ion Mass Spectrometry In a product ion scan (Product Ion), the system generates mass spectrometric information by selecting a particular precursor ion window in Q1, fragmenting in Q2 (a collision cell) and pulsing the ions (fragment ions) into a flight tube and recording their precise arrival time at the detector. Product ions can provide information on the molecular structure of the original (precursor) ions.
Basic Acquisition Methods 1 3 4 2 Figure 4-1 Ion optics path and parameters Item Parameter Parameter type Use Scan type 1 Ion Source Source and Gas 1 (Gas 1) Gas The GS1 parameter controls the All nebulizer gas. The nebulizer gas helps generate small droplets of sample flow and affects spray stability and sensitivity. 1 Ion Source Source and Gas 2 (Gas 2) Gas The GS2 parameter controls the flow of All heater gas, which helps evaporate the solvent to produce gas phase sample ions.
Basic Acquisition Methods Figure 4-1 Ion optics path and parameters (Continued) Item Parameter Parameter type Use Scan type 1 IonSpray Voltage Floating (ISVF) Source and Gas The ISVF parameter affects the stability of the spray and hence the signal sensitivity. This is the voltage applied to the needle that sprays the sample.
Basic Acquisition Methods Figure 4-1 Ion optics path and parameters (Continued) Item Parameter Parameter type Use 1 Compound The DP parameter controls the voltage TOF MS/MS on the orifice, which affects the ability to decluster ions between the orifice and QJet® ion guide. It is used to minimize the solvent clusters that may remain on the sample ions after they enter the vacuum chamber, and, if required, to fragment ions. The higher the voltage, the higher the energy imparted to the ions.
Basic Acquisition Methods Figure 4-1 Ion optics path and parameters (Continued) Item Parameter Parameter type Use 3 Compound The amount of time in milliseconds MS/MS only, before the ion pulse. The default (11 Enhanced msec) is calculated based on the TOF masses and can be adjusted by the operator. The range is typically 6 msec to 333 msec. Ion Release Delay (IRD) Scan type This parameter is optimized using Instrument Optimization if the Enhanced Ion option is selected in the Advanced options.
Basic Acquisition Methods Table 4-2 Acquisition Method Editor Icons (Continued) Icon Name Function Syringe Pump Opens the Syringe Pump Properties tab. Column Oven Opens the Column Oven Properties tab. Valve Opens the Valve Properties tab. DAD Opens the DAD Method Editor. For more information about the DAD, refer to View DAD Data on page 74. ADC Opens the ADC Properties tab. For more information about the ADC, refer to Show ADC Data on page 67.
5 Batches A batch is a collection of information about the samples to be analyzed. Batches tell the software the order in which to analyze the samples. For information about importing batches, refer to the Advanced User Guide. Create and Submit a Batch Use this workflow to create and submit a batch.
Batches Figure 5-1 Queue Options dialog 3. In the Max Num Waiting Samples field, set the maximum number of samples to a value that is greater than the number of samples that will be submitted to the queue. 4. In the Max Idle Time field, type the length of time the queue will wait after acquisition is completed before going into Standby mode. The preset value is 60 minutes. If using gas cylinders, adjust this time to make sure that the gas in the cylinders is not depleted.
Batches Add Sets and Samples to a Batch A set can consist of a single sample or multiple samples. 1. In the Navigation Bar, under Acquire, double-click Build Acquisition Batch. Figure 5-2 Batch Editor 2. In the Sample tab, in the Set list, type a name. 3. Click Add Set. 4. Click Add Samples to add samples to the new set. Figure 5-3 Add Sample dialog 5. In the Sample name section, in the Prefix field, type a name for the samples in this set. 6.
Batches For example, if 3 is typed, the sample names would be samplename001, samplename002, samplename003. 8. In the Data file section, in the Prefix field, type a name for the data file that will store the sample information. 9. Select the Set name check box to use the set name as part of the data file name. 10. Select the Auto Increment check box to increment the data file names automatically. Note: AB SCIEX recommends using a new .wiff file for each sample. 11. In the Sub Folder field, type a name.
Batches 2. In the Calibrate Every _ Samples field, type the number of samples to be acquired between calibration samples. 3. From the Calibrant Reference Table, select a table from the list of all calibrant reference tables available for the current polarity. Ensure that the selected reference table has the correct Calibrant Valve Position. 4. Set the CDS Inject Flow Rate. When the batch is submitted, the calibration samples are inserted into the queue. Each set starts with a calibration sample.
Batches 2. Click View > Sample Queue. The Queue Manager opens with all submitted samples. 2 3 1 Figure 5-4 Queue Manager Item Description 1 The Tune icon should not be pressed in. 2 Queue status. 3 Queue Server should be in Normal mode. For more information, refer to Queue States on page 59. 3. Click Acquire > Start Sample. 4. Next steps: After the data has been acquired, it can be analyzed.
Batches Select Vial Positions using the Locations Tab (Optional) 1. In the Batch Editor, click the Locations tab. 2. In the Set list, select the set for which sample locations need to be specified. 3. In the Autosampler list, select the autosampler. The appropriate number of rack spaces for the autosampler is shown in the graphic rack display. 4. In the space associated with the rack, right-click and then select the rack type. The plates or trays are shown in the rack. 5.
Batches Batch and Acquisition Method Editor Tips Table 5-1 Tips To do this... ...do this To change all the values in a column click a column heading and then right-click. From the simultaneously menu, use the Auto Increment and Fill Down commands to change the values in the column. This also works for multiple cells in the same column. To change an existing acquisition method from the list, select the method and then click Method Editor.
Batches Figure 5-5 Batch right-click menu (Continued) Menu Function Hide/Show Column Hides or shows a column. Save Column Settings Saves the batch column settings. Add Custom Column Adds a custom column. Delete Custom Column Deletes a custom column. Fill Down Fills the same data into the selected cells. Auto Increment Automatically increments data into the selected cells. Delete Samples Deletes the selected row. Select Autosampler Selects an autosampler.
Batches Table 5-2 Queue States (Continued) Icons State Definition Standby In the Standby state, the hardware profile has been activated, but all devices are idle. Pumps are not running and gases are turned off. Warming Up In the Warming Up state, the instrument and devices are equilibrating, columns are being conditioned, the autosampler needle is being washed, and column ovens are reaching temperature. The period of equilibration is selected by the operator.
Batches Table 5-3 Instrument and Device Status (showing the instrument icon) Status Icon Background color Description Idle Green or yellow The device is not running. If the background color is yellow, the device should be equilibrated before it is ready to run. If the background color is green, the device is ready to run. Equilibrating Green or yellow The device is equilibrating.
Batches Queue Right-Click Menu Right-click in the Queue table to access the following options. Figure 5-8 Queue Manager right-click menu Menu Function Sample Details Opens the Sample Details dialog. Reacquire Acquires a sample again. Insert Pause Inserts a pause, in seconds, between two samples. Delete Deletes either the batch or the selected samples. Move Batch Moves the batch within the queue. Sort Sorts by the preselect column. Column Settings Changes the column settings.
Batches Table 5-4 Acquire Mode Icons (Continued) Icon Name Function Stop Sample Stops the sample in the queue. Abort Sample Aborts a sample acquisition in the middle of the processing of that sample. Stop Queue Stops the queue before it has completed processing all the samples. Pause Sample Now Inserts a pause in the queue. Insert Pause before Selected Sample Inserts a pause before a specific sample. Continue Sample Continues acquiring the sample. Next Period Starts a new period.
Batches AB SCIEX TripleTOF® 5600/5600+ Instruments 64 of 116 System User Guide D5033331 A
Analyzing and Processing Data 6 Use the sample files installed in the Example folder to learn how to view and analyze data using the most common analysis and processing tools. For more information about the following topics, refer to the Advanced User Guide.
Analyzing and Processing Data 4. To skip to the next sample in the data file, click the icon with the arrow pointing to the right. 5. To skip to a non-sequential sample, click the icon with the arrow curving to the right. 6. In the Select Sample dialog, in the Sample list, select the sample. 7. To go to the previous sample in the data file, click the icon with the arrow pointing to the left.
Analyzing and Processing Data Show Experimental Data in Tables • With a data file open, click Explore > Show > Show List Data. The data is shown in a pane below the graph. Table 6-2 Right-Click Menu for the Spectral Peak List Tab Menu Function Column Options Opens the Select Columns for Peak List dialog. Save As Text Saves the data as text file. Delete Pane Deletes the pane.
Analyzing and Processing Data 3. Click Explore > Show > Show ADC Data. Figure 6-1 Select ADC Channel dialog 4. In the Channel list, select a channel. 5. Click OK. The ADC data opens in a new pane below the active pane. Show Basic Quantitative Data 1. With a data file open, click Explore > Show > Show List Data. 2. In the Peak List tab, right-click and select Show Peaks in Graph. Peaks appear in two colors. 3.
Analyzing and Processing Data Table 6-4 Chromatograms (Continued) Types of chromatograms Purpose BPC (Base Peak Chromatogram) Chromatographic plot that shows the intensity of the most intense ion within a scan versus time or scan number. TWC (Total Wavelength Chromatogram) A chromatographic display created by summing all of the absorbance values in the acquired wavelength range and then plotting the values against time.
Analyzing and Processing Data There are several methods for extracting ions to generate an XIC, depending on whether chromatographic or spectral data is used. Table 6-5 contains a summary of methods that can be used with chromatograms and spectra. Table 6-5 Summary of XIC Generation Methods Method Use with chromatogram Use with spectrum Extraction Selected range No Yes The selected range method extracts ions from a selected area in a spectrum.
Analyzing and Processing Data To generate an XIC using the maximum peak 1. In the Navigation Bar, under Explore, double-click Open Data File. The Select Sample dialog opens. 2. In the Data Files list, select a file containing spectra. 3. Click OK. 4. Select a range. The selection is highlighted in blue. 5. Click Explore > Extract Ions > Use Maximum. An XIC of the maximum peak specified selection opens below the spectrum pane.
Analyzing and Processing Data 2. Click Explore > Extract Ions > Use Dialog. Figure 6-2 Extract Ions dialog 3. Type the values for each XIC to be created. If a stop value is not typed, then the range is defined by the start value. • In the Start field, type the start value (lower value) for the mass range to be extracted. • In the Stop field, type the stop value (higher value) for the mass range to be extracted. 4. Click OK. An XIC of the selection opens below the chromatogram pane.
Analyzing and Processing Data 2. Click Explore > Show > Show Base Peak Chromatogram. The selections are shown in the Start Time and End Time fields. Figure 6-3 Base Peak Chromatogram Options dialog 3. In the Mass Tolerance field, type the value to dictate the mass range used to find a peak. The software finds the peak using a value twice the typed range (± the mass value). 4. In the Minimum Intensity field, type the intensity below which peaks are ignored by the algorithm. 5.
Analyzing and Processing Data Generate XWCs The user can extract up to three ranges from a DAD spectrum to generate the XWC. For more information about using the available icons, refer to Table 6-8. 1. Open a data file that contains a DAD spectrum. 2. Anywhere in the pane, right-click and then click Extract Wavelengths. Figure 6-4 Extract Wavelengths dialog 3. Type start and stop values. 4. Click OK. The XWC opens in a pane below the DAD spectrum.
Analyzing and Processing Data The TWC opens in a pane below the DAD spectrum. Tip! Right-click inside the pane containing the DAD spectrum and then click Show DAD TWC. Adjust the Threshold The threshold is an invisible line drawn parallel to the x-axis of a graph that sets a limit below which the software will not include peaks in a spectrum. The line has a handle, represented by a blue triangle to the left of the y-axis. Click the blue triangle to view a dotted line that represents the threshold.
Analyzing and Processing Data Table 6-6 Right-Click Menu for Chromatogram Panes (Continued) Menu Function Save Explore History The Explore History File records changes to processing parameters, also called Processing Options, when a .wiff file is processed in Explore mode. The processing history is stored in a file with an .EPH (Explore Processing History) extension. Add Caption Adds a caption at the cursor point in the pane. Add User Text Adds a text box at the cursor point in the pane.
Analyzing and Processing Data Data Processing Graphical data can be processed many ways. This section provides information and procedures for using some of the most commonly used tools. The user can zoom in on part of a graph to view a particular peak or an area in greater detail in both spectra and chromatograms. The user can also zoom in repeatedly to view smaller peaks. Graphs The same data can be examined in different ways.
Analyzing and Processing Data Table 6-8 Graph Options (Continued) To do this... use this menu option... Move a pane • Select the graph. Click Window > Move Pane. ...or click this icon • Select the pane or window and then drag it to the new position. This position can be within the same window or within another window. A four-headed arrow is shown when the cursor is on the boundary of the active window or pane.
Analyzing and Processing Data Zoom in on the y-axis 1. Position the pointer to the left of the y-axis and then drag vertically away from the starting point. A box is drawn along the y-axis representing the new scale. Note: Take care when zooming in on the baseline. Zoom in too low and the zoom-in box disappears. 2. Release the mouse button to draw the graph to the new scale. Tip! To return the graph to the original scale, double-click on either axis.
Analyzing and Processing Data Table 6-9 Explore Quick Reference: Chromatograms and Spectrum (Continued) Icon Name Function Show Base Peak Chromatogram Generates a BPC. Show Spectrum Generates a spectrum from a TIC. Copy Graph to new Window Copies the active graph to a new window. Baseline Subtract Opens the Baseline Subtract dialog. Threshold Adjusts the threshold. Noise Filter Click to use the Noise Filter Options dialog to define the minimum width of a peak.
Analyzing and Processing Data Table 6-9 Explore Quick Reference: Chromatograms and Spectrum (Continued) Icon Name Function Show History View a summary of data processing operations performed on a particular file, such as smoothing, subtraction, calibration, and noise filtering. Open Compound Database Opens the compound database. Set Threshold Adjusts the threshold. Show Contour Plot Shows selected data as either a spectrum graph or an XIC.
Analyzing and Processing Data AB SCIEX TripleTOF® 5600/5600+ Instruments 82 of 116 System User Guide D5033331 A
7 DuoSpray™ Ion Source User Reference The DuoSpray™ ion source combines a TurboIonSpray® probe and an APCI (atmospheric pressure chemical ionization) probe in a single ion source housing. WARNING! Toxic Chemical Hazard: Use the ion source only if you have knowledge of and training in the proper use, containment, and evacuation of poisonous or injurious materials used with the ion source.
DuoSpray™ Ion Source User Reference Figure 7-1 Parts of the ion source (Continued) Item Description 4 Y-axis adjustment knob for the APCI probe, used to position the probe on the vertical axis for ion source sensitivity adjustments 5 Corona discharge needle, which ionizes the trace species, or sample gas. Primary ions, which are formed as a result of the discharge, are converted by collisional processes to final ion-molecule reaction products.
DuoSpray™ Ion Source User Reference 1 2 3 Figure 7-2 Parts of the TurboIonSpray probe Item Description 1 Electrode adjustment nut (black collar) that adjusts the extension of the electrode tip 2 Bronze retaining ring that fastens the probe to the probe tower on the ion source housing 3 Electrode tip through which samples are sprayed into the sample inlet area of the ion source APCI Probe The APCI probe is suitable for: • Ionization of compounds that do not readily form ions in solution.
DuoSpray™ Ion Source User Reference 1 Figure 7-3 2 3 Parts of the APCI probe Item Description 1 Electrode adjustment nut (black collar) that adjusts the extension of electrode tip 2 Bronze retaining ring that fastens the probe to the probe tower on the ion source housing 3 Electrode tip through which samples are sprayed into the sample inlet area of the ion source Gas and Electrical Connections Gas and high-voltage electrical connections enter through the front plate of the interface and connect
DuoSpray™ Ion Source User Reference WARNING! Toxic Chemical Hazard: Vent the source exhaust system to an external fume hood or an external vent to prevent hazardous vapors from being released into the laboratory environment. A pressure switch mounted on the source exhaust pump measures the pressure in the source exhaust line. If the pressure in the line rises above the set point while the probes are installed, the high-voltage power supply is turned off.
DuoSpray™ Ion Source User Reference 1. Adjust the black electrode adjustment nut on the probe to move the electrode tip inside the electrode tube. For optimum stability and performance, the electrode tip should extend between 0.5 mm and 1.0 mm from the end of the probe.Insert the probe into the tower. 2. Insert the APCI probe into the tower that is on the left side of the ion source when the glass window is facing you, inserting the raised plastic post into the groove on the probe. 3.
DuoSpray™ Ion Source User Reference 2. Install the sample tubing nut on the fitting at the top of the TurboIonSpray probe. 3. Tighten the sample tubing nut until it is finger-tight. 4. Connect red PEEK tubing from the sample supply device to the grounding union on the ion source. 5. Connect the other end of the red PEEK tubing to the grounding union. Tip! Refer to Figure 7-1 on page 83 for a picture on the parts referenced in this procedure.
DuoSpray™ Ion Source User Reference Tip! It is easier to optimize signal and signal-to-noise with FIA or on-column injections. Optimize the TurboIonSpray Probe Optimize performance while injecting a known compound and monitor the signal of the known ion. Adjust the parameters to maximize the signal-to-noise ratio and signal stability.
DuoSpray™ Ion Source User Reference For LC pumps, use a value between 30 and 50 for GS2. Note: Gas 2 is used with higher flow rates typical with an LC system and in conjunction with increased temperature. 3. Type a starting value for IonSpray Voltage Floating (ISVF). 4. In the Curtain Gas (CUR) field, type 20. 5. On the Compound tab, in the Declustering Potential (DP) field, type 80. Adjust the TurboIonSpray Probe Position At low flow rates, the probe can be adjusted to its lowest Y-axis position.
DuoSpray™ Ion Source User Reference especially if the probe is too low, resulting in signal instability and a high chemical background noise. Similarly, a high heater gas flow could produce a noisy or unstable signal. 1. Adjust GS1 and GS2 in increments of 5 to achieve the best signal or signal-to-noise ratio. 2. Increase CUR until the signal starts to decrease. Note: To prevent contamination, use the highest value for CUR possible without sacrificing sensitivity. Do not set CUR lower than 20. 3.
DuoSpray™ Ion Source User Reference Caution: Potential Equipment Damage: If the LC system connected to the mass spectrometer is not controlled by the Analyst TF software, then do not leave the mass spectrometer unattended while in operation. The LC system can flood the ion source housing when the mass spectrometer goes into Standby mode. 1. Start the Analyst TF software. 2. In the Analyst TF software, in Tune and Calibrate mode, double-click Manual Tuning. 3.
DuoSpray™ Ion Source User Reference Adjust the Corona Discharge Needle When using the APCI probe, make sure that the corona discharge needle is pointing toward the orifice. WARNING! Electrical Shock Hazard: Follow this procedure to avoid contact with the high voltages applied to the corona discharge needle and the curtain plate. 1. Use the slotted screwdriver to rotate the plastic screw on the top of the needle. 2.
DuoSpray™ Ion Source User Reference Optimize the APCI Probe Temperature The quantity and type of solvent affects the optimal APCI probe temperature. At higher flow rates, the optimal temperature increases. WARNING! Toxic Chemical Hazard: Vent the source exhaust system to an external fume hood or an external vent to prevent hazardous vapors from being released into the laboratory environment. • Adjust the TEM parameter in increments of 50°C to achieve the best signal or signalto-noise ratio.
DuoSpray™ Ion Source User Reference 1. Switch to a mobile phase that is 50:50 water:acetonitrile or 50:50 water:methanol. 2. Adjust the position of the TurboIonSpray and APCI probes so that they are as far from the orifice as possible. 3. In the Analyst TF software, set Temperature (TEM) to between 500 and 600, Ion Source Gas 1 (GS1) and Ion Source Gas 2 (GS2) to at least 40, and Curtain Gas (CUR) to the highest setting possible. 4. Wait until the TEM setpoint is reached. 5.
DuoSpray™ Ion Source User Reference Clean the Electrode Tube Clean the electrode tube periodically, or when performance decreases. This procedure applies to both the TurboIonSpray and APCI probes. Use this procedure to remove the electrode tube for cleaning. If the electrode tube cannot be cleaned, then use this procedure to replace it with a new part. WARNING! Electrical Shock Hazard: Remove the ion source from the mass spectrometer before starting any maintenance procedures. 1.
DuoSpray™ Ion Source User Reference 6. Remove the electrode tube from the retaining nut. 7. Clean the electrode tube with a 50:50 methanol:water solution, by running the solution through the electrode tube or by soaking the tube in an ultrasonic bath. Replace the Electrode 1. Insert the electrode tube into the retaining nut and then into the PEEK union fitting. Make sure that the electrode tube is inserted as far into the PEEK union fitting as it will go.
DuoSpray™ Ion Source User Reference 1 2 0.5 - 1 mm Figure 7-5 Electrode tip extension adjustment Item Description 1 Probe 2 Electrode Remove the Corona Discharge Needle The corona discharge needle tip may become so corroded that it must be cut off from the corona discharge needle. If this occurs, replace the corona discharge needle. WARNING! Electrical Shock Hazard: Remove the ion source from the mass spectrometer before starting any maintenance procedures.
DuoSpray™ Ion Source User Reference Figure 7-6 Corona discharge needle tip at rear of ion source 5. Turn the corona discharge needle adjustment knob counter-clockwise until it is loose. 6. Gently pull the corona discharge needle adjustment knob to remove the corona discharge needle from the ceramic sleeve, taking care not to break the sleeve. 7. Remove the corona discharge needle adjustment knob from the corona discharge needle. 8.
DuoSpray™ Ion Source User Reference 5. Install the ion source. Refer to Install the Ion Source on page 88. 6. Resume the sample flow. Troubleshooting Table 7-4 Troubleshooting Symptom Possible cause Solution ® • The probe is not installed. The Analyst TF software reports that the mass spectrometer is in Fault state. (The mass spectrometer icon on the Analyst TF software status • The probe is not connected bar is red.) securely. • Install the probe.
DuoSpray™ Ion Source User Reference Table 7-4 Troubleshooting (Continued) Symptom Possible cause Solution Arcing or sparks occur. The position of the corona discharge needle is incorrect. Turn the corona discharge needle toward the curtain plate, and away from the stream of Gas 2. Refer to Adjust the Corona Discharge Needle on page 94. Consumables The following tables list the orderable parts for the DuoSpray™ ion source.
8 Cleaning and Maintenance Regularly clean and maintain the instrument for optimal performance. Table 8-1 provides a recommended schedule for cleaning and maintaining the instrument. Contact your Qualified Maintenance Person to order consumable parts. Table 8-1 System Maintenance Tasks Component Frequency Task For more information, refer to... Curtain plate As needed Clean Clean the Curtain Plate QJet® As needed Clean Contact an AB SCIEX FSE.
Cleaning and Maintenance • Avoid ignition sources when working with flammable materials, such as isopropanol, methanol, and other flammable solvents. • Take care in the use and disposal of any chemicals. Potential risk of personal injury if proper handling and disposing of chemicals are not followed. • Avoid skin contact with chemicals during cleaning, and wash hands after use. • Comply with all local regulations for the handling of biohazard, toxic, or radioactive materials.
Cleaning and Maintenance 1 2 3 4 Figure 8-1 Item Vent connections Description 1 Roughing pump exhaust connection to vent 2 Source exhaust output connection to vent 3 Vacuum hose 4 Source exhaust drain bottle 3. Unscrew the cap and dispose of the waste. 4. Replace the cap and connect the tubes. Front-End Cleaning Clean the instrument front-end using the routine cleaning method, to: • Minimize unscheduled instrument downtime. • Maintain optimum sensitivity.
Cleaning and Maintenance This section provides instructions for performing routine cleaning without breaking vacuum and full cleaning under atmospheric pressure, after venting the instrument. Note: Follow all applicable local regulations. For health and safety guidelines, refer to Health and Safety Precautions for more information.
Cleaning and Maintenance • Prepare and store all organic solutions and organic-containing solutions in very clean glassware only. Never use plastic squirt bottles. Contaminants can leach from these bottles and further contaminate the mass spectrometer. • Allow only the center area of the wipe to contact the instrument surface. Cut edges can leave fibers behind. Tip! Figure 8-2 Wrap the wipe around a thermally-bonded polyester swab.
Cleaning and Maintenance 1 Figure 8-3 Source drain on the vacuum interface Item Description 1 Source drain Clean the Curtain Plate Caution: Potential Instrument Damage: Do not rest the curtain plate on the orifice. Make sure that the conical side faces up. 1. Remove the curtain plate and then place it, conical side up, on a clean, stable surface. Figure 8-4 Interface with curtain plate removed 2. Using wipes and water, clean both sides of the curtain plate. 3.
Cleaning and Maintenance 6. Inspect the curtain plate for solvent stains or lint, removing any residue with a clean, slightly damp lint-free wipe. Note: Persistent spotting or filming is an indicator of contaminated solvent. Clean the Front of the Orifice Plate Note: If the standard orifice plate has a removable interface heater, do not remove the heater during cleaning. 1. When cleaning a NanoSpray orifice plate, remove the interface heater and clean it: i.
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9 Basic System Troubleshooting This appendix contains basic information for troubleshooting basic system issues. Certain activities may be carried out by the AB SCIEX trained Qualified Maintenance Person in the laboratory. For advanced troubleshooting, contact an AB SCIEX Field Service Employee (FSE).
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A Recommended Calibration Ions The following tables list the standards recommended by AB SCIEX for calibrating the AB SCIEX TripleTOF® 5600/5600+ Instruments. For information about tuning solutions, refer to Chapter 3: Instrument Tuning and Calibrating. Table A-1 Q1 PPG Positive Calibration Ions Masses 59.04914 175.13286 442.3374 674.50484 906.67228 1196.88158 Table A-2 Q1 PPG Negative Calibration Ions Masses 44.99819 411.25991 585.38549 933.63665 1165.
Recommended Calibration Ions Table A-5 APCI Negative Calibration Solution: TOF MS TOF MS Masses 7-aminoheptanoic acid 144.103 amino-dPEG 4-acid 264.14526 sulfinpyrazone fragment 277.09825 amino-dPEG 6-acid 352.19769 sulfinpyrazone 403.11219 amino-dPEG 8-acid 440.25012 amino-dPEG 12-acid 616.35498 amino-dPEG 16-acid 792.45984 Table A-6 APCI Negative Calibration Solution: MSMS (Sulfinpyrazone) MSMS (Sulfinpyrazone) Masses C6H5O 93.0344 C6H5OS 125.0067 C10H8NO 158.
B Exact Masses and Chemical Formulas PPG Table B-1 contains the exact monoisotopic masses and charged species (positive and negative) observed with the PPG (polypropylene glycol) calibration solutions. The masses and ions were calculated using the formula M = H[OC3H6]nOH, while the positive ion MSMS fragments used the formula, [OC3H6]n(H+). In all calculations, H = 1.007825, O = 15.99491, C = 12.00000, and N = 14.00307.
Exact Masses and Chemical Formulas Reserpine (C33H40N2O9) Table B-2 Reserpine Exact Masses Description Mass Molecular Ion C33H41N2O9 609.28066 Fragment C23H30NO8 448.19659 Fragment C23H29N2O4 397.21218 Fragment C22H25N2O3 365.18597 Fragment C13H18NO3 236.12812 Fragment C10H11O4 195.06519 Fragment C11H12NO 174.09134 Taurocholic Acid (C26H45NO7S) Table B-3 Taurocholic Acid Exact Masses Description Mass Molecular Ion C26H44NO7S 514.28440 Fragment C2H3O3S 106.98084 Fragment C2H6NO3S 124.