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1. File name RS Save as type TOF tofpar Cancel Operator Manual Version 1 0 Volume II Operation 5 19 5 BioTOF Window Figure 5 13 Save As Dialog Box for Saving a Method 2 Select an existing method in the dialog box to update the file with the current parameters or enter an unused name to create a new method 3 Click Save If you save to an existing name a dialog box prompts you to confirm the action Click Yes to overwrite the previous file or No to cancel the action and select a new name 5 7 Tools Menu The Tools menu includes diagnostic functions and a link to the HyStar application if it is installed on the BioTOF workstation 5 7 1 Get Actual Values F5 Note This feature is available with Version 3 0 and greater The values displayed in the Parameter tabs may differ from the values being sent by the controllers to the BioTOF hardware For example the tabs may be displaying ESI Source settings from a just loaded methods file but the ESI Source controller has not yet apply those settings To display the controller values in the BioTOF window Choose Tools gt Get Actual Values from the menu bar or click in the tool bar 5 7 2 Open HyStar NT The Tools Open HyStar NT menu selection is active when the HyStar Software for Chromatography and Hyphenated Experiments application is installed on the BioTOF system host enabling you to launch the HyStar application and run hyphe
2. Instruction Gauge Figure 10 2 Vacuum Pump Motor and Oil Gauge 2 Follow the instructions on the tag attached to the pump 3 If a refill is indicated contact Bruker Daltonics customer service for assistance and to ensure that you have the correct oil Note Use only the specified oil Operator Manual Version 1 0 Volume II Operation 10 5 10 Maintenance 10 3 BioTOF Reflectron Tuning This procedure describes tuning of the BioTOF time of flight analyzer for resolution and peak sensitivity This procedure is completed during final test and reproduced during installation and acceptance and method files with the default parameters are stored on the BioTOF workstation Furthermore the components of the BioTOF time of flight analyzer are configured and mechanically aligned in the factory or at installation by a trained field service engineer Once set the tuning of the BioTOF system is stable and does not require user interaction The procedure is described here for reference and for the purpose of retuning the instrument after extensive periods of use or shutdown of the instrument Note Before re tuning the instrument save the current parameters and calibration in a method file Changing any of the TOF voltages will affect the instrument calibration and re calibration is required after the instrument tuning 10 3 1 Setup for Tuning To set up the BioTOF system to tune Reflectron voltages 1 Save the current par
3. Computer workstation that controls operation of the system stores and displays spectra and provides additional analytical software In the basic BioTOF configuration the sample is introduced to the ESI source by means of a syringe pump Figure 1 1 The sample can also be delivered to the BioTOF via a liquid chromatography LC system Mass Spectrometer Electrospray Fl Flight Tube ss lon Source D BioTOF Figure 1 1 Basic BioTOF System Configuration Getting Started Version 1 0 1 1 1 BioTOF Operation 1 1 Overview Figure 1 2 shows the relationship of the ESI source to the TOF spectrometer and the orthogonal interface between these two components The interface contains an accelerator electrode structure that orthogonally deflects the ions 90 into the TOF ion drift region and also houses the detector that senses the reflected time dispersed ions Orthogonal Interface Accelerator Reflectron Drift Region See Figure 1 3 Detector ESI Source Spray Chamber Figure 1 2 ESI and TOF Operation 1 2 ESI Source In the ESI spray chamber a fine spray of charged droplets is generated from the sample solution by means of a sprayer under the influence of an electric field only or an electric field with the assistance of a pneumatic nebulizer When the solvent evaporates from the droplets their charge per volume increases resulting in electrostatic Coulomb forces that break up the droplets into ever smalle
4. Eile Edit View Method Tools Format Help a Di lose T tl _ gt t RMR A A Figure 5 10 BioTOF Menu Bar and Tool Bar 5 12 Operator Manual Version 1 0 Volume II Operation 5 BioTOF Window 5 4 File and Edit Menus Exit is the only active item in the File menu the Edit menu is not used in he BioTOF application To close the BioTOF window Choose File gt Exit from the menu bar or click C31 in the upper right corner of the window 5 5 View Menu 5 5 1 Tool Bar Choose View gt Tool Bar to toggle the display of the BioTOF tool bar Figure 5 10 on and off The tool bar includes functions for controlling the Spectra Display properties and for quick access to functions in the Method and Tools menu The icons are described in Table 5 4 Table 5 4 BioTOF Tool Bar Icons Icon Function Description el Select Method Displays a Windows Open dialog box for loading a ie previously saved method See Load Method on page 5 18 Start Starts spectra acquisition Stop Stops spectra acquisition Get Active Values Retrieves parameter values from the BioTOF drivers so that tab displays reflect values actually being sent to the system controllers Operator Manual Version 1 0 Volume II Operation 5 13 5 BioTOF Window Table 5 4 Icon Function Print Autoscale X Axis BioTOF Tool Bar Icons Continued Description Opens a Print dialog box for selecting a printer setting print properti
5. When Instrumentation is set to MS Only or FIA MS the Spectra Display window includes a single pane displaying the accumulated records for each m z value Figure 5 6 Units for the horizontal axis can be set to m z flight time or point see Chromatogram Tab on page 7 6 Int BioT OF Spectrum 50000 Points 200 300 400 500 600 m z Figure 5 6 Spectra Display Window for MS Only and FIA MS When Instrumentation is set to Chromatography MS the window also includes the Chromatogram pane that displays ion counts on a time scale Figure 5 7 In this mode the display units in the MS pane can only be m z nt BioTOF Spectrum 54000 Points viv 10 0 10 5 11 0 115 120 125 Time min Figure 5 7 Spectra Display Window in Chromatography MS Mode Operator Manual Version 1 0 Volume II Operation 5 7 5 BioTOF Window 5 2 Spectrum Display Pop up Menu Several of the Spectrum Display functions in the tool bar are also available from a pop up menu in the Spectrum Display Window To access the menu Right click anywhere in the Spectrum Display The context menu displays the functions listed in Table 5 3 Table 5 3 Spectrum Display Window Pop Up Menu Items Function Description Mouse Zoom Rectangle Enables you to define an area and then zoom in to display that area by dragging the cursor from one corner of the area you want to zoom in on to the opposite corner and then releasing the
6. 2 3 2 3 N nospray ER 2 9 2 4 E ENEE 2 10 25 APGI S urce Operation RE 2 10 3 TOF MASS SPECTROMETER OPERATION uunuuusssssnnnnnnnnonnnnnnnnnnnnnnnnnnnennnnnnn 3 1 3 1 Orthogonal Interfaeg EE 3 2 3 2 Flight Tube ee EHRT 3 5 3 3 Detector es ae 3 7 3 4 Bol rra MERLO c I PEE TES 3 8 3 5 Time of Flight Calculation tien aaa teen 3 9 3 6 Space EE ee an Rense 3 11 3 7 MUllipass 22 22 eine 3 12 iv Getting Started Version 1 0 OPERATOR MANUAL VOLUME II OPERATION Il el lee EE ll PREFACE E HN Vil SAFETY LABELS 00 ee VIII 4 SYSTEM SSTARTUP coincida 4 1 4 1 Startfhe Systelias o head etie uS leaden rea SL itd a Boia deen 4 1 4 2 Check the Spray Chamber and Sprayer ccccceceeceeeeeeeeeeeeneeeeeeeeeeeeeseneeees 4 5 4 3 Start the BioTOF Sollware nennen 4 6 QA T rmthe Gases Onnan e nee AEA Ea AG 4 6 S BIOTOF MIN D O E 5 1 5 1 Window E LEE 5 1 5 2 Spectrum Display Pop up Menta a 5 8 5 3 Menus and Tool Ba ran Eege EE EE 5 12 5 4 File a d Edit Menus lt td m 5 13 5 5 View MENU A o O ee 5 13 5 6 Method Menu 5 18 5 7 o kenn 5 20 5 8 Forniar Men tec 5 26 5 9 Help Mena 5 26 6 INTRODUCING SAMPLES iret reete hen rettet 6 1 6 1 Sample Delivery to Electrospray AE 6 1 6 2 Liquid Chromatography EG ae ee a 6 7 6 3 Nanospray star REAT Enana 6 10 6 4 A ne EE 6 16 6 5 Configuring the APCI Spray Chamber AANEREN 6 21 Getting Started Version 1 0 V T METHODS ee 7 1 7
7. C BRUKER CX BioTOF Series Getting Started IRR EU BioTOF BRUKER DALTONICS Copyright Copyright 2003 Bruker Daltonics Inc All Rights Reserved Reproduction adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws Document History First edition May 2003 Printed in U S A Warranty The information contained in this document is subject to change without notice Bruker Daltonics Inc makes no warranty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a particular purpose Bruker Daltonics Inc is not liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Bruker Daltonics Inc assumes no responsibility for the use or reliability of its software on equipment that is not furnished by Bruker Daltonics Inc Safety Information Safety Class The instrument is a Safety Class instrument and has been designed and tested in accordance with IEC Publication 1010 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Class 1 LED Product WARNING Connecting an instrument to a power source that is not equipped with a protective earth contact creates a shock hazard for the operator and can damage the instrume
8. Figure 8 4 Indicators and Controls on the Left Side of the BioTOF Window To run spectra acquisition 1 Click the Start button Data acquisition and transfer begin and the Spectra Display plots the results The Spectrum Counter indicates the total number of records accumulated in the buffer The Start button changes to a Stop button enabling you to manually stop acquisition when a sufficient number of records have been stored in the buffer 8 4 Operator Manual Version 1 0 Volume Il Operation 8 Acquiring Data 2 Click the two checkboxes in the lower left corner of the Spectra Display to enable autoscaling for both axes of the display Figure 8 5 The BioTOF continues to accumulate records until the buffer contains the number of records specified in PreSet Spectra f Manual Stop is selected the buffer is cleared and a new acquisition is started If Auto Stop is selected acquisition stops and the data in the buffer is maintained Int JBioTOF Spectrum 50000 Points 600 500 400 300 vw 300 400 500 600 m z Figure 8 5 Spectra Display 3 Click the Stop button to manually stop the acquisition when there sufficient records The Save button becomes active Operator Manual Version 1 0 Volume II Operation 8 5 8 Acquiring Data 4 Click Save in the control panel The BioTOF software opens a dialog box for setting the file options Figure 8 6 File Save Options Path D data 04110
9. Spectra Display Pane Window Command Buttons Pane lt gt Parameter Tabs System Option Buttons Figure 5 1 Layout of the BioTOF Window Operator Manual Version 1 0 Volume II Operation 5 1 5 BioTOF Window The BioTOF window displays information and controls in five different panes The Spectra Display plots the spectra captured by the system There are a variety of user selected formats For example the graph in Figure 5 2 shows the intensity of ion signals from the detector for each m z value The two checkboxes in the bottom left corner of the display control autoscaling for the vertical axis left and horizontal axis right The pane on the upper left side of the window has system status indicators The pane on the lower left side has command buttons for starting and stopping spectra acquisition The four groups immediately to the right of the command buttons includes option button and checkboxes for controlling system operation To the right of these controls are seven tabs for setting system parameters for acquisition and display of the spectra Figure 5 2 shows the location of the Autoscaling checkboxes Ud 2 07 oF Last Good Method Eile Edit View Method Tools Format Help los Ex em E EE Instrumentation Int BioT OF Spectrum 50000 Points MS ONLY 600 500 400 Automation MANUAL 300 Current method 200 Last Good Me
10. The APCI Source accommodates flow rates up to 2 mL min Reversed Phase LC Proteins and peptides exist as amphoteric species which have charged or chargeable sites available on relatively hydrophobic molecules Both charged sites and hydrophobic characteristics are present Proteins and peptides are usually separated using mobile phase modifiers such as trifluoracetic acid TFA TFA inhibits positive ion formation in Electrospray This problem can be overcome through the post column addition of high concentrations of volatile weak organic acids such as formic or acetic acids Normal Phase LC Normal phase LC can be used with Electrospray Solvents used are typically non aqueous and non polar The compounds separated are relatively nonpolar To overcome this compounds can be protonated M H if a proton donor is present in the solution Alternatively alkali metal cation adducts can be formed by the addition of sodium acetate potassium acetate or other metal acetate salts as well as some organic compounds Operator Manual Version 1 0 Volume II Operation 6 7 6 Introducing Samples 6 2 1 Operating With Liquid Chromatography A liquid chromatograph LC can serve several functions in an Electrospray interface As a Pump In this mode of operation only the LC pump is used The sample is injected manually into a sample loop on the multiport valve The valve is switched manually to transport the sample into the Electrospra
11. 1 6 T Temperature 7 8 Time of Fight TOF mass spectrometer 1 2 Time of Flight Calculation 3 9 Timing Sequence Tab 7 11 TOF Mass Spectrometer 3 1 Tool Bar 5 12 Tools Menu 5 20 Trap 2 8 Troubleshooting 10 11 Typical Needle Configurations 6 14 V Vacuum 1 4 Gauge Controller 1 3 4 3 Pumps Button 4 2 Vacuum Pump Motor 10 5 View Menu 5 13 Tool Bar 5 13 Voltage High 1 4 Index 8 Operator Manual Version 1 0 Volumes and Il
12. 1 BioTOF Operation 1 6 Getting Started Version 1 0 2 System Controls 2 System Controls The principal means for controlling and monitoring the BioTOF system is a Windows based application configured on the workstation The software can operate on both Windows NT and Windows XP platforms By means of a single multi pane window you can Start the BioTOF system Set the system parameters Acquire and display spectra Save the data for use with the data processing software included with the system An array of hardware controls and indicators are located on four panels behind a hinged door on the front of the BioTOF cabinet These controls include the On Off switches for the mass spectrometer and ESI source components Many of the controls and indicators are replicated in the BioTOF software and can be set from the BioTOF window An additional panel on the upper right of the BioTOF cabinet displays the status of the ESI source 2 1 BioTOF Window This section gives a brief description of the main window for the BioTOF application Refer to Chapter 5 in the BioTOF Operator Manual Volume Il Operation for a detailed description of the window features including the menus tool bars and options To view the BioTOF window 1 Turn the workstation on 2 Do one of the following when the system is initialized Double click the BioTOF desktop or tool bar icon Tor Choose Start gt All Programs gt BIOTOF gt BioTO
13. rast Good Method rt LRS LER ES TR PRs Int EGTOF Spectrum 50000 Ponts System Status Pane Spectra Display Parameter Tabs Command Buttons Pane Parameter Settings v System Option Buttons Checkboxes Figure 1 4 Layout of the BioTOF Window Operator Manual Version 1 0 Volume I Theory 1 5 1 System Components The BioTOF window displays information and controls in five different panes Spectra Display Plots the spectra captured by the system in a variety of user selected formats The graph in Figure 1 4 on page 1 5 shows the intensity of ion signals from the detector for each mass value m z The two checkboxes in the bottom left corner of the display control autoscaling for the vertical axis left and horizontal axis right System Status Pane Contains the system status indicators Command Buttons Pane Contains command buttons for starting and stopping data acquisition and for saving data System Option The four vertical groups immediately to the right of the Buttons Checkboxes command buttons control system operation Parameter Settings Parameter settings for acquisition and display of the spectra 1 6 Operator Manual Version 1 0 Volume I Theory 2 Source Operation 2 Source Operation This chapter describes the various means of introducing sample to the ESI source the operation of the source using the Electrospray and t
14. 0 3 3 3 Quick Start 9 Use the arrow button below the LCD display to position the display cursor on End Figure 3 4 so that the Source Lens Current gauge on the ESI Source reads the current on the Endplate Electrode CON edit active Remote Menu 1500 3500 4000 Cyl Ela Cap Endplate Electrode Select End for the Source Lens Current Reading Figure 3 4 Setting the Source Lens Current Reading 10 Set the ion Polarity and Enable switches on the Instrument HV unit to the middle position When the two 3 position switches on the Instrument HV panel Figure 3 3 on page 3 3 are in the middle position the unit is remotely controlled from the BioTOF window 11 Check the nitrogen supply 3 4 Getting Started Version 1 0 3 Quick Start 3 2 Check the Spray Chamber and Sprayer The next step is to check the Sprayer Assembly on the front of the ESI Source The assembly is attached to the ESI Source with hinges on the right and locked with a latch on the left A sample is introduced to the spray chamber via an off axis spray needle which is held in position by a bayonet connection Figure 3 5 shows the sprayer assembly unlatched and open and the off axis sprayer removed from the assembly Spray Shield Off Axis Sprayer Nebulizer Gas Nebulizer Gas Valve Figure 3 5 Off axis Sprayer Assembly To check the Spray Chamber 1 Release the latch on the left side of the assembly and swing the assembly o
15. 6 23 6 Introducing Samples 11 The vacuum side voltages should be set for normal operation The recommended default values are Capillary Exit 130 V Skimmer 1 40 V Skimmer 2 30 V Offset 15V Trap 45 V Lens 50 V RF Amplitude 600 V 12 Set the ampere meter range switch on the source controller to High 13 On the Source Control Panel select CNdl for the Source Lens Current reading 14 Increase the Corona needle voltage until a stable current appears on the current meter of the source controller Using the above values for End and Cap this should be reached at 1200 V to 1300 V The current should be in the 10 A range Move or turn the corona needle to establish contact if no current is observed at 1400 V 15 Set mass range to 100 to 700 and start data acquisition Observe the spectrum and tune the CNal voltage to get a stable signal Note Do notuse excessively high differential voltages between CNdl on one side and Endplate Capillary on the other side Keep End to Cap differential at or below 500 V since higher voltages can lead to source arcing 16 Switch to Chromatography mode and verify the performance with manual sample injections e g Reserpine m z 609 5 uL injections 0 5 uM 1 uM 2 uM 4 uM series 17 Lower the dry gas flow carefully to optimize and stabilize the signal but not lower than 5 units 6 24 Operator Manual Version 1 0 Volume II Operation 7 Methods 7 Methods In the BioT
16. Installation of the APCI source requires that you open the panels of the instrument Before installing APCI Exit the BioTOF acquisition software and then turn off all electronics modules with the power switch labeled Electronics Operator Manual Version 1 0 Volume II Operation 6 21 6 Introducing Samples 6 5 1 Installation To install the APCI chamber 1 2 OA CM Se 11 12 13 14 Remove the right front panel section Disconnect the ESI source high voltage connector large black round plug from the cable plug coming from the source controller This connection is found inside the frame by tracing the cable from the source body down to the connector Disconnect the Nebulizer gas from the ESI spray chamber Remove the ESI spray chamber from the ESI source body Place APCI spray chamber on the source hinges and close the latch Connect the Nebulizer gas to the APCI sprayer Connect the ESI source high voltage cable to the outlet on the left side of the APCI spray chamber Connect the high voltage cable from the APCI source to the above mentioned HV outlet cable of the Source controller inside the cabinet These plugs are keyed Make sure the connector are seated and locked properly Connect the 6 pin APCI heater cable to the mating cable plug in the source controller cable tree Install the APCI drain and connect it to the outside vent Close the panel Open the APCI spray chamber and pull
17. Neb Gas V DG Heat Figure 3 15 Instrument Control Group The instrument goes from Standby to Operation The Enable LED on the Instrument HV unit lights up Figure 3 16 The high voltages ramp up All ESI source voltages turn on Getting Started Version 1 0 3 13 3 Quick Start You may also be prompted to press the Voltage On button on the Source Control unit Figure 3 16 This switch lights up to indicate that the source voltages have been turned on Voltage On Button LED indicates when high voltages are enabled Figure 3 16 Location of the Voltage On Button Source Control Unit and Instrument HV Panel 2 Set the syringe pump flow rate to 120 uL h 2 uL min and start the pump 3 Press and hold the two right buttons on the syringe pump to fast forward the solution to the ESI source When sample is flowing through the tip of the spray needle assembly and nebulizer gas and spray chamber voltages are turned on the current meter Figure 3 17 of the ESI Source gauges indicates the spray current If there is no reading verify that the meter is set to read the endplate electrode as described in Setting the Source Lens Current Reading on page 3 4 4 Return the syringe pump to the 120 uL h 2 uL min rate when the meter indicates current EEE For EL 1 HGH VOLTAGE em VACUUM mua Power P Figure 3 17 Source Lens Current Reading 3 14 Getting Started Version 1 0 3 Quick St
18. Note Pull the capillary straight out until it is completely removed from the assembly 3 Once removed flush the capillary with an appropriate electrospray solvent Wiping with a lint free cloth Kim Wipe or similar moistened with solvent will sufficiently clean the metal coated ends of the glass capillary 4 Reinstall the Capillary reverse the procedure above Cleaning Skimmer 1 does not require removing it from the source Operator Manual Version 1 0 Volume II Operation 10 3 10 Maintenance To clean Skimmer 1 1 Moisten a lint free cloth with solvent and gently polish the exposed face of the Skimmer Depending on the amount of material deposited on the skimmer it may be necessary to perform this step a few times The round metal shroud in which the capillary exit resides should also be cleaned in a similar fashion Inspect the skimmer and remove any fibers left from the cleaning 3 Close the hinged door and tighten the two housing screws The source is now ready to be pumped down and turned on for operation 10 4 Operator Manual Version 1 0 Volume II Operation 10 Maintenance 10 2 3 Checking the Oil Supply At least monthly check the oil supply for the vacuum pump The pump is located in the right rear of the BioTOF system The gauge is on the front of the pump To check the oil 1 Open the door in the lower right corner of the right side of the cabinet to access the pump motor Figure 10 2
19. Operation along with the various options for sample introduction Receives ions from the ESI source redirects them into the flight tube using high voltage pulses and records the flight times The recorded data are delivered to the BioTOF computer workstation for display and analysis Operation of the TOF mass spectrometer is described in Chapter 3 TOF Mass Spectrometer Operation Controls the source and mass spectrometer operation stores and displays mass spectra and provides additional analytical software Mass Spectrometer Electrospray Flight Tube ge lon Source 5 BioTOF Figure 1 1 Basic BioTOF System Configuration 1 2 Operator Manual Version 1 0 Volume I Theory 1 System Components 1 1 Front Control Panels The cabinet door on the front of the BioTOF console provides access to four control panels Figure 1 2 The Main Control Panel on the top left includes the main power switch and switches for turning on the vacuum pumps system electronics and additional electrical outlets on the power supply labeled Computer These pushbutton switches light up when they are engaged The Vacuum Gauge Controller on the top right includes an LCD display of the vacuum pump gauges and lights indicating when pressures are below threshold Function buttons allow you to select gauges set units of measurement and specify threshold settings The Source Control Unit is used to set power for the ESI source and to con
20. Reflectron before turning around than those with lower kinetic energy Space Focusing 2 Ro J Figure 3 8 Space Focusing in Single Pass Mode Initially a packet of ions of identical m z in the accelerator is dispersed over a small but finite width in the direction of the flight tube with some ion being further back 2 and others 3 being further ahead in the accelerator When the high voltage pulse is applied the ions starting further back gain slightly higher kinetic energies than the ions which started further down As a result these ions will have a higher drift velocity in the field free region of the spectrometer and catch up with the slower ions at a point in the drift region For a single stage accelerator this point is at a distance 2S away from the accelerator A similar process is repeated in the Reflectron The ions with higher kinetic energy and velocity penetrate deeper into the Reflectron than the ions with lower kinetic energy and thus spend more time in the Reflectron Longitudinal space focusing is achieved if the total drift length D measured from the first point of passing at 2S is equal to four times the mean Reflectron penetration depth D 4R Operator Manual Version 1 0 Volume l Theory 3 11 3 TOF Mass Spectrometer Operation 3 7 Multipass Multipass is a BioTOF method that enables greater resolution for a specified mass range In multipass the Repeller pote
21. Select Gauge 3 When the inlet to the vacuum system is open the pressure should be between 1 5 and 1 9 mbar When the inlet to the vacuum system is closed the pressure should be less than 10 mbar The indicators labelled 1 through 6 in the upper right corner of the Vacuum Gauge Controller are lit when the vacuum levels are below the factory set thresholds When indicators 1 2 and 3 are on you can safely turn the BioTOF system on Indicators 4 5 and 6 are not used Refer to the separate Vacuum Gauge Controller documentation for information on the controller menu for changing units of measure and making other adjustments to the controller display Operator Manual Version 1 0 Volume II Operation 4 3 4 System Startup 8 Check the Power Switch in the lower right corner of the Source Control to make sure that the controller is turned on Figure 4 4 The Source Control unit LCD displays the default startup menu Source Lens Current Reading Source Control Power Switch lon Polarity Switch HV Enable Switch Figure 4 4 Source Control Unit top and Instrument HV Panel 9 Use the arrow button below the LCD display to position the display cursor on End Figure 4 5 so that the Source Lens Current gauge on the ESI Source reads the current on the Endplate Electrode CON edit active Remote Menu 1500 3500 4000 Cyl aila Cap Endplate Electrode Select End for the Source Lens Current Reading Figure 4 5 S
22. To i Prepare an ESI solvent of 1 1 methanol water mixture with 0 1 Acetic Acid Prepare a peptide stock solution in ESI solvent at a concentration of 100 uM which is equal to 100 pmole uL Dilute the stock solution in the ESI solvent to a ratio of 1 100 to 1 20 to obtain a sample solution at an analyte concentration of 1 to 5 uM ntroduce the sample to the system Fill a 100 to 250 uL syringe with the ESI solvent and position it in the syringe pump Figure 3 8 Set the syringe pump to a flow of 120 uL h 2 L min Connect the syringe with the sample inlet of the off axis sprayer using low volume clean tubing and fittings Figure 3 8 H To ESI N x Syringe Fill Port Low Volume Tubing Press and hold these two buttons for fast forward Cole Parmer Series 74900 Syringe Pump Figure 3 8 Connecting the Syringe Pump to the ESI Source 3 8 Getting Started Version 1 0 3 Quick Start 3 6 Check BioTOF Parameters When the BioTOF control software is started it automatically loads the Last Good Method used If this is the first use of the system since installation the parameters saved in Last Good Method are the factory defaults applied in the setup process You can acquire data using the parameters from the Last Good Method or you can modify the method by changing one or more parameters You can also load a previously saved method In this Quick Start the Last Good Method is used but to check the sett
23. a spectrum and check resolution with the data processing software 8 Start again with the voltage from the maximum peak again not clearing the spectrum between acquisitions 9 Tune in steps of 50 V up and down over a range of 150 V to 150 V from previous optimum 10 Follow the envelope of the tuning peaks and pick a voltage which correspond to the visual maximum of the envelope This may be in between the recorded steps 11 Clear and fine tune Multideflector High in 1 V steps as described above optimizing peak shapes and resolution without giving up intensity by more than 10 12 Fine tune Reflectron Lens in steps of 50 V optimizing peak shapes and resolution without giving up intensity by more than 10 then repeat from step 8 Operator Manual Version 1 0 Volume II Operation 10 9 10 Maintenance 13 Clear and fine tune Reflectron voltage in steps of 25 V from previous best value Figure 10 3 shows a Spectra Display with 1000 spectra each for seven different Reflectron voltages The maximum at 9350 V corresponds to the best tuning conditions Figure 10 3 Determining Reflectron Voltage Providing Maximum Resolution 14 Re calibrate the instrument as described in Chapter 9 Calibration 15 Save spectrum and save parameters and calibration in a new method file 10 10 Operator Manual Version 1 0 Volume II Operation 10 Maintenance 10 4 Basic Troubleshooting Figure 10 4 describes an algorithm f
24. accumulate in the RF ion guide by means of a trapping voltage applied to the guide s exit gate electrode Then the voltage on the gate electrode drops for a specified time and ions drift out of the RF ion guide Thus the ions form packets of a spatial length determined by the pulse width and ion velocity At the end of the gate pulse the exit electrode potential is raised starting another cycle of ion accumulation in the RF ion guide Determination of the pulse width and repetition rate is explained in the next section Figure 2 3 shows an ESI source configuration Hexapole lon Guide Offset Voltage Nebulizer Gas Skimmer Trap Gate Off Axis Sprayer 1 SES PUN Capillary 7 N i Spray Chamber N f Time of Flight m Analyzer Cylinder Endplate Figure 2 3 ESI Source Operation 2 4 Operator Manual Version 1 0 Volume l Theory 2 Source Operation The following table lists the system components and how they are used for ESI Table 2 1 ESI Source Operation by Component Component Function Off Axis Sprayer Receives the solution of sample and solvent from a syringe pump or liquid chromatograph passes the solution through a very fine needle and sprays the liquid into the spray chamber The needle consists of two concentric tubes the outer tube carrying the nebulizing gas and the inner tube carrying the sample liquid The liquid emerges from the needle into the
25. allowed under the Safety Requirements for the manufacturer listed have no part numbers copyright laws Electrical Equipment for and are not available from Measurement Control Noise Declaration Bruker Daltonics Inc Most Document History and Laboratory Use 70 dB operator position of these can be obtained First edition May 2003 normal operation per from laboratory supply Printed in U S A Class 1 LED Product ISO 7779 1988 companies Warranty WARNING Instrument Identification Manual part number A5001 The information contained in Connecting an instrument Each instrument is this document is subject to to a power source that is identified by a unique change without notice not equipped with a serial numbers These protective earth contact serial number is located on Bruker Daltonics Inc makes creates a shock hazard a label on the lower left side no warranty of any kind with for the operator and can near the rear of the regard to this material damage the instrument instrument including but not limited to Likewise interrupting the the implied warranties of protective conductor inside When corresponding with merchantability and fitness or outside the instrument Bruker Daltonics Inc about for a particular purpose or disconnecting the your instrument be sure to protective earth terminal include the model and the Bruker Daltonics Inc is not creates a shock hazard for full serial number liable for errors contained the operator and can
26. difference between any two of the spray chamber voltages to a maximum of 2000 V You may need to increase the voltages sequentially and in steps 4 Verify that the seven voltages in the Lens group are in line with the values shown in Figure 3 10 On the BioTOF offset must be 15 V 3 10 Getting Started Version 1 0 3 Quick Start 5 Click the Timing Sequence tab to display the timing parameters and verify that the values are those shown in Figure 3 11 Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Advanced Spectrometer Timing Cycle Period 25 H ms Repeller 10 us Gate 100 u us Transfer 100 us Deflector off Delay 25 EZ us Figure 3 11 Timing Sequence Tab 6 Click the Spectrometer tab to display the spectrometer voltages Figure 3 12 and verify that the voltage levels are those set during installation Note Changing the parameters in the Spectrometer tab alters the mass calibration The advanced parameters are set during installation and are specific to each instrument Modifying them can adversely affect system performance Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition High Voltage Volts Repeller ot Post Acceleration 2000 Detector 1600 ET Reflectron 3300 Multi Deflector Low 5 0 ET Multi Deflector High 71 0 Reflectron Lens 100 Figure 3 12 Spectrometer Tab Getting Started Versio
27. exist within this instrument or specific area Handle this biohazard with appropriate caution and protection viii Operator Manual Version 1 0 Volume I Theory 1 System Components 1 System Components The Bruker Daltonics atmospheric pressure ionization time of flight systems provide high resolving power accuracy and mass range for today s mass spectrometry needs They all have high performance ESI Electrospray APCI atmospheric pressure chemical ionization and Nano Electrospray sample introduction and ionization sources highly efficient hexapole ion guide transfer technology state of the art orthogonal acceleration time of flight spectrometers and analog to digital ADC detection systems for the highest dynamic range and true isotope pattern measurement Like all Bruker Daltonics mass spectrometers these time of flight systems share the common COMPASS data processing and analysis software tools for viewing printing calibration mass and chromatogram analysis library search quantitative analysis metabolite search peptide annotation and database search The series includes BioTOF The highest performance ESI TOF instrument with unprecedented mass resolution and accuracy The built in Multipass feature further enhances mass resolution Ideal instrument for accurate mass measurement of small molecules The BioTOF s versatility and mass range make it the best choice for the study of intact proteins polymers
28. flow and drying heat on at all times However if is necessary to conserve your source of drying gas turn the gas off and seal the spray chamber inlet when it has cooled You do not need to exit the BioTOF software simply minimize the application 3 20 Getting Started Version 1 0 Getting Started Index A Accelerator 1 3 Accept Button 3 19 Acquisition 3 12 Tab 3 12 Autoscaling 2 3 AutoStop 3 15 Basic BioTOF System Configuration 1 1 BioTOF Control Panels 2 4 Operation 3 13 Parameters 3 9 Software 3 6 System Configuration 1 1 Window 2 1 3 13 C Calibration 3 18 Tab 3 18 D Data Acquisition 1 4 E ELECTRONICS Button 3 2 Enable switch 3 4 ESI Operation 1 2 Sample 3 8 Source 1 2 Source tab 3 6 3 10 F File Save Options Dialog Box 3 17 Front Control Panels 2 4 Getting Started Version 1 0 Index 1 G Gases 3 6 Gauges 3 3 H High Voltage Electrodes 3 10 Instrument HV Panel 2 4 Instrumentation Mode 3 12 lonization 3 9 Polarity 3 9 L Last Good Method 3 9 Lens Settings 3 10 Linear Button 3 19 M Main Button 3 2 Control Panel 2 4 Manual Stop 3 15 Mode Tab 3 12 N Nebulizer and Drying Gas Controls 3 7 O Off axis Sprayer Assembly 3 5 On Off Button 3 3 P Polarity Switch 3 4 Positive Polarity 3 9 Power Switch 3 3 Q Quick Start 3 1 Index 2 Getting Started Version 1 0 S Sample Preparation 3 7 Source Control Unit 2 4 3 14 Control
29. from 100 uL min to 1 mL min The actual dilution is simply a ratio of the flow rates Dividing the initial sample concentration by the dilution factor gives the final concentration at the nebulizer Concentrations as low as 1 pg uL after dilution can give good results in infusion experiments The factors that limit this sensitivity are background contamination that may exist in the LC system solvents or sample When properly configured the sample solution is metered into the LC flow stream Because the flow rates are higher it is important that all connections are correct and tight Small leaks may dramatically effect the small infusion amounts coming from the syringe pump Operator Manual Version 1 0 Volume Il Operation 6 9 6 Introducing Samples 6 3 Nanospray For analyzing sample with low concentration and volumes the ESI source can be configured with the Nanospray assembly in place of the standard Electrospray spray chamber The Nanospray operates at a flow rate of less than 20 nL min by delivering the analyte to the source via a small inner diameter needle positioned at the center of the source For Nanospray nebulizing gas is not used and drying gas is applied at lower temperatures than in Electrospray applications High voltage is applied to the capillary only There are two assemblies in use The On Axis Nanospray assembly left in Figure 6 4 includes an XYZ stage immediately in front of the spray chamber that a
30. herein or for incidental or damage the instrument Write the serial number consequential damages of the instrument here for in connection with the WARNING reference furnishing performance The instrument must be or use of this material disconnected from its power Model Source before any cover is removed or the system is opened Bruker Daltonics Inc Serial Number assumes no responsibility for the use or reliability of its software on equipment that is not furnished by Bruker Daltonics Inc BioTOF Series Operator Manual Version 1 0 Volume I Theory Copyright Bruker Daltonics Inc Support 978 663 3660 Ext 1445 Manning Park FAX 978 667 5993 Billerica MA 01821 U S A Email support bdal com Internet www bruker daltonics com ii Operator Manual Version 1 0 Volume I Theory Contents GETTING STARTED GOPYRIGHT etes litio ll PHEFADE 5 deae os ae a deor VII SAFETY llblqu Vill 1 BIOTOP OPERATION 1 1 1 1 EEN ee 1 2 1 2 Eege E ee E E ES 1 2 1 3 Time of Flight Mass Gpechomeier EEN 1 3 1 4 Dat ACQUISITION EE 1 4 2 SYSTEM CONTROLS ERU ee 2 1 2 1 EIS TO aTe ON Soa cae cedida 2 1 2 2 Eront E eine Panels een 2 4 231 System Status Indica 2 5 3 X QUIGK START EE 3 1 3 1 Start the SySt EE 3 2 3 2 Check the Spray Chamber and Gpraver AAA 3 5 3 3 Start the BioTOF ein 3 6 3 4 Ji d INS Gases WEE 3 6 3 5 Prepare the Sample ege geed deer eebe deen St
31. in the lower right section of the Parameter Setting Spectrometer dialog box See Diagnostics on page 5 21 To manually trigger a signal to a connected device 1 Choose Tools gt Diagnostics gt Spectrometer to open the Parameter Setting Spectrometer dialog box 2 Click External Out 1 to send the ready signal or External Out 2 to send the start signal Operator Manual Version 1 0 Volume II Operation 7 5 7 Methods 7 3 Chromatogram Tab Use the Chromatogram tab Figure 7 5 to define mass ranges from the LC system These will appear in the Spectra Display window When the Instrument mode is Chromatography MS the Chromatography pane displays a trace for each mass range specified in the tab Those ranges always include TIC MS Total lon Chromatogram Mass Spectrum The control buttons on the right side of the table enable definition of Extracted lon Chromatograms so you can focus on specific ranges When you select a range in the tab a color bar at the base of the mass spectrum pane identifies the ions as determined by the mass spectrometer Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition B ricus a EIC 100 0 120 0 MS Add Ml EIC 100 0 500 0 M Change B EIC 100 0 235 1 M Delete Delete All Reset Type Extracted lon Chrom v Masses 100 0 120 0 Color GM Dark Magenta Filter INS Y Enable Figure 7 5 Chromatogram Tab To define an Ex
32. lonization Option Button 7 2 L Last Good Method Indicator 5 4 LC Flow Rates 6 9 With a Second Detector 6 8 LC MS 5 3 Lens 2 8 Lens Volts 7 9 Liquid Chromatograph 6 8 Chromatography 6 7 Load Method 5 18 Low Flow Direct Infusion Setup 6 2 M Main Button 4 2 Control Panel 1 3 Power Supply 4 2 Switch Panel 4 2 Index 4 Operator Manual Version 1 0 Volumes and Il Maintenance 10 1 Manual Stop 5 5 8 5 Masses Field 7 7 Menu Bar 5 12 Method ESI Source Mass Spectrometer Settings 7 1 Files 7 13 Menu 5 18 MicroTOF 1 1 Mode Tab 7 3 7 6 Monthly Maintenance 10 2 MS Only 5 3 7 3 Multideflector 3 4 3 5 High Low 3 5 Multipass 3 12 N Nanospray 2 9 6 10 Assembly 6 17 Assembly Installation 6 11 Needle 6 17 Nanospray Settings 6 20 Neb Gas Checkbox 4 6 Nebulizer 2 2 4 7 Gas 2 5 Needle For Online Nanospray 6 13 Position 6 4 nESI 7 2 Not Ready Indicator 5 4 O Off Axis Nanospray Assembly 6 10 Sprayer 2 5 Sprayer Assembly 4 5 Off Line Introduction 2 2 Oil Gauge 10 5 Oil Supply 10 5 On Axis Nanospray Assembly 6 10 Operator Manual Version 1 0 Volumes and Il Index 5 Open Dialog Box 5 18 7 15 Open HyStar NT 5 20 Operation Option Button 8 1 Parameter Settings 7 2 Orthogonal Interface 3 2 P Parallel path 6 9 Parameter Settings 1 6 5 22 7 2 Settings Spectrometer Dialog Box 5 22 Tabs 5 12 Polarity 1 4 4 4 Option Buttons 7 2 Power 1 4 PreSet Spectra Spectrum Count
33. max cursor snapped to the wrong mass To restart a calibration 1 Click Clear in the bottom left corner of the tab to erase values in the Current Mass column and clear the three constants in the Fit group Repeat steps 4 through 8 Click the Accept button in the Fit group The new values appear in the Constants group to the right and the mass scale in the Spectra Display changes accordingly To calibrate using a curved line Snap at least three peaks and click Quadratic To temporarily add a reference mass to a table 1 2 3 Enter a name in the field immediately to the right of the Add button Enter a mass in the next field Click Add The new reference mass is listed in the table However the mass is deleted when you Click Clear select a new table or exit the BioTOF software Note The Preset button is a service tool that allows a rough calibration based mainly on instrument voltages Use Preset if you do not have any calibration and need a starting point 9 4 Operator Manual Version 1 0 Volume Il Operation 9 Calibration 9 3 Creating a Custom Reference File You can set up a custom reference file by copying one of the reference files and adding reference masses for items of interest To create a new file 1 Use Notepad or a similar text editor to open one of the supplied reference files 2 Choose File gt Save As from the menu bar and save the file in the same directory with a differe
34. mouse button Mouse Shift Changes the cursor to a hand icon m in the Spectra display enabling you to scroll the display by dragging the hand vertically or horizontally Mouse Zoom Shift Mouse Max Cursor Changes the cursor to an arrow 4 and snaps to the next highest peak to the left with each left mouse click 5 8 Operator Manual Version 1 0 Volume Il Operation 5 BioTOF Window Table 5 3 Spectrum Display Window Pop Up Menu Items Function Description Mouse Data Cursor Changes the cursor to an arrow if The arrow rides along the trace as you move the mouse to the right or left Freeze Stops updates to the display while data is still collected in the background Choosing Freeze a second time refreshes the display with current data and resumes real time updates Background Color Opens a standard Windows dialog box for selecting the background for the Spectrum Display Figure 5 8 Color HHNNENHM NM Custom colors Figure 5 8 Spectrum Display Background Dialog Box Operator Manual Version 1 0 Volume II Operation 5 9 5 BioTOF Window Table 5 3 Spectrum Display Window Pop Up Menu Items Function Properties Description Opens the Properties Profile dialog box for setting characteristics for the Spectra Display The properties dialog box Figure 5 9 for the profile display sets various display characteristics in the Spectra Display window Properties Profile Display Cr
35. non covalent protein protein or protein ligand complexes or proteomics applications BioTOF Q New Quadrupole TOF Q q TOF mass spectrometer for sensitive and highly accurate MS MS analysis It combines state of the art quadrupole and TOF technologies and offers fully automated precursor detection and ms ms switching and scan functions The Bio TOF Q is the ideal instrument for peptide analysis and de novo sequencing Includes Multipass TOF for highest mass resolution microTOF An easy to use bench top package for powerful and exact mass measurement It concentrates the performance of a floor standing instrument in a format that fits on a bench The microTOF is specifically designed for extreme calibration stability which makes it the ideal instrument for accurate mass analysis in all LC MS applications This manual describes operation of BioTOF systems Operator Manual Version 1 0 Volume Theory 1 1 1 System Components The BioTOF system Figure 1 1 consists of Electrospray ion ESI source Time of Flight TOF mass spectrometer Computer workstation Generates molecular ions from liquid samples and facilitates ion transport from atmospheric pressure into the high vacuum of the mass analyzer The liquid sample is dispersed into a fine spray of charged droplets using a sprayer under the action of an electric field and pneumatic nebulization Operation of the source is described in Chapter 2 Source
36. shown in Table 8 1 Table 8 1 Spectral Range for Multiple Passes Passes Ratio of High m z to Low m z 2 4 4 3 2 3 4 1 9 5 1 6 Operator Manual Version 1 0 Volume II Operation 8 7 8 Acquiring Data 8 3 1 Using a Predefined Multipass Method Multipass methods are saved on the BioTOF workstation during final test in the factory and re confirmed during installation These methods are defined typically for a standard mass range To change the mass range in multipass mode the digitizer acquisition delay and the Repeller timing must be reconfigured as described below To set up multipass using a predefined method 1 Click Standby in the Instrument group load the predefined multipass method and then click Operation in the Instrument group 2 Select the Mode tab and enter the low end of the desired mass range in the Mass Range m z group 3 Calculate the high end of the new range using the ratios in Table 8 1 on page 8 7 and enter the value in the Mass Range m z group 4 Click the Acquisition tab and find the new Digitizer Delay in the Acquisition Window group 5 Click the Timing Sequence tab and set the set Repeller to 5 ps lower than the new Digitizer Delay value 6 Test the new settings with a standard sample 7 Fine tune Multideflector High Multideflector Low and Reflectron voltages as necessary 8 Recalibrate the instrument 9 Save the parameters as a new method 8 8 Op
37. the Instrument HV panel and the Source Control Unit 3 When both lights are off allow the source to cool down and seal the inlet of the capillary 4 Select gauge 1 on the gauge controller and press ON OFF Note It is recommended but not required that you turn off all electronics by pressing the ELECTRONICS switch on the main switch panel This may also require shutdown of the BioTOF software and a restart the next time the system is turned on 5 Release VACUUM PUMPS switch on the main switch panel As soon as the Turbo Pumps have slowed sufficiently the vacuum system automatically vents with air filtered through an air dryer cartridge 6 Allow approximately 10 to 15 minutes for the vacuum pump to spin down and for the system to vent to atmosphere completely 10 2 Operator Manual Version 1 0 Volume II Operation 10 Maintenance 10 2 2 Cleaning the Capillary and Skimmer 1 To clean the Capillary 1 Raise the hood on the ESI source and use a screwdriver to loosen the two holding screws on the left side of the source housing and swing the housing open See Figure 5 KIT Figure 10 1 Skimmer 1 and Capillary Exit in the ESI Source 2 Open the spray chamber door and remove the end cap that covers the face of the glass capillary The glass capillary is removed from the source by unscrewing the knurled nut that is behind the capillary exit and pulling the capillary out from the front spray chamber side
38. the spray shield forward approximately 1 mm from the backstop position If necessary lock the position with the screw 6 22 Operator Manual Version 1 0 Volume Il Operation 6 Introducing Samples 6 5 2 APCI Parameters 10 Turn on the power by pressing the Electronics switch on the Main Control panel Restart the BioTOF software Make sure the instrument is in Standby mode and the voltages are off The Voltage On button on the left side of the Source Control Unit is off The green Enable LED on the Instrument HV Panel is off Set to positive ion mode if not already done Set the source mode to APCI and verify that the source controller mode switches are set to APCI The Corona Needle voltage is automatically set to 500 V Set the Endplate and Capillary to 2500 and 3000 V respectively The polarity is not displayed in the ESI Source tab but can be seen on the source controller display In APCI mode the polarity of Capillary and Endplate are opposite of the polarity of Corona Needle Turn on the nebulizer and the drying gas and set the nebulizer pressure to 60 psi on the Source Control unit Set dry gas flow to 10 units and dry gas temperature to 250 C Connect solvent sample flow of MeOH H20 or CH3CN H20 from the LC system or syringe pump at 20 to 150 uL min With the liquid flowing set APCI Heater to 400 C and allow it to warm up for 10 minutes Operator Manual Version 1 0 Volume Il Operation
39. 1 GOntro BUTS ee EE 7 2 7 2 Mode Tab een 7 3 7 3 Chromatogram Lab es de ae xe mdr cei ad 7 6 7 4 Galibration Tabu ee 7 7 7 5 ES SOUufee Lab EE 7 8 7 6 Spectrometer Tab EE 7 10 7 7 T m nga Seguenee Tab EE 7 11 7 8 Acquisition Tabea ee eek euren 7 12 7 9 Method Files EE 7 13 8 ACQUIRING DATA 2 8 1 8 1 Starting System ODerallohi id 8 1 8 2 Acquiring and Saving Spectra 8 4 8 3 DUNT DSS Cm 8 7 9 GALIBRATION lt lt ainia 9 1 9 1 Reference Tables for Calibration AAA 9 1 9 2 Calibrating the System emer ee 9 3 9 3 Creating a Custom Reference File 9 5 10 MAINTENANGE un SEES BEE 10 1 10 1 Daily En Eu ne 10 1 10 2 Monthly Mairitenange eret RR ret ER da 10 2 10 3 BIOTOF Reflectron Tuning soe od Eas cena tee serre aea Ea ee 10 6 10 4 Basic Troubleshooting 2er 10 11 10 5 Advanced Troubleshooting x 10 12 OPERATOR MANUAL INDEX vi Getting Started Version 1 0 Preface This document is an introduction to the BioTOF system It includes An overview of BioTOF operation A brief description of the system indicators and controls Instructions on acquiring spectra from a known sample The purpose of this book is to introduce the system and familiarize you with key concepts More detailed information on system functions and test sequences is provided in BioTOF Operator Manual Volume 1 Theory BioTOF Operator Manual Volume 2 Operation Three additional manuals in this document set provide info
40. 3 Sample Name Sample_1 ExperimentNo f1 Experiment ID Comment Free format comments Comment Free format comments Comment3 Free format comments Apply and Save Cancel Figure 8 6 File Save Options Dialog Box The BioTOF application creates two files one formatted for the DataAnalysis application the other compatible with the XMass To set the application to use either or both formats Choosing Format gt DA format and or Format gt Xmass format from the menu bar 8 6 Operator Manual Version 1 0 Volume Il Operation 8 Acquiring Data 8 3 Multipass Multipass is a patented technology available in Bruker s BioTOF Series Time of Flight mass spectrometers Multipass effectively multiplies the length of the flight tube by sending the ions through the tube multiple times creating a much longer instrument with enhanced mass resolution Multipass is accomplished by keeping the Repeller voltage up when the ions return from the Reflectron thereby sending them back into the flight tube for another turn After the desired number of turns the Repeller voltage is dropped and ions pass through the Accelerator and on to the Detector As ions move at different velocities through the flight tube the timing of the Repeller voltage depends on the mass range of interest High and low m z ions may separate so far that they are no longer in the same cycle and the free spectral range of ions with the same number of turns is limited as
41. 3660 Ext 1445 Manning Park FAX 978 667 5993 Billerica MA 01821 U S A Email support bdal com Internet www bruker daltonics com ii Getting Started Version 1 0 Contents GETTING STARTED GOPYRIGHT etes litio ll PHEFADE 5 deae os ae a deor VII SAFETY llblqu Vill 1 BIOTOP OPERATION 1 1 1 1 EEN ee 1 2 1 2 Eege E ee E E ES 1 2 1 3 Time of Flight Mass Gpechomeier EEN 1 3 1 4 Dat ACQUISITION EE 1 4 2 SYSTEM CONTROLS ERU ee 2 1 2 1 EIS TO aTe ON Soa cae cedida 2 1 2 2 Eront E eine Panels een 2 4 231 System Status Indica 2 5 3 X QUIGK START EE 3 1 3 1 Start the SySt EE 3 2 3 2 Check the Spray Chamber and Gpraver AAA 3 5 3 3 Start the BioTOF ein 3 6 3 4 Ji d INS Gases WEE 3 6 3 5 Prepare the Sample ege geed deer eebe deen Stee 3 7 3 6 Check Bio TOF Parameters 2 dei la 3 9 3 7 Start BIO TOR Operation EE 3 13 3 8 Acquire elo 3 15 3 9 Check CG AID PAU ON EE 3 18 3 10 Place the System on Standby nee 3 19 GETTING STARTED INDEX Getting Started Version 1 0 iii OPERATOR MANUAL VOLUME I THEORY RL 1 EE ll PREFA CE E Vil SAFETY LABELS a ses een VIII 1 SYSTEM COMPONENTS 2 22 22 en 1 1 1 1 Front Control Panels uio eon ee 1 3 1 2 Computer ee 1 5 1 3 BIO TOE SOIDWAl6 cys cect eS eaten sad 1 5 2 SOURCE OPERATION ee taa 2 1 2 1 Sample Introduct n screen iaa 2 1 2 2 ESI SOURGE
42. A biohazard may exist within this instrument or specific area Handle this biohazard with appropriate caution and protection viii Operator Manual Version 1 0 Volume Il Operation 4 System Startup 4 System Startup This chapter describes how to start the BioTOF system After installation and calibration the system is normally placed in standby mode with the pumps running rather than being completely powered down as it can take several hours to pump down the TOF chamber below the pressure threshold If this system is already in standby mode use the instructions in this chapter section to verify that the system is ready for use 4 1 Start the System To start the BioTOF system 1 Open the front cabinet door to access the BioTOF control panels Figure 4 1 Main Control Vacuum Gauge Panel Controller Source Control Unit ESI Power Switch Source HV Switch Instrument HV Panel Figure 4 1 BioTOF Control Panels Operator Manual Version 1 0 Volume Il Operation 4 1 4 System Startup The Main Control panel Figure 4 2 on the upper left contains four pushbutton switches which are illuminated when they are engaged MAIN The main power supply for the ESI Source and TOF Spectrometer VACUUM PUMPS Pump controller ELECTRONICS All system electronics COMPUTER Three optional power connections on the bottom left side of the console one of which can be used to supply power to the computer workstatio
43. BioTOF Sollware nennen 4 6 QA T rmthe Gases Onnan e nee AEA Ea AG 4 6 S BIOTOF MIN D O E 5 1 5 1 Window E LEE 5 1 5 2 Spectrum Display Pop up Menta a 5 8 5 3 Menus and Tool Ba ran Eege EE EE 5 12 5 4 File a d Edit Menus lt td m 5 13 5 5 View MENU A o O ee 5 13 5 6 Method Menu 5 18 5 7 o kenn 5 20 5 8 Forniar Men tec 5 26 5 9 Help Mena 5 26 6 INTRODUCING SAMPLES iret reete hen rettet 6 1 6 1 Sample Delivery to Electrospray AE 6 1 6 2 Liquid Chromatography EG ae ee a 6 7 6 3 Nanospray star REAT Enana 6 10 6 4 A ne EE 6 16 6 5 Configuring the APCI Spray Chamber AANEREN 6 21 Operator Manual Version 1 0 Volume I Theory V T METHODS ee 7 1 7 1 GOntro BUTS ee EE 7 2 7 2 Mode Tab een 7 3 7 3 Chromatogram Lab es de ae xe mdr cei ad 7 6 7 4 Galibration Tabu ee 7 7 7 5 ES SOUufee Lab EE 7 8 7 6 Spectrometer Tab EE 7 10 7 7 T m nga Seguenee Tab EE 7 11 7 8 Acquisition Tabea ee eek euren 7 12 7 9 Method Files EE 7 13 8 ACQUIRING DATA 2 8 1 8 1 Starting System ODerallohi id 8 1 8 2 Acquiring and Saving Spectra 8 4 8 3 DUNT DSS Cm 8 7 9 GALIBRATION lt lt ainia 9 1 9 1 Reference Tables for Calibration AAA 9 1 9 2 Calibrating the System emer ee 9 3 9 3 Creating a Custom Reference File 9 5 10 MAINTENANGE un SEES BEE 10 1 10 1 Daily En Eu ne 10 1 10 2 Monthly Mairitenange eret RR ret ER da 10 2 10 3 BIOTOF Reflectron Tuning soe od Eas cena tee serre aea E
44. Ca 0 0000 Y 31955 0000 3 18 Getting Started Version 1 0 3 Quick Start Drag the cursor immediately to the right of the most intense peak of the reference compound and click the left mouse button The cursor snaps to the peak and the current mass appears in the reference table in the Calibration tab Use the same technique to snap the line to each calibration peak A minimum of two peaks is required for linear calibration A minimum of three peaks is required for quadratic calibration Click the Linear button in the Fit group Figure 3 21 to display the new calibration constants Check for obvious errors such as negative constants Errors can occur if the peaks selected are not the proper compounds or the max cursor was snapped to the wrong mass Click the Accept button in the Fit group The new values appear in the Constants group to the right and the Spectrum Display calibration changes accordingly With the system calibration verified using the known sample you can now proceed to acquiring spectra from the material of interest Refer to Chapter 6 in the BioTOF Operator Manual Volume Il Operation for instructions on flushing the syringe and ESI unit and introducing the new sample 3 10 Place the System on Standby Take the following steps when you have finished with spectra acquisition 1 2 Click Standby in the Instrument group to bring the instrument from operation mode to standby mod
45. F Getting Started Version 1 0 2 1 2 System Controls The BioTOF window appears Figure 2 1 Once you have started the application you can leave it running minimizing the window when you are not using the BioTOF system System Status st Good Method EIS Method Tools Format Help oli s le TE gt tt RO ke EA In BIGTOF Spectrum 50000 Points soo 500 Pane Spectra Display a 5h Em Parameter Tabs tese su ee qnd Command TET SE ES Ea eee Buttons Pane E E Ecc HE Longen Tabs B 6 eege eet System Option Buttons Figure 2 1 Layout of the BioTOF Window The BioTOF window displays information and controls in five different panes The Spectra Display plots the spectra captured by the system There are a variety of user selected formats For example the graph in Figure 2 2 shows the intensity of ion signals from the detector for each m z value The two checkboxes in the bottom left corner of the display control autoscaling for the vertical axis left and horizontal axis right The pane on the upper left side of the window has system status indicators The pane on the lower left side has command buttons for starting and stopping spectra acquisitions The four groups immediately to the right of the command buttons include option button and checkboxes for controlling system operation To the right of these controls are seven tabs for setting system parameters for a
46. L syringe suitable for most applications Syringe Clamp Advance Plate Release Button Figure 6 1 Syringe Pump 6 1 2 Setup for Low Flow Direct Infusion The simplest configuration is a direct connection of the syringe pump to the Electrospray The flow is started by the keypad on the front of the syringe pump To set up the direct infusion to the Electrospray 1 Fill the syringe with the ESI solvent and sample liquid avoiding any bubbles 2 Connect the syringe with the sample inlet of the off axis sprayer using low volume clean tubing and fittings Figure 6 2 3 Position the syringe in the pump Lift the syringe clamp on the pump place the syringe barrel in the holder as shown in Figure 6 1 and place the clamp on the syringe 6 2 Operator Manual Version 1 0 Volume II Operation 6 Introducing Samples 4 Release Button Press the release button on the front of the drive plate and advance the plate so that it rests against the syringe plunger Figure 6 2 Advance Plate Syringe Clamp I ww To ESI ES Syringe Fill Port Low Volume Tubing Press and hold these two buttons for fast forward Cole Parmer Series 74900 Syringe Pump Figure 6 2 Syringe Pump Controls 5 Turn the syringe pump on using the rocker switch on back of the pump just above the power cord connection Press Select on the pump keypad to access a menu on the small display to the left of the select button The
47. OF system the method consists of the ESI Source and mass spectrometer operational settings When the system is started it uses the Last Good Method that is the setting in use when the system was shutdown You can adjust these settings using the option buttons and the parameter tabs below the Spectra Display window as described in this chapter Additionally you can save the current settings to a file for use later on similar projects or restore settings after using variations on a setup The Current method is identified in the status group in the upper left of the BioTOF window Figure 7 1 Instrumentation MS ONLY System Automation Instrumentation MS ONLY System READY Automation MANUAL Current method Current method after loading method file Current method Current method IntactProtein_0305 Last Good Method at startup 03 tofpar Figure 7 1 Current Method Identified in the System Status Group This chapter describes how to adjust a method using the option buttons and parameter tabs and how to save and load methods Operator Manual Version 1 0 Volume Il Operation 7 1 7 Methods 7 1 Control Buttons The lonization and ion Polarity option buttons Figure 7 2 to the left of the parameter tabs set the basic parameters for method Instrument C Standby Operation lonization ESI nESI APCI Polarity Positive C Negative Gas Heater Control Iw Dry G
48. Operator Manual Version 1 0 Volume Theory 2 Source Operation 2 3 Nanospray For analyzing small amounts of sample with low concentration the ESI source can be configured with the Nanospray assembly in place of the standard BioTOF Electrospray For on line Nanospray The flow rate during operation is less than 200 nL min For off line Nanospray The flow rate during operation is lower than 20 nL min The analyte is delivered to the source via a small inner diameter needle positioned at the center of the source Typically nebulizing gas is not used Small amount of drying gas may be used at lower temperatures The high voltage is applied to the capillary only For Nanospray the spray chamber with the off axis Electrospray is removed from the ESI Source assembly and is replaced by a Nanospray spray chamber Figure 2 4 Figure 2 4 Nanospray Spray Chamber Assembly The assembly includes an XYZ stage mounted on the front of the spray chamber For on line Nanospray The XYZ stage is connected to a NanoLC or the syringe pump For off line Nanospray The XYZ stage accepts disposable Nanospray tips See Nanospray on page 6 10 for information on setup and operation Operator Manual Version 1 0 Volume Theory 2 9 2 Source Operation 2 4 E Z Nanospray The E Z Nanospray enables you to set up off line nanospray without removing the standard spray chamber assembly A nanospray needle is filled with 1 to 3 uL of the a
49. TOF Window Table 5 1 Command Button Descriptions Function Description Start Stop The Start button is enabled when the BioTOF system is in Save Auto Stop Manual Stop PreSet Spectra Spectrum Counter Clear Operation mode Click Start to begin spectra acquisition The button changes to Stop when the system is acquiring data and changes back to Start when acquisition has been stopped either manually or automatically In MS Only mode you can stop acquisition and the click Start again to continue acquisition In Chromatography MS mode and FIA MS mode stopping and restarting acquisition clears the buffer and Spectra display Save is active when data acquisition has been stopped Click Save to save spectra from the current session The spectra are saved in the file formats selected from Format menu as described in Format Menu on page 5 26 When AutoStop is selected the spectra acquisition stops automatically once the system has acquired the number of spectra specified in PreSet Spectra When Manual Stop is selected data acquisition continues until you click the Stop button When the system has collected the number of PreSet Spectra incoming records replace the oldest records in the buffer Auto Stop and Manual Stop buttons are only active when the Instrumentation mode is MS Only Use PreSet Spectra to specify the number of records to be accumulated The Spectrum Counter shows the actual number of records acqui
50. TOF is measured in m the experiment Tess Cp de where Cg represents the fixed internal electronic propagation delay ofthe transistor gates switches and cables and Lest Cy is the first order coefficient 28U This equation establishes the basic relationship between m z ratio and the ion s time of flight and is used for calibration of the spectrometer The coefficients cy and c4 can be determined experimentally by measuring the time of flight of two ions with different m z ratios See Calibration on page 9 1 3 10 Operator Manual Version 1 0 Volume I Theory 3 TOF Mass Spectrometer Operation 3 6 Space Focusing The accelerator and the Reflectron are tuned to compensate for the fact that ions in a given packet do not travel the same distance in the mass spectrometer When the ions arrive in the orthogonal interface from the ESI source they are at different distances from the Repeller lons closer to the Repeller have a longer round trip in the mass spectrometer than those further away from the Repeller To ensure that all ions of a given m z arrive at the detector at the same time the field in the accelerator is shaped so that ions closer to the Repeller exit the accelerator with higher kinetic energy than those further away Figure 3 8 The field in the Reflectron mirrors the accelerator field When the ions enter the Reflectron assembly those with the greater kinetic energy travel further into the
51. Temp and APCI Neb Temp are active Typical settings are 250 C and 400 C respectively Dry Gas Flow and Neb Gas Flow are not active in the BioTOF software as these flow rates are set with control on the Source Control Unit panel 7 8 Operator Manual Version 1 0 Volume Il Operation 7 Methods High Voltage Electrodes Volts When Instrumentation is ESI or nESI high voltage is applied to three electrodes Cylinder End Plate Capillary When Instrumentation is APCI high voltage is applied to Corona Needle End Plate Capillary When lon Polarity is Positive the actual ESI spray chamber voltages in the High Voltage Electrodes group are negative Note The software limits the difference between any two of the spray chamber voltages to a maximum of 2000 V You may need to increase the voltages sequentially and in steps Lens Volts The voltages in the Lens group in Figure 7 7 are typical settings The Offset must be 15 V Refresh button Click the Refresh button to send the displayed gas and voltage values to the ESI controller Operator Manual Version 1 0 Volume Il Operation 7 9 7 Methods 7 6 Spectrometer Tab The High Voltage settings on the Spectrometer tab Figure 7 8 are set during installation and configuration of the system and should not be modified Note Changing the parameters in the Spectrometer tab changes the mass calibration These advanced parameters were set during installa
52. Unit and Instrument HV Panel 3 3 3 14 Lens 3 4 3 14 Spectra Display 2 2 3 16 Spectrometer Tab 3 11 Voltages 3 11 Voltages Tab 3 11 Spectrometer tab 3 11 Spectrum 1 4 Spray Chamber 3 5 Sprayer 3 5 Standby 3 19 Control Settings 3 20 Start button 3 15 Syringe Pump ESI Source Connection 3 8 System Configuration 1 1 Control Settings for Standby 3 20 Controls 2 1 Startup 3 2 Status Indicators 2 5 T Time of Flight Mass Spectrometer 1 3 Timing Sequence Tab 3 11 TOF Operation 1 2 V Vacuum Gauge Controller 2 4 3 2 Pumps Button 3 2 Voltage On button Button Voltage On 3 14 Getting Started Version 1 0 Index 3 Index 4 Getting Started Version 1 0 C BR K R CX BioTOF Series Operator Manual Volume I Theory BioTOF BRUKER DALT ONICO Copyright Copyright 2003 Safety Information WARNING Part Numbers Bruker Daltonics Inc Safety Class All connections of the In this manual All Rights Reserved instrument must be used in Bruker Daltonics Inc The instrument is a Safety correct way The instrument part numbers are generally Reproduction adaptation Class I instrument and has should only be used with listed in parentheses after or translation without prior been designed and tested the wires and cables the name of the part written permission is in accordance with IEC delivered with the system prohibited except as Publication 1010 or otherwise provided by A few tools and supplies
53. Voltage DC3kV DC 3 kV DC 50V DC 10V DC5V Pulsed O 10V 2 2 2 2 2 2 m 5 kV 5 kV 200 V 20 V 10 V 50V 40 V Extreme caution should be used when measuring these voltages Interlock must be defeated for measuring high voltages Set all high voltages to 700 V before measuring 10 14 Operator Manual Version 1 0 Volume II Operation Operator Manual Index A Accelerator 3 3 Acquire Data 8 1 Spectra and Saving 8 4 Acquisition Tab 7 12 Window 7 12 Adding Reference Mass to a Table 9 4 Advanced Data Handling 7 13 Digitizer Setting 7 13 Advanced Troubleshooting 10 12 APCI 7 2 Chamber Installation 6 22 Parameters 6 23 Source Operation 2 10 Spray Chamber Configuration 6 21 Auto Stop 8 5 Stop Manual Stop 5 5 Autoscaling 5 2 AutoStop 5 5 Basic Troubleshooting 10 11 BioTOF 1 1 Components 3 1 Control Panels 1 3 4 1 Edit Menu 5 13 File Menu 5 13 Menu Bar 5 12 Software 4 6 System Configuration 1 2 Tool Bar 5 12 Window 5 1 Window Indicators and Controls 8 4 Operator Manual Version 1 0 Volumes and Il Index 1 BioTOF Reflectron Tuning 10 6 BioTOF Q 1 1 C Calibration 9 1 9 3 Tab 7 7 9 3 Using a Curved Line 9 4 Capillary 2 7 Cap 6 12 Cleaning 10 3 Entrance 2 6 Exit 2 7 Checking the Oil Supply 10 5 Chromatogram Tab 7 6 Chromatography MS 7 3 Cleaning Capillary and Skimmer 1 10 3 ESI Source 10 1 Clear 5 5 Color Field 7 7 Command But
54. a ee 10 6 10 4 Basic Troubleshooting 2er 10 11 10 5 Advanced Troubleshooting x 10 12 OPERATOR MANUAL INDEX vi Operator Manual Version 1 0 Volume I Theory Preface This document provides detailed information on using the BioTOF system It is consists of two volumes BioTOF Operator Manual Volume 1 Theory BioTOF Operator Manual Volume 2 Operation Three additional manuals in this document set provide information on using the analytical software included with the BioTOF system DataAnalysis User Manual QuantAnalysis User Manual Library Editor User Manual For technical assistance contact Bruker Daltonics customer service In the U S 1 978 663 3660 Ext 1445 In Germany 49 0 421 2205 430 In Japan 81 0 45 440 0471 Local Service Operator Manual Version 1 0 Volume Theory vii Safety Labels These labels are placed on the instrument to indicate the respective hazard possibility SEPP Hazard Note Refer to the Operator Manual for details on this hazard Warning High Voltage A dangerous high voltage or shock hazard exists within this area Only trained service persons should perform work in this area Warning High Temperature Hot parts and surfaces are present in this area Allow the part or surface to cool before performing work in this area This may require turning off power to selected components or to the entire instrument Warning Biohazard A biohazard may
55. ally in steps of 2 V note the peak intensity and then click the Clear button For the BioTOF Il the Maximum intensity should be achieved somewhere between 63 and 72 V 3 Change the Multideflector High to optimal voltage and acquire a new spectrum 10 8 Operator Manual Version 1 0 Volume Il Operation 10 Maintenance 4 Optimize peak intensity by tuning Reflectron Lens voltage in steps of 100V noting the peak intensity then clear change the voltage and restart acquisition Check peak shapes for splits bumps or other irregularities For good signal to noise ratio always check intensities resolution and peak shapes on the most intense peak of the isotope distribution 5 Go back to step 1 and repeat procedure starting from the Reflectron voltage determined in the first round 6 Zoom in on the most intense peak turn on the data point markers and check resolution by estimating the fractional number of data points across the width of the peak at half height At the 1 Gsa sampling rate the time between data points is 1ns Mass resolution is equal to the flight time divided by 2 times the peak width in the same time units For example at a flight time of 70 us or 70 000 ns a peak width of 3 5 ns or 3 5 data points corresponds to a mass resolution of 10 000 At this point in the tuning resolution should be at least 8 500 to 9 000 or approximately 4 data points for the Reserpine or Leucine Enkephalin peaks Save
56. ameters and calibration to a method file as described in Method Files on page 7 13 2 Click Operation in Instrument Group and Positive in the ion Polarity group and infuse a standard sample from the syringe pump 3 Select the ESI Source tab to verify that the Offset in the Lens group is set to 15 V and that standard values are set for all other source parameters 4 Select the Spectrometer tab and set the Detector to 1600 V Post Acceleration may be set to 2000 V but is not required at this point 5 Use the Acquisition tab to verify that the Digitizer offset is set properly to give only a 1 2 count electronic noise band per scan and set the Sample Rate to 1 Gsa 10 6 Operator Manual Version 1 0 Volume Il Operation 10 Maintenance 6 Set the voltages in the Spectrometer tab as follows Repeller 8500 V Multideflector High 67V BioTOF II or the default value Multideflector Low 5V that is no secondary deflection Reflectron Lens 300 V Reflectron 9100 V 7 Start data acquisition The instrument should show good ion signal of at least 1000 counts for 200 spectra at the expected flight times Leucine Enkephaline or Reserpine around 70 us With good signal proceed as described below otherwise enter the troubleshooting procedure If the ion signal is not strong enough set Reflectron Lens to 500 V and then to 700 V f signal is better with the higher Reflectron Lens voltage proceed Note Make sure t
57. an electrical pulse in the electronic detection system MICROCHANNEL MICROCHANNEL PLATE PLATE Anode ef 1 lon collides with channel wall emitting an electron 2 Electron collides with wall emitting multiple electrons 3 Reactions continue causing an avalanche of electrons 4 Electrons bombard anode Note lons can be generating an electrical pulse positive or negative Figure 3 6 Microchannel Amplification Operator Manual Version 1 0 Volume Theory 3 7 3 TOF Mass Spectrometer Operation 3 4 Digitizer Recording of the detector signal starts after a preset digitizer delay The digitizer delay determines the lowest m z value in the recorded spectrum and shifts the recording window along the mass scale The electrical signal from the detector is converted from analog to digital for each scan Multiple scans are aggregated to form a single higher density record The records are summed in the digitizer to form a spectrum that is transferred to the workstation In MS Only mode the spectra are further accumulated in a single buffer and the buffer contents are displayed in the BioTOF window At the end of the data acquisition the contents of the buffer can be saved as a single mass spectrum In Chromatography MS mode the transferred spectra are stored and displayed consecutively and can be saved as time stamped mass spectra The width of the recording time window is specified by either Selecting a mass
58. are possible Serial path The sample flows first through a flow cell in a non destructive detector and then to the Electrospray or APCl In a parallel path configuration the sample is split usually with a standard tee with part of the flow going to each detector A serial configuration provides maximum sensitivity to the alternate detector because it receives the full sample flow It is also easier to do post column addition in this configuration The disadvantages are that the detector must be non destructive and its flow cell must tolerate high pressures Also the flow cell can cause peak broadening that will affect soectrometer data Parallel path A parallel configuration eliminates peak broadening problems and makes sample fraction collection easy It also allows destructive detectors Sensitivity is reduced slightly for the alternate detector because it does not receive all of the sample A parallel configuration also complicates post column additions and is prone to split ratio variations 6 2 2 Using the Syringe Pump at LC Flow Rates The higher flow LC compatible configuration is very similar to the low flow configuration as viewed from the syringe pump The inclusion of the LC pump is easily accomplished by simply adding a tee directly above the connection made to the Electrospray nebulizer Typically the syringe pump is operated at flow rates from 1 uL min 60 uL h to 10 uL min 600 uL h The LC pump is typically operated
59. art 3 8 Acquire Spectra Controls and indicators for starting and stopping spectrum acquisition are located in the two panes on the left side of the BioTOF window Figure 2 1 on page 2 2 You can start acquisition when the status bar is green and displays Ready Figure 3 18 Instrumentation MS ONLY uit Stat System You can start READY Auto Stop acquisition when TENE the status bar acea le Manual Stop is green MANUAL Current method PreSet Spectra Last Good Method 500 Spectrum Counter Shows the number of records stored X Clear in the buffer Figure 3 18 Indicators and Controls on the Left Side of the BioTOF Window To run spectra acquisition 1 Click the Start button Data acquisition and transfer begin and the Spectra Display plots the results The Spectrum Counter indicates the total number of records accumulated in the buffer The Start button changes to a Stop button enabling you to manually stop acquisition when a sufficient number of records have been stored in the buffer 2 Click the two checkboxes in the lower left corner of the Spectra Display Figure 2 2 on page 2 3 to enable autoscaling for both axes of the display Figure 3 19 The BioTOF continues to accumulate records until the buffer contains the number of records specified in PreSet Spectra f Manual Stop is selected the buffer is cleared and a new acquisition is started SH AutoStop is selected acquisition stops and th
60. art the System After installation and calibration of the BioTOF system the system is normally placed in standby mode rather than being completely powered down as it can take several hours to pump down the TOF chamber below the pressure threshold The instructions in this section involve the use of the control panels on the front of the BioTOF cabinet to verify that the system is ready for use To start the BioTOF system 1 Open the front cabinet door to access the BioTOF control panels Figure 2 3 on page 2 4 2 On The Main Switch Panel Figure 3 1 verify The MAIN pushbutton is illuminated red The VACUUM PUMPS pushbutton is illuminated green VACUUM PUMPS ELECTRONICS COMPUTER Figure 3 1 Main Switch Panel 3 Press the VACUUM PUMPS button if it is not already lit When this button is pressed it lights up and the pumps start sequentially However the system will not be ready until the pressure in the vacuum system has dropped below threshold This may take several hours 4 Press the ELECTRONICS button to initialize the system electronics including the vacuum gauge controller Figure 3 2 The buttons illuminate when pressed to engage the switches gg Lights indicate when the vacuum the selected the factory set gauge m thresholds mm Press this button to TA enable the selected CEN gauge EJ Press these buttons MI EDWARDS Active Gauge Controller to select a gauge Figure 3 2 Vacuum Gauge Con
61. as Y Neb Gas Y DG Heat Figure 7 2 lonization and Polarity Groups To set basic operating parameters 1 Click the lonization button that is appropriate from the ESI source to be used ESI for the standard Electrospray lon source nESI if you are using the low flow rate Nano Electrospray source APCI if you are using atmospheric pressure chemical ionization 2 Select either Positive or Negative for lon Polarity The appropriate voltages are applied to the ESI Source and Spectrometer The ion polarity can be set remotely if the three position Polarity switch on the Instrument HV Voltage Panel is in the middle position 7 2 Operator Manual Version 1 0 Volume II Operation 7 Methods 7 2 Mode Tab Use the Mode tab Figure 7 3 to set the instrument mode display units and mass range Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Instrumentation Mode Chromatographic Setting C Chromatography MS C FIA MS Total Run Time mins r Solvent Delay mins Data Display mz C Flight Time C Points MS Acq Rate 1 lt 70 Spectra min Mass Range m z External Start m z from 147 to 874 Enable Configure Figure 7 3 Mode Tab Instrument Modes There are three instrumentation modes MS Only Mass spectrometry only Chromatography MS for when the sample is supplied from an LC system and you want to display a chromatogram and the mass spectra The Spectra displ
62. ats for use with the data processing applications DataAnaylsis XMass To specify formats for saving data 1 Choose Format from the menu bar 2 Click either or both of the formats listed A format is selected when a checkmark appears to the left of the menu option To deselect an option click a checkmarked menu option and the checkmark is removed 5 9 Help Menu The Help menu has two selections Help Topics provides information on the BioTOF application using a the Windows Help system About TOF Control displays the version and copyright information 5 26 Operator Manual Version 1 0 Volume Il Operation 6 Introducing Samples 6 Introducing Samples This chapter describes how to set up sample inlets for Electrospray ionization using the syringe pump and or a liquid chromatography LC system The chapter then describes the use of the Nanospray and Atmospheric Pressure Chemical lonization APCI options 6 1 Sample Delivery to Electrospray Many analytical methods can be accomplished by directly pumping the analyte in solution to the Electrospray using the supplied syringe pump Often the sample has already undergone some separation or purification process Direct infusion can be accomplished at a low flow rate using the syringe pump only and at higher flow rates using the LC system In low flow mode the outlet of the syringe needle is coupled through the necessary fittings to the union on the inlet of
63. ay window is divided into two panes the top one showing the spectra and the lower one showing ion counts from the connected LC system FIA MS Flow injection analysis and mass spectrometry to specify a run time With this mode you set up the run in the Chromatographic Setting group by specifying the total run time in minutes and a wait time for the solvent to clear Data Display The default data display horizontal scale is m z When Instrumentation Mode is set to MS Only or FIA MS you can also specify Flight Time or Points When Instrumentation Mode is Chromatography MS the scale must be m z Mass Range The Mass Range group specifies in m z in Dalton to be recorded displayed and saved Operator Manual Version 1 0 Volume II Operation 7 3 7 Methods Chromatographic Setting When the Instrument mode is either Chromatography MS or FIA MS you specify the duration of the data acquisition by entering Total Run Time and Solvent Delay in minutes The Solvent Delay allows you to flush previous samples from the system before the start of the run Solvent Delay is not included in Total Run Time When the Instrument mode is Chromatography MS you set a mass spectra acquisition rate in the Chromatographic Setting group The rate is between 1 and 70 spectra per minute 7 2 1 External Start Use the External Start group in the lower right corner to enable and configure the start of spectra acquisition on a signal from an e
64. button for each server 5 24 Operator Manual Version 1 0 Volume Il Operation 5 BioTOF Window 2 Click a server button to open a separate window to display the message traffic Figure 5 17 is an example of message traffic for the ESI Controller ESIcon1 3 Click in the upper right corner of the window to minimize the display to the task bar Note Do not use to close the window as this action stops the server program 4 Choose Tools gt Diagnostics gt Show Servers in the menu bar a second time to uncheck the option and clear the server buttons from the task bar BRUKER ESI Controller ESIcon1 File Edit View Window Standalone Test Debug Help D c WP ESIcon1 set ESIController EsiApci value set set E set E set set fl set set f set set set ESIController ESIController ESIController ESIController ESIController ESIController ESIController ESIController ESIController ESIController ESIController ESIController 8 EsilonMode value 1 EsiDgOnoff value 1 EsiNgOnoff value 1 EsiRFAmplitude value 600 EsilonMode value 1 EsiCapillary value 4000 EsiCapillaryExit value 120 EsiCoronaNeedle value 500 EsiCylinder value 1500 EsiOffset value 15 EsiEndplate value 3500 EsiSkimmer value 40 Figure 5 17 ESI Server Window Operator Manual Version 1 0 Volume Il Operation 5 25 5 BioTOF Window 5 8 Format Menu By default the BioTOF application saves spectra to two file form
65. by Component Continued Component Function Skimmers Transport and focus the ions into a beam while neutral gas is pumped away in separate sequential pumping stages The voltages established for these ion optical components determine the ion transport efficiency The two skimmers have separate voltage settings which along with the Lens voltage setting are found in the Lens group of the ESI Source tab RF lon Guide Six parallel rods that serve as an ion guide and a trap for the accumulation of ions As the hexapole RF ion guide transports ions the neutral gas is pumped away see pumping stage II and Ill in Figure 2 3 on page 2 4 Two voltages are applied to the hexapole RF Amplitude typically set at 600 V peak to peak with a frequency of 5 MHz Offset a constant voltage applied for bias The RF Amplitude and Offset are set in the Lens group in the ESI Source tab Trap Held at a higher voltage than the Offset voltage to prevent ions from emerging from the RF ion guide The accumulation of ions in the RF ion guide increases the efficiency of the instrument After a pre set interval the Trap voltage drops automatically to 0 and the accumulated ions pass through the Lens and Exit plates and into the TOF spectrometer Lens Concentrates and focuses the analyte ions in the final ESI source pumping stage Exit The final aperture in the ESI Source The exit is held at ground potential 2 8
66. ccepts a needle assembly for on line or off line Nanospray applications The unit features a camera and LCD screen for monitoring spray operation The Off Axis Nanospray assembly right in Figure 6 4 delivers the analyte via an off axis needle assembly The Off axis Nanospray assembly includes a microscope for monitoring J f WM gg Z MOO Figure 6 4 On Axis left and Off Axis right Nanospray Assemblies 6 10 Operator Manual Version 1 0 Volume II Operation 6 Introducing Samples 6 3 1 Installing the Nanospray Assembly This section describes how to install the On axis Nanospray Assembly For detailed instructions refer to Apollo On Line NanoElectrospray lon Source User Manual To replace the Electrospray with the On axis Nanospray 1 At the computer workstation click Standby in the Instrument group to bring the instrument from operation mode to standby mode The ESI voltages are turned off 2 Double check to ensure that the ESI voltages and the TOF voltages are off The light on the high voltage indicator on the ESI front panel must be off and the Instrument HV unit is disabled Enable LED is off 3 Click the ESI Source tab and set the Dry Gas Temp to 30 C or below 4 Uncheck Neb Gas and DG Heat in the Gas Heater Control Group 5 Disconnect the nebulizing gas from the Electrospray 6 Release the latch on the left side of the spray chamber and swing the assembly out so that the spray shield is visib
67. ce pass through the Reflectron lens that provides for ion beam collimation and enter the Reflectron at the far end of the tube A potential higher than the Repeller accelerator voltage is applied to the far end of the Reflectron the ions gradually slow down and eventually reverse direction The reflected ions travel back through the flight tube In order to prevent a second deflection in the multideflector the main steering voltage is now turned off The ions penetrate freely through the accelerator and through the Repeller electrode finally striking the detector and generating an electrical signal The arrival time and amplitude of this signal constitute the raw output data of the BioTOF and both are recorded by the data acquisition system 1 4 Data Acquisition The ion flight time through the tube is proportional to the square root of the ion s m z ratio Thus during the flight through the mass spectrometer the ions separate according to their m z ratio Lighter ions arrive at the detector before the heavy ions with higher m z values Conversely this ratio can be determined from measurement of the total flight time through the mass spectrometer from the Repeller to the Reflectron and then to the detector Measurement of the flight time starts synchronously with the Repeller high voltage pulse Recording of the detector signal starts after a digitizing delay The digitizer delay determines the lowest m z value in the recorded spectrum and shi
68. cquisition and display of the spectra 2 2 Getting Started Version 1 0 2 System Controls Figure 2 2 shows the location of the Autoscaling checkboxes E 2 07 oF Last Good Method Eile Edit View Method Tools Format Help EE AS alii o 1 i e gt t1ifR0 Lar En ee Instrumentation Int BioT OF Spectrum 50000 Points MS ONLY System READY Automation Current method Last Good Method na Start B f Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition yndby Operation Acquisition Window Advanced Digitizer Setting Auto Stop Ionization IG lem ER was Spectrum Offset 0 0350 V EndofScanDeadtime 0 5440 us Menta D ESI C NSI C APCI Spectrum Size 50000 pts 200 kg PreSet Spectra Gan Digitizer Delay 35 us Adv Data Handing 500 m S c C NN SC Spectrum Counter ad Nenie Sample Rate Digitizer Summing 20 Spectra Gas Heater Control ol E Ez GS s D z Iw Dry Gas 590 X Clear Mass Range For Help press F1 X 347 1343 Y 1845 0000 Autoscaling Checkboxes Figure 2 2 BioTOF Window and Autoscaling Checkboxes Getting Started Version 1 0 2 3 2 System Controls 2 2 Front Control Panels The cabinet door on the front of the BioTOF console provides access to four control panels Figure 2 3 The Main Control Panel on the top left includes the main power switch and switches for turning on the vacuum pump
69. d increase the Transfer delay 8 10 Operator Manual Version 1 0 Volume II Operation 9 Calibration 9 Calibration As part of the BioTOF installation the system was calibrated using a table of reference masses included in the software The Calibration tab enables you to check the calibration after acquiring spectra from a known sample See Time of Flight Calculation on page 3 9 for any explanation of the calibration formula 9 1 Reference Tables for Calibration The following tables are supplied with the BioTOF software Table 9 1 Reference Tables for Calibration Reference File Peptides ref Negative_lons ref NA TFA_neg ref NA TFA_pos ref PEG_Na ref HPESIPOS ref Description Positive ions Peptides Negative lons Peptides Assorted Sodium TFA trifluoracetic acid negative ions Sodium TFA positive ions Sodiated Polyethylene Glycol positive ions Agilent ESI Tune mixture positive ions Operator Manual Version 1 0 Volume Il Operation 9 1 9 Calibration The reference files are ASCII text file consisting of a two column list of elements and their masses Figure 9 1 shows the file format amp j Peptides ref Notepad lol xj File Edit Search Help Valine_ M H Caffeine_ M H tri Tyrosine_ M H Leucine Enkephalin_ M H Methionine Enkephalin_ M H Na Leucine Enkephalin H Na Reserpine_ M H hexa Tyrosine_ M H Leucine Enkephalin Dimer 2H H Angiotensin I_ M H Substa
70. d previously saved methods Operator Manual Version 1 0 Volume II Operation 7 13 7 Methods 7 9 1 Save Method Use the Save Method function to create a new method or to update a previously saved method This menu function does not save mass spectrum data Click GP Save in the Command Buttons pane on the left side of the window to store data for analysis To save the current method for later use 1 Choose Method gt Save As to open a Save As dialog box Figure 7 11 Save As Save in E methods e a 1x neg 030503 tofpar E IntactProtein 022503 tofpar 2 1x_pos_030503 tofpar 2 mbi tofpar B 1x_pos_033103 tofpar test tofpar 2 3x_pos_030503 tofpar a bugtest_1 tofpar bugtest 2 tofpar Type TOFPAR File Date Modified 3 5 2003 6 30 PM Size 3 23 KB Save as type TOF tofpar Cancel Figure 7 11 Save As Dialog Box for Saving a Method The dialog box displays the methods that have been saved in Data Methods folder the files in the folder are filtered to include only those with the extension tofpar You can change the path but you must use the tofpar extension 2 Select an existing method in the dialog box to update file with the current parameters or enter an unused name to create a new method 3 Click Save If you save to an existing name a dialog box prompts you to confirm the action Click Yes to overwrite the previous file or No to cancel the actio
71. dcap Weak signal for standard Unusual settings needed for Cap Exit or Skimmers Low signal intensity through the entire range of voltage settings of Cap Exit Low signal intensity through the entire range of voltage setting on Skimmer 1 Advanced Troubleshooting Table Possible Cause Air bubbles Drying gas flow temperature and nebulizing gas not optimized Source out of tune Skimmer may be dirty Bad electrical contact or bad cable Bad electrical contact or bad cable Dirty Capillary or Endcap Solution Purge the solvent Adjust drying gas nebulizing gas and temperature Tune the Source Clean the Skimmer Check the continuity of the Capillary Exit spring contact inside the 45 door and cable Check the continuity between pin 6 of the 6 pin feedthrough and the first skimmer Clean the Capillary and Endcap 10 12 Operator Manual Version 1 0 Volume II Operation 10 Maintenance Table 10 1 Advanced Troubleshooting Table Continued Symptom Diagnostic Possible Cause Solution Error 151 Check the Spray Chamber is Close the Spray Chamber not closed properly Spray Chamber Check the HV cable is not Plug in or change HV cable plugged in or has the cable a defect Error 120 Check RF box RF box and cable Fix the connection and cable not connected properly Check the Damaged hexapole Call for service RF feedthrough itis a short circuit to ground or to each o
72. de the spray chamber is shaped by potentials applied to three electrodes the cylinder the end plate and the metal coated entry of the glass capillary tube When the solvent evaporates from the droplets the droplets charge per volume increases resulting in electrostatic Coulomb forces that break up the droplets into ever smaller particles until in the final stage de solvated sample ions emerge The charged droplets contain analyte solvent and a surplus of either positive or negative ions The type of ion formed depends on the composition of the sample liquid sprayed If for example the solution contains the sample in acetic acid with a relative positive potential on the needle the predominant positive ions are H4O and positively charged molecular analyte ions of the form MH Operator Manual Version 1 0 Volume Theory 2 3 2 Source Operation De solvation of the charged droplets is facilitated by a heated drying gas flowing counter to the stream of droplets and ions as they are electrostatically attracted towards the capillary tube that is the inlet orifice of the vacuum system lons enter the vacuum system of the mass spectrometer through the dielectric glass capillary stream through a skimmer and then enter into the RF ion guide which is biased at an appropriate offset voltage Neutral gas molecules that enter with the sample ions are pumped away in differential pumping stages at successively lower pressures lons
73. ding data This delay establishes the low end of the m z range When you change the delay the mass range is shifted accordingly Sample Rate There are three sampling rates 0 5 1 0 and 2 0 gigasamples per second The higher the rate the greater the digitizer resolution that is the number of data points across the ion peak However as you increase the sampling rate the upper end of the m z range is decreased If you increase resolution you may need to adjust either Spectrum Size or Digitizer Delay to capture the correct m z range 7 12 Operator Manual Version 1 0 Volume Il Operation 7 Methods Advanced Digitizer Setting Spectrum Offset adjusts the zero point of the vertical scale in the Spectra Display window Increasing the value excludes data points that may be noise The offset should be in the range 0 4 to 0 4 End of Scan Deadtime is a delay at the end of a cycle before the next cycle begins This value cannot be changed from the BioTOF window Advanced Data Handling Digitizer Summing sets the number of records the digitizer accumulates before it sends the summed records to the BioTOF software as a single Spectrum A setting of 20 is usually appropriate 7 9 Method Files When the BioTOF window is opened the application loads the Last Good Method that is the system settings and parameters that were in use when the system was closed The Method menu allows you to save the current method to a file and loa
74. displays show three items at a time To select an item Use the arrow keys to scroll through the menu and highlight the item and then press Select Select Table to display a list of syringe manufacturers highlight the syringe manufacturer and press Select Choose the Size Dia table select the syringe and press Select Choose the Rate from the menu and use the arrow keys to set the syringe pump to a flow rate and press Select Note that the rate is specified in volume per hour not per minute Operator Manual Version 1 0 Volume II Operation 6 3 6 Introducing Samples 6 1 3 Adjusting the Electrospray Needle The Electrospray needle consists of two concentric cylinders the outer one supplying the nebulizing gas and the inner one carrying the sample liquid The inner needle should protrude from the outer barrel by about 0 5mm To optimize spray performance for some low flow rate application it may be necessary to adjust the needle outward The Electrospray needle is mounted off axis in the top of the spray chamber The Electrospray needle is held in position by a bayonet connection To check the Electrospray Needle 1 Remove the off axis sprayer assembly from the top of the spray chamber 2 Verify that the tip of the inner needle protrudes about 0 5 mm from the body of the sprayer To adjust the needle position 1 Loosen the lock nut shown in Figure 6 3 Turn the needle assembly clockwise to the extend needl
75. dually slow down and eventually reverse direction Reflectron Lens gt Reflectron Figure 3 5 Reflectron and Reflectron Lens The reflected ions travel back through the flight tube In order to prevent a second deflection in the accelerator the main steering voltage in the tube is now turned off The ions penetrate freely through the accelerator and through the Repeller electrode as well finally striking the detector The Reflectron and Reflectron Lens voltage values are set on the Spectrometer tab 3 6 Operator Manual Version 1 0 Volume I Theory 3 TOF Mass Spectrometer Operation 3 3 Detector The detector consists of dual microchannel plates and an anode The dual microchannel plates are shown in Figure 3 3 on page 3 3 The plates are configured as a chevron that is the microchannels in the first plate are at an angle to the ion flight path while the channels in the second plate are at the same angle but in the opposite direction Figure 3 6 The microchannels are lined with a semiconducting layer When an ion enters a microchannel it collides with the channel wall causing it to release an electron The electron then collides with the opposite wall emitting multiple electrons As the electrons pass through the channel and the opposite angle channel in the next plate they create an avalanche of electrons When the electrons emerge from the microchannel plates they are collected with a metal anode generating
76. e Figure 3 22 The ESI source voltages are turned off the light in the high voltage indicator on the ESI front panel must be off the high voltages are turned off and the Instrument HV unit is disabled Enable LED off Double check that the ESI voltages and the TOF voltages are off Note For this procedure the high voltages must be off Getting Started Version 1 0 3 19 3 Quick Start 3 Uncheck DG Heat in the Gas Heater Control Group Figure 3 21 on page 3 18 to allow the spray chamber to cool Click Standby to exit Instrument Operating mode and e Standby Operation shut down the ESI and TOF voltage sources EE f ESI nESI APCI Polarity Ze Positive Negative Gas Heater Control Y Dry Gas Before cleaning the V Neb Gas After cleaning the spray chamber e spray chamber uncheck DG Heat to DG Heat recheck DG Heat allow the unit to cool Figure 3 22 System Control Settings for Standby 4 Stop the syringe pump 5 Fill the syringe with ESI solvent or a plain methanol water mixture and flush both the transfer line and ESI sprayer several times 6 Make sure the liquid flows easily through the plumbing 7 Uncheck Neb Gas in the Gas Heater Control group 8 After the spray chamber has cooled down wipe clean the spray shield endplate and the face of the inlet capillary 9 Close the spray chamber 10 Return to the BioTOF window and recheck DG Heat Note You should leave the drying gas
77. e aea Ea ee 10 6 10 4 Basic Troubleshooting 2er 10 11 10 5 Advanced Troubleshooting x 10 12 OPERATOR MANUAL INDEX vi Operator Manual Version 1 0 Volume II Operation Preface This document provides detailed information on using the BioTOF system It is consists of two volumes BioTOF Operator Manual Volume 1 Theory BioTOF Operator Manual Volume 2 Operation Three additional manuals in this document set provide information on using the analytical software included with the BioTOF system DataAnalysis User Manual QuantAnalysis User Manual Library Editor User Manual For technical assistance contact Bruker Daltonics customer service In the U S 1 978 663 3660 Ext 1445 In Germany 49 0 421 2205 430 In Japan 81 0 45 440 0471 Local Service Operator Manual Version 1 0 Volume Il Operation vii Safety Labels These labels are placed on the instrument to indicate the respective hazard possibility SEPP Hazard Note Refer to the Operator Manual for details on this hazard Warning High Voltage A dangerous high voltage or shock hazard exists within this area Only trained service persons should perform work in this area Warning High Temperature Hot parts and surfaces are present in this area Allow the part or surface to cool before performing work in this area This may require turning off power to selected components or to the entire instrument Warning Biohazard
78. e data in the buffer is Getting Started Version 1 0 3 15 3 Quick Start maintained Figure 3 19 Spectra Display 3 Set PreSet Spectra to 500 and click AutoStop Spectra acquisition automatically stops when 500 spectra have been accumulated The Save button becomes active 3 16 Getting Started Version 1 0 3 Quick Start 4 Click Save in the control panel The BioTOF software opens a dialog box for setting the file options Figure 3 20 File Save Options Path D data 041103 Sample Name Sample_1 Experiment No f1 Experiment ID Commenti Free format comments Comment2 Free format comments Comment3 Free format comments Apply and Save Cancel Figure 3 20 File Save Options Dialog Box The BioTOF application creates two files one formatted for the DataAnalysis application the other compatible with the XMass program You can set the application to use either or both formats by choosing Format gt DA format and or Format gt Xmass format from the menu bar Getting Started Version 1 0 3 17 3 Quick Start 3 9 Check Calibration As part of the BioTOF installation the system was calibrated using a table of reference masses included in the software The Calibration tab enables you to check the calibra tion after acquiring spectra from the known sample See Chapter 8 in the BioTOF Operator Manual Volume Il Operation for detailed instructions on checking and adjust ing BioTOF calibration To ch
79. e rather than displaying the legend data in the top left corner of the MS pane Data Lists the possible colors for the traces and data points Axes Lists the possible colors for the axes Background Color Opens a Windows Color dialog box for selecting the background color for both panes in the Spectra Display window Show Peak Width Displays the width of peaks in Dalton Note Some functions are not available in earlier versions of the BioTOF software Operator Manual Version 1 0 Volume Il Operation 5 11 5 BioTOF Window 5 2 1 Parameter Tabs The seven tabs below the Spectra Display window set the parameters for the collection method Tab Description Mode Sets the instrument mode display units and mass range Chromatogram Defines a range of ion counts to mark the chromatogram display when Chromatography MS mode is selected Calibration Calibrates the BioTOF system using reference tables ESI Source Specifies gas temperatures and voltage levels for the ESI Source Spectrometer Sets voltages applied to various components in the mass spectrometer Timing Sequence Sets advanced timing parameters for the mass spectrometer Acquisition Sets sampling rate spectrum size and other data acquisition parameters For more information see Methods on page 7 1 5 3 Menus and Tool Bar This section lists the BioTOF functions available from the menu bar and tool bar Figure 5 10 H 2 07 OF Last Good Method
80. e tip beyond the outer cylinder Figure 6 3 Adjusting the Electrospray Needle 2 Hold the bayonet connection with one hand and turn the needle assembly clockwise to extend the inner needle outward or counterclockwise to draw the needle back into the assembly 3 Tighten the lock nut and reinstall the assembly in the spray chamber 6 4 Operator Manual Version 1 0 Volume Il Operation 6 Introducing Samples 6 1 4 Starting the Syringe Pump To operate the syringe pump 1 Press Run to start the pump 2 Press the right arrow key and Run simultaneously to push the liquid to Electrospray This is called Fast Forward mode 3 Release the buttons to stop the pump 4 Press Run to operate at the specified rate After the flow has reached the source end of the nebulizer some additional optimization may be required for low flow rates such as adjusting the amount of nebulizing gas drying gas and in some cases a slight re adjustment of the extension of the nebulizer needle To adjust the spray needle on line 1 Loosen the lock nut 2 Rotate the body of the needle clockwise or counterclockwise 3 Make quarter turns and observe the result with the Source Lens Current in the System Status indicators on the ESI Source 4 When the spray is optimized tighten the lock nut Operator Manual Version 1 0 Volume Il Operation 6 5 6 Introducing Samples 6 1 5 Flushing the Syringe Pump Unlike FIA or liquid chroma
81. eck calibration 1 Click the Calibration tab to display the table of reference masses 2 Use the pull down list at the top of the tab to select Peptides ref to display the Reference list 3 Select the first known sample used in acquiring the spectra Angiotensin Il in Figure 3 21 A red vertical line appears in the Spectra Display at the calculated position of the selected compound on the m z scale and the cursor in the Spectra Display changes to the Max cursor Jf Int 7 BioT OF Spectrum 54000 Points Max Cursor Calculated Position Instrument C Standby Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Fit Operation Reference List Peptides ref lonization ESI nESI APCI Polarity Positive C Negative Gas Heater Control IV Dry Gas V Neb Gas Y DG Heat Figure 3 21 Name Leucine Enkephalin Dim Angiotensin M H Angiotensin M 2H Angiotensin M 3H Angiotensin ll M H gt Angiotensin l M 2H Angiotensin I M 3H Reference Mass 1111 545877 1296 684770 548 845023 432 899774 1046 541794 523 774535 349 518782 Current Mass Error ppm xx v lt X Clear Calibrating the BioTOF System Linear Quadratic Preset Constants 475 1750 y 476 1743 c 1194363184 1194358164 0 0000 12 t em c m Accept X 523 7703
82. ee 3 7 3 6 Check Bio TOF Parameters 2 dei la 3 9 3 7 Start BIO TOR Operation EE 3 13 3 8 Acquire elo 3 15 3 9 Check OallBEAUOTY EE 3 18 3 10 Place the System on Standby t tn tact exem RE ERE EE 3 19 GETTING STARTED INDEX Operator Manual Version 1 0 Volume I Theory iii OPERATOR MANUAL VOLUME I THEORY RL 1 EE ll PREFA CE E Vil SAFETY LABELS a ses een VIII 1 SYSTEM COMPONENTS 2 22 22 en 1 1 1 1 Front Control Panels uio eon ee 1 3 1 2 Computer ee 1 5 1 3 BIO TOE SOIDWAl6 cys cect eS eaten sad 1 5 2 SOURCE OPERATION ee taa 2 1 2 1 Sample Introduct n screen iaa 2 1 2 2 ESI SOURGE 2 3 2 3 N nospray ER 2 9 2 4 E ENEE 2 10 25 APGI S urce Operation RE 2 10 3 TOF MASS SPECTROMETER OPERATION uunuuusssssnnnnnnnnonnnnnnnnnnnnnnnnnnnennnnnnn 3 1 3 1 Orthogonal Interfaeg EE 3 2 3 2 Flight Tube ee EHRT 3 5 3 3 Detector es ae 3 7 3 4 Bol rra MERLO c I PEE TES 3 8 3 5 Time of Flight Calculation tien aaa teen 3 9 3 6 Space EE ee an Rense 3 11 3 7 MUllipass 22 22 eine 3 12 iv Operator Manual Version 1 0 Volume I Theory OPERATOR MANUAL VOLUME II OPERATION Il el lee EE ll PREFACE E HN Vil SAFETY LABELS 00 ee VIII 4 SYSTEM SSTARTUP coincida 4 1 4 1 Startfhe Systelias o head etie uS leaden rea SL itd a Boia deen 4 1 4 2 Check the Spray Chamber and Sprayer ccccceceeceeeeeeeeeeeeneeeeeeeeeeeeeseneeees 4 5 4 3 Start the
83. en Dialog Box for Loading a Method 2 Ifthe method is stored in another location use the dialog box tools to browse to the other folder 5 18 Operator Manual Version 1 0 Volume Il Operation 5 BioTOF Window 3 Select the method and click Open The BioTOF system displays a progress gauge as it loads the method Figure 5 12 It may take several minutes for the data to be loaded When it is finished the method file name is shown in the Current Method box on the left side of the BioTOF window Loading Method Figure 5 12 Progress Gauge as a Method is Being Loaded 5 6 2 Save Method Use the Save As function to create a new method or to update a previously saved method This menu function does not save data Use the Save button in the Command Buttons pane on the left side of the window to store data for analysis To save the current method for later use 1 Choose Method gt Save As to open a Save As dialog box Figure 5 13 The dialog box displays the methods that have been saved in Data Methods folder the files in the folder are filtered to include only those with the extension tofpar You can change the path but you must use the tofpar extension Save As Save in methods yv e Ers 2 1x_neg_030503 tofpar 2 IntactProtein_022503 tofpar sj 1x pos 030503 tofpar a mbi tofpar 8 1x pos 033103 tofpar a test tofpar 2 3x_pos_030503 tofpar a bugtest_1 tofpar DN bugtest_2 tofpar
84. en Stee 3 7 3 6 Check Bio TOF Parameters 2 dei la 3 9 3 7 Start BIO TOR Operation EE 3 13 3 8 Acquire elo 3 15 3 9 Check CG AID PAU ON EE 3 18 3 10 Place the System on Standby nn en ee 3 19 GETTING STARTED INDEX Operator Manual Version 1 0 Volume Il Operation ili OPERATOR MANUAL VOLUME I THEORY RL 1 EE ll PREFA CE E Vil SAFETY LABELS a ses een VIII 1 SYSTEM COMPONENTS 2 22 22 en 1 1 1 1 Front Control Panels uio eon ee 1 3 1 2 Computer ee 1 5 1 3 BIO TOE SOIDWAl6 cys cect eS eaten sad 1 5 2 SOURCE OPERATION ee taa 2 1 2 1 Sample Introduct n screen iaa 2 1 2 2 ESI SOURGE 2 3 2 3 N nospray ER 2 9 2 4 E ENEE 2 10 25 APGI S urce Operation RE 2 10 3 TOF MASS SPECTROMETER OPERATION uunuuusssssnnnnnnnnonnnnnnnnnnnnnnnnnnnennnnnnn 3 1 3 1 Orthogonal Interfaeg EE 3 2 3 2 Flight Tube ee EHRT 3 5 3 3 Detector es ae 3 7 3 4 Bol rra MERLO c I PEE TES 3 8 3 5 Time of Flight Calculation tien aaa teen 3 9 3 6 Space EE ee an Rense 3 11 3 7 MUllipass 22 22 eine 3 12 iv Operator Manual Version 1 0 Volume Il Operation OPERATOR MANUAL VOLUME II OPERATION Il el lee EE ll PREFACE E HN Vil SAFETY LABELS 00 ee VIII 4 SYSTEM SSTARTUP coincida 4 1 4 1 Startfhe Systelias o head etie uS leaden rea SL itd a Boia deen 4 1 4 2 Check the Spray Chamber and Sprayer ccccceceeceeeeeeeeeeeeneeeeeeeeeeeeeseneeees 4 5 4 3 Start t
85. er 5 5 Progress Gauge 5 19 7 16 Properties Profile Display Dialog Box 5 10 Pulse ON OFF Button 7 11 Pump and Sample Inlet 6 8 R Ready Indicator 5 4 Reference Tables for Calibration 9 1 Trajectory 3 9 Reflectron 3 6 Reflectron Tuning 10 6 10 8 Refresh Button 7 9 Resize Dialog Option 5 17 RF lon Guide 2 8 Running Indicator 5 4 S Sample 2 1 6 1 Delivery to Electrospray 6 1 Rate 7 12 Index 6 Operator Manual Version 1 0 Volumes and Il Save As Dialog Box 5 20 7 14 Save Method 5 19 7 14 Serial path 6 9 Setup for Tuning 10 6 Show Servers 5 24 Signals from the external device 7 5 Single Pass Mode 3 11 Skimmer 1 Cleaning 10 3 Skimmers 2 8 Source Control Unit 1 3 Control Unit Instrument HV Panel 4 4 8 2 Lens Current 1 4 4 4 6 12 8 3 Operation 2 1 Space Focusing 3 11 Spectra Display 1 6 5 2 8 5 Spectra Display Window 5 7 Chromatography MS Mode 5 7 MS Only and FIA MS 5 7 Spectrometer 5 22 Spectrometer Tab 7 10 Spectrum Display Background Dialog Box 5 9 Display Pop up Menu 5 8 Spray Chamber 4 5 Shield 2 6 Sprayer 4 5 Standby 6 11 Standby Indicator 5 4 Start Button 5 5 Starting BioTOF 8 1 Startup 4 1 Status Bar 5 17 Indicators 5 3 Operator Manual Version 1 0 Volumes and Il Index 7 Stop Button 8 5 Stopping the Vacuum System 10 2 Syringe Pump 2 1 6 2 Pump Controls 6 3 Pump Operation 6 5 System Components 1 1 Option Buttons Checkboxes 1 6 Startup 4 1 Status Indicators 1 4
86. erator Manual Version 1 0 Volume Il Operation 8 Acquiring Data 8 3 2 Creating a Multipass Method The flight times multiply approximately with the number of turns in Multipass mode that is for two passes flight times approximately double for three passes they triple and so on However the focusing conditions change and it is necessary to retune the Reflectron voltage in order to achieve best performance In general the Reflectron voltage increases with the number of turns Set up Multipass methods sequentially for two three or more passes To set up multipass without a predefined method 1 10 11 Start in normal mode single pass positive ions using a strong sample with only one or two well known peaks chosen in accordance with the Table 8 1 on page 8 7 Click the Mode tab and select Flight Times in the Data Display group Note the flight times for the peaks of interest Click the Acquisition tab and set the Digitizer Delay 5 to 10 us below the doubled flight time of the low m z peak Change to the Timing Sequence tab and set the Repeller duration to 5 us lower than the new Digitizer Delay Record a spectrum Out of focus peaks will appear at approximately doubled flight times Retune the Reflectron Reflectron Lens and multideflector voltages for optimized performance as described in BioTOF Reflectron Tuning on page 10 6 Click the Mode tab and select m z in the Data Display group Record a
87. es and printing the current spectra See Load Method on page 5 18 Automatically scales the horizontal axis in the Spectra Display to show the full range of values Equivalent to checking the left checkbox in the bottom left corner of the Spectra Display 1 Autoscale Y Axis Automatically scales the vertical axis in the Spectra Display to show the full range of values Scale increases as additional records are accumulated Equivalent to checking the right checkbox in the bottom left corner of the Spectra Display Ex Autoscale Both Axes Automatically scales both axes of the Spectra Display Equivalent to checking both checkboxes in the bottom left corner of the Spectra Display Increase X Range Increases the scale of the horizontal axis without changing the vertical axis Decrease X Range Decreases the scale of the horizontal axis without changing the vertical axis Increase Y Range Increases the scale of the vertical axis without changing the horizontal axis i Decrease Y Range Decreases the scale of the vertical axis without changing the horizontal axis Scroll Left Scrolls the Spectra Display to the left Scroll Right Scrolls the Spectra Display to the right t Scroll Up Scrolls the Spectra Display up 5 14 Operator Manual Version 1 0 Volume Il Operation 5 BioTOF Window Table 5 4 BioTOF Tool Bar Icons Continued Icon Function 1 Scroll Down Q Zoom A Shift Max Cursor 4 Perpendicular C
88. etal disk centered around the entrance of the glass capillary High voltage is applied to the endplate which forms the electrostatic gradient in the spray chamber Spray Shield Removable metal cap that is part of the Endplate An opening in the center of the spray shield which is immediately in front of the capillary allows ions to enter the capillary and forces counter current flow of the drying gas Capillary Entrance The third of the spray chamber electrodes The metallized entrance to the capillary carries a potential with the following function In positive ion mode the entrance to the capillary is at 3 kV to 5 kV relative to the needle and approximately 500 V relative to the endcap The electrostatic gradient created by these voltages supports the production of charged droplets and helps them to migrate towards the capillary entrance Once near the entrance the analyte ions are pushed through the capillary by the pressure gradient between the spray chamber about 1013 mbar or 760 Torr and the first pumping stage approximately 1 to 2 mbar The ion Polarity is set with option buttons in the BioTOF window just to the left of the Parameter tabs 2 6 Operator Manual Version 1 0 Volume Theory 2 Source Operation Table 2 1 ESI Source Operation by Component Continued Component Function Capillary The capillary is A sampling orifice that transfers ions from the spray chamber at atmo
89. etting the Source Lens Current Reading 10 Set the ion Polarity and Enable switches on the Instrument HV unit to the middle position When the two 3 position switches on the Instrument HV panel Figure 4 4 are in the middle position the unit is remotely controlled from the BioTOF window 11 Check the nitrogen supply 4 4 Operator Manual Version 1 0 Volume Il Operation 4 System Startup 4 2 Check the Spray Chamber and Sprayer The next step is to check the Sprayer Assembly on the front of the ESI Source The assembly is attached to the ESI Source with hinges on the right and locked with a latch on the left A sample is introduced to the spray chamber via an off axis spray needle which is held in position by a bayonet connection Figure 4 6 shows the sprayer assembly unlatched and open and the off axis sprayer removed from the assembly Spray Shield Off Axis Sprayer Nebulizer Gas Nebulizer Gas Valve Figure 4 6 Off Axis Sprayer Assembly To check the Spray Chamber 1 Release the latch on the left side of the assembly and swing the assembly out so that the spray shield Figure 4 6 is visible Remove the spray shield remove any plug or rubber seal from the end of the capillary tube and re install the spray shield Remove the off axis sprayer assembly from the top of the spray chamber verify that the tip of the inner needle protrudes about 0 5 mm from the body of the sprayer and reinstall the spraye
90. fts the recording window along the mass scale The electrical signal from the detector is converted from analog to digital for each scan Multiple scans are aggregated to form a single higher density record The records are summed in the digitizer to form a spectrum that is transferred to the workstation In MS Only mode the spectra are further accumulated in a single buffer and the buffer contents are displayed in the BioTOF window At the end of the data acquisition the contents of the buffer can be saved as a single mass spectrum In Chromatography MS mode the transferred spectra are stored and displayed consecutively and can be saved as time stamped mass spectra 1 4 Getting Started Version 1 0 1 BioTOF Operation You specify the width of the recording time window by using one of two methods Selecting a mass range in which case the BioTOF software automatically determines digitizer delay in keeping with the other timing parameters Specifying the number scans per record and a sampling speed The recording window is given by the time per data point multiplied by the number of points to be accumulated in each scan In this case you must set the appropriate digitizer delay to assure that the mass range includes ion masses of interest The methods used for determining the sampling rates for the various BioTOF Series models are described in BioTOF Operator Manual Volume I Theory Getting Started Version 1 0 1 5
91. hat the spray and the spray current are stable Check spray regularly during the tuning procedure 8 Pre tune Multideflector High voltage in steps of 2 V up and down from the default value for maximum signal intensity Operator Manual Version 1 0 Volume II Operation 10 7 10 Maintenance 10 3 2 Reflectron Tuning Procedure The goal of the Reflectron tuning procedure is to optimize the Reflectron voltage for mass resolution and simultaneously balance Multideflector High and Reflectron Lens voltages for peak intensity Note Do not change Repeller voltage during the tuning leave it set at 8500 V 1 Tune the Reflectron voltage as follows Acquire a decent spectrum Typically 200 to 500 scans should be sufficient with standard sample Set data acquisition to AutoStop with acquisition counter preset to an appropriate number Acquire a spectrum and DO NOT CLEAR Set Reflectron voltage in steps of 100 V higher and lower from the starting value covering the range from 8600 to 9600 V This procedure generates an envelope of peaks with each peak or group of isotope peaks representing a Reflectron tuning voltage Settle for the voltage that gives the highest intensity in that envelope which coincides with the narrowest peak shape squeezing all the ions into a narrower package on the detector Click the Clear button 2 Optimize peak intensity by tuning the Multideflector High deflector voltage for highest intensity initi
92. he BioTOF Sollware nennen 4 6 QA T rmthe Gases Onnan e nee AEA Ea AG 4 6 S BIOTOF MIN D O E 5 1 5 1 Window E LEE 5 1 5 2 Spectrum Display Pop up Menta a 5 8 5 3 Menus and Tool Ba ran Eege EE EE 5 12 5 4 File a d Edit Menus lt td m 5 13 5 5 View MENU A o O ee 5 13 5 6 Method Menu 5 18 5 7 o kenn 5 20 5 8 Forniar Men tec 5 26 5 9 Help Mena 5 26 6 INTRODUCING SAMPLES iret reete hen rettet 6 1 6 1 Sample Delivery to Electrospray AE 6 1 6 2 Liquid Chromatography EG ae ee a 6 7 6 3 Nanospray star REAT Enana 6 10 6 4 A ne EE 6 16 6 5 Configuring the APCI Spray Chamber AANEREN 6 21 Operator Manual Version 1 0 Volume II Operation V T METHODS ee 7 1 7 1 GOntro BUTS ee EE 7 2 7 2 Mode Tab een 7 3 7 3 Chromatogram Lab es de ae xe mdr cei ad 7 6 7 4 Galibration Tabu ee 7 7 7 5 ES SOUufee Lab EE 7 8 7 6 Spectrometer Tab EE 7 10 7 7 T m nga Seguenee Tab EE 7 11 7 8 Acquisition Tabea ee eek euren 7 12 7 9 Method Files EE 7 13 8 ACQUIRING DATA 2 8 1 8 1 Starting System ODerallohi id 8 1 8 2 Acquiring and Saving Spectra 8 4 8 3 DUNT DSS Cm 8 7 9 GALIBRATION lt lt ainia 9 1 9 1 Reference Tables for Calibration AAA 9 1 9 2 Calibrating the System emer ee 9 3 9 3 Creating a Custom Reference File 9 5 10 MAINTENANGE un SEES BEE 10 1 10 1 Daily En Eu ne 10 1 10 2 Monthly Mairitenange eret RR ret ER da 10 2 10 3 BIOTOF Reflectron Tuning soe od Eas cena tee serr
93. he use of the APCI Source 2 1 Sample Introduction There are three basic methods of delivering sample to the BioTOF system Syringe pump Flow from a liquid chromatography LC system Off line Nanospray 2 1 1 Syringe Pump A syringe pump Figure 2 1 is included with the instrument system to provide for the introduction of sample directly to the Electrospray Nanospray or APCI ion source Tubing from the syringe can be connected directly to the spray or tee d to the flow from a LC system The pump holds syringes from 5 uL to 5 mL A 100 uL syringe is supplied with the pump Function buttons and a small display on the front of the pump enable you to set flow rate and stop and start the pump motor Figure 2 1 Syringe Pump Included with the BioTOF System Operator Manual Version 1 0 Volume Theory 2 1 2 Source Operation The following are typical syringe pump flow rates for various source configurations Electrospray 1 to 1 000 uL 60 uL h to 60 mL h On line Nanospray 50 to 500 nanoL min 3 to 30 uL h e APCI 10 to 1 000 uL min 600 uL h to 60 mL h Note The units of measure on the syringe pump display are volume per hour See Setup for Low Flow Direct Infusion on page 6 2 for additional information on setup of the syringe pump 2 1 2 LC System Sample can be introduced to the Electrospray Nanospray or APCI directly from a Liquid Chromatography LC system In LC applications the ionization
94. hold the two right buttons on the syringe pump to fast forward the solution to the ESI When sample is flowing through the tip of the spray needle assembly and nebulizer gas and spray chamber voltages are turned on the current meter Figure 8 3 of the ESI source controller indicates the spray current If there is no reading Check to make sure that the meter is set to read the endplate electrode as described in Start the System on page 4 1 Adjust the needle Adjusting the Electrospray Needle on page 6 4 Verify that the spray shield is installed 4 Release the two syringe pump buttons to stop fast forward and press Run the pump at the specified rate OURCE LENS CURRENT ESS Poan E BP HIGH VOLTAGE Jee Vacuum mp POWER Figure 8 3 Source Lens Current Reading Operator Manual Version 1 0 Volume II Operation 8 3 8 Acquiring Data 8 2 Acquiring and Saving Spectra Controls and indicators for starting and stopping spectrum acquisition are located in the two panes on the left side of the BioTOF window Figure 8 4 You can start acquisition when the status bar is green and displays Ready Instrumentation MS ONLY St System You can start ESSHEADYSSS Auto Stop acquisition when E the status bar Manual Stop is green MANUAL Current method PreSet Spectra Last Good Method 500 lt Spectrum Counter Shows the number records stored Clear in the buffer
95. ings and familiarize yourself with the controls browse through the parameter tabs Note The parameter values shown in the examples that follow are typical values appropriate for the samples used in this Getting Started guide To ensure that the Last Good Method is appropriate 1 Check the option buttons in the lonization and ion Polarity groups to make sure that the lonization is set to ESI and that the Polarity is set to Positive Figure 3 9 Instrument Standby C Operation lonization f ESI nESI APCI Polarity Positive Negative Gas Heater Control Iw Dry Gas v Neb Gas Iw DG Heat Figure 3 9 lonization and Polarity Groups Getting Started Version 1 0 3 9 3 Quick Start 2 Click the ESI Source tab to display the ESI control parameters Figure 3 10 Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Gas Flow T emperature Dry Gas Temp Eer E C High Voltage Electrodes Volts Lens Volts Cylinder 20001 Capillary Exit 140 Trap 153 Skimmerl 40 3 Lens 50 End Plate 3500 Skimmer2 30 RF Amplitude 600 Canil 15 apillary 4000 gt Offset Refresh Figure 3 10 ESI Source Tab 3 Set the ESI spray chamber voltages in the High Voltage Electrodes group to the values shown in Figure 3 10 With ion Polarity set to Positive the actual spray chamber voltages are negative Only the absolute values are displayed Note The software limits the
96. is optimized to accept flow rates up to 1 ml min with the Electrospray or with the APCI Source The nebulization process for both of these ion sources is assisted with nebulizing gas and countercurrent drying gas The LC system can be operated in several modes in conjunction with the BioTOF system including standard LC analysis and analysis without LC separation flow injection analysis FIA The BioTOF system can be quickly adapted to the different instrumentation modes using the BioTOF window Parameters tabs described in Methods on page 7 1 2 1 3 Off Line Introduction In off line Nanospray applications the sample can be introduced directly into the disposable Nanospray tip itself using a micro pipet See Nanospray on page 6 10 2 2 Operator Manual Version 1 0 Volume I Theory 2 Source Operation 2 2 ESI Source This section describes operation of the source configured with the Electrospray spray chamber Subsequent sections cover use of the Nanospray and the APCI Source Figure 2 2 shows ESI Source with the spray chamber opened to show some of the source components Spray Removable Spray Shield Chamber Cylinder Endplate Figure 2 2 ESI Source With Open Spray Chamber In the ESI spray chamber a fine spray of charged droplets is generated from the sample solution by means of a sprayer under the influence of an electric field or an electric field assisted by a pneumatic nebulizer The electric field insi
97. le left in Figure 6 5 7 After the spray chamber has cooled remove the spray shield from the end plate and capillary entrance right in Figure 6 5 Figure 6 5 Endplate with Spray Shield in Place left and with Spray Shield Removed right Operator Manual Version 1 0 Volume Il Operation 6 11 6 Introducing Samples 8 Insert the gas diverter disk into the endplate left in Figure 6 6 and place the capillary cap over the capillary entrance right in Figure 6 6 Figure 6 6 Placement of the Gas Diverter left and Capillary Cap right 9 Lift the spray chamber door from the hinges on the ESI Source assembly 10 Place the Nanospray assembly on the hinges and close and latch the spray chamber For Nanospray operations high voltage is applied to the capillary only After installing the Nanospray assembly you should use the Source Control Unit to set Source Lens Current display to read the current on the Capillary 11 Use the arrow buttons below the Source Control Panel LCD display to position the display cursor on Cap Figure 6 7 CON edit active Remote Menu 0 0 1000 Cyl End Capillary Electrode Figure 6 7 Select Cap for the Source Lens Current Reading 6 12 Operator Manual Version 1 0 Volume II Operation 6 Introducing Samples 6 3 2 On Line Nanospray For On Line Nanospray use Nanospray needles such as 8 micron inner diameter or 15 micron inner diameter New Objective Tips To ins
98. lowed under the Safety Requirements for the manufacturer listed have no part numbers copyright laws Electrical Equipment for and are not available from Measurement Control Noise Declaration Bruker Daltonics Inc Most Document History and Laboratory Use 70 dB operator position of these can be obtained First edition May 2003 normal operation per from laboratory supply Printed in U S A Class 1 LED Product ISO 7779 1988 companies Warranty WARNING Instrument Identification Manual part number A5001 The information contained in Connecting an instrument Each instrument is this document is subject to to a power source that is identified by a unique change without notice not equipped with a serial numbers These protective earth contact serial number is located on Bruker Daltonics Inc makes creates a shock hazard a label on the lower left side no warranty of any kind with for the operator and can near the rear of the regard to this material damage the instrument instrument including but not limited to Likewise interrupting the the implied warranties of protective conductor inside When corresponding with merchantability and fitness or outside the instrument Bruker Daltonics Inc about for a particular purpose or disconnecting the your instrument be sure to protective earth terminal include the model and the Bruker Daltonics Inc is not creates a shock hazard for full serial number liable for errors contained the operator and can he
99. lume II Operation 8 Acquiring Data 8 Acquiring Data After introducing the sample to the ESI Source and selecting the acquisition method the next step is to acquire and save spectra Topics covered by this chapter include Starting system operation Acquiring and saving spectra Acquiring spectra in MS Chromatogram mode Multipass 8 1 Starting System Operation To start BioTOF operation 1 In the BioTOF window click the Operation option button in the Instrument group Figure 8 1 Instrument C Standby Operation lonization f ESI C nESI C APCI Polarity Positive Negative Gas Heater Control Y Dry Gas v Neb Gas jw DG Heat Figure 8 1 Instrument Control Group Operator Manual Version 1 0 Volume II Operation 8 1 8 Acquiring Data The instrument goes from Standby to Operation The Enable LED on the Instrument HV unit lights up Figure 8 2 and the high voltages ramp up All ESI source voltages turn on You may also be prompted to press the Voltage On button on the Source Control unit Figure 8 2 This switch lights up to indicate that the source voltages have been turned on Voltage On Button LED indicates high voltages enabled Figure 8 2 Source Control Unit and Instrument HV Panel 2 Start the sample flow from the syringe pump or LC system 8 2 Operator Manual Version 1 0 Volume II Operation 8 Acquiring Data 3 If using the syringe pump press and
100. m an LC system and you want to display a chromatogram and the mass spectra The Spectra display window is divided into two panes the top one showing the spectra and the lower one showing ion chromatograms njector MS for time controlled mass spectrometry FIA MS Flow injection analysis mass spectrometry has been selected in the Mode tab to specify a run time With this mode you set up the run in the Chromatographic Setting group by specifying the total run time in minutes and a wait time for the solvent to clear Operator Manual Version 1 0 Volume II Operation 5 3 5 BioTOF Window Table 5 1 Status Indicator Descriptions Continued Function Description System The system modes are indicated by Automation Current method Yellow STANDBY Green READY Red Not Ready Blue Running Automatic when the instrument is in Injector MS mode Otherwise Manual Identifies the method in use Initially the field displays Last Good Method the settings in use when the BioTOF application was closed When you load a saved method the Current method displays the method file name See Methods on page 7 1 5 1 2 Command Buttons Group Figure 5 4 describes the buttons and fields in the Command Buttons group AN Start Auto Stop Manual Stop PreSet Spectra 500 Spectrum Counter Ces x Clear Figure 5 4 Command Buttons 5 4 Operator Manual Version 1 0 Volume Il Operation 5 Bio
101. n MAIN VACUUM PUMPS ELECTRONICS COMPUTER Figure 4 2 Main Switch Panel 2 Press MAIN pushbutton The MAIN is illuminated red to indicate the main power is On 3 Press the VACUUM PUMPS pushbutton to start the vacuum pumps When this button is pressed it lights up and the pumps start sequentially However the system will not be ready until the pressure in the vacuum system has dropped below threshold This may take several hours The buttons illuminate when pressed to engage the switches 4 2 Operator Manual Version 1 0 Volume II Operation 4 System Startup 4 Press the ELECTRONICS button to initialize the system electronics including the vacuum gauge controller Figure 4 3 ma Lights indicate umm when the vacuum the selected oy An the factory set gauge Esc o thresholds D Press this button to O8 ER enable the selected EN Press these buttons MI EDWARDS Active Gauge Controller to select a gauge Figure 4 3 Vacuum Gauge Controller 5 Press the 4 and w buttons on the vacuum gauge controller to cycle through the readouts for the gauges used in the BioTOF system Gauge 1 reads the flight tube pressure this gauge is initially off Gauge 3 reads the pressure in the first pumping stage of the ESI source 6 Select Gauge 1 and press the ON OFF button on the gauge controller The flight tube pressure must be below 5 10 mbar to engage the pressure interlock of the high voltage supplies 7
102. n 1 0 3 11 3 Quick Start 7 Click the Acquisition tab and ensure that the spectrum acquisition parameters are in line with those shown in Figure 3 13 Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Acquisition Window Advanced Digitizer Setting m z from to era Spectrum Offset 0 0350 V End of Scan Deadtime 05440 us Spectrum Size 50000 pts 200 kB Digitizer Delay 35 Zus Advanced Data Handling Sample Rate Digitizer Summing Spectra o5 E 2 ass Mass Range Resolution Figure 3 13 Acquisition Tab 8 Click the Mode tab Figure 3 14 and set the Instrumentation Mode to MS Only Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Instrumentation Mode Chromatographic Setting C Chromatography MS FIA MS Total Run Time If mins Solvent Delay l mins Data Display mz Flight Time Paints MS Acq Rate 1 lt lt 70 Spectra min Mass Range m z External Start m z from 147 to 874 Enable Configure Figure 3 14 Mode Tab 3 12 Getting Started Version 1 0 3 Quick Start 3 7 Start BioTOF Operation To start BioTOF operation Te In the BioTOF window Figure 3 6 on page 3 6 click the Operation option button in the Instrument group Figure 3 15 Instrument C Standby Operation lonization f ESI nESI C APCI Polarity Positive Negative Gas Heater Control V Dry Gas Iv
103. n and select a new name The Current method field changes to the name of the saved file When you exit the BioTOF window the system saves the parameters and presents them as the Last Good Method next time the window is opened 7 14 Operator Manual Version 1 0 Volume Il Operation 7 Methods 7 9 2 Load Method To load a previously saved method 1 Choose Method gt Load Method from the menu bar or click pl in the tool bar An Open dialog box displays the methods that have been saved in Data Methods folder Figure 7 12 The files in the folder are filtered to include only those with the extension tofpar Look in IS Methods B3 e E3 2 1x_neg_030503 A 3x_pos_030503 IntactProtein_022503 File name 1x pos 030503 Files of type TOF tofpar D Cancel Figure 7 12 Open Dialog Box for Loading a Method 2 If the method is stored in another location use the dialog box tools to browse to the other folder Operator Manual Version 1 0 Volume II Operation 7 15 7 Methods 3 Select the method and click Open The BioTOF system displays a progress gauge as it loads the method Figure 7 13 It can take several minutes for the data to be loaded When finished the method file name is shown in the Current Method box on the left side of the BioTOF window Loading Method Figure 7 13 Progress Gauge as a Method is Being Loaded 7 16 Operator Manual Version 1 0 Vo
104. nalyte solution The needle is then placed inside the E Z Nanospray assembly and a needle shroud Main Nanospray Assembly Body Collet Nanospray Needle u mi a Funnel Needle Shroud Figure 2 5 E Z Nanospray The assembly is inserted into the front of the ESI spray chamber See E Z Nanospray on page 6 16 for information on setup and operation 2 5 APCI Source Operation Atmospheric Pressure Chemical lonization APCI is a combined liquid chromatography and mass spectrometer technique closely related to Electrospray ionization With the APCI Source the ESI spray chamber is removed from the front of the source assembly and replaced with the APCI spray chamber Figure 2 6 See Configuring the APCI Spray Chamber on page 6 21 for information on changing the spray chambers 2 10 Operator Manual Version 1 0 Volume I Theory 2 Source Operation Figure 2 6 Source Configured with APCI Spray Chamber The APCI technique involves passing LC eluent through a nebulizing needle thus creating a fine spray The spray is then passed through a heated ceramic tube where the eluent droplets are fully vaporized The resulting gas vapor mixture is then passed over a corona discharge needle where the solvent vapor and carrier gas are ionized to create reagent gas ions These reagent ions in turn ionize the eluent sample molecules via a chemical ionization process APCI is an LC gas phase chemical ionization mechanism APCI re
105. nated experiments that include BioTOF with the current loaded method 5 20 Operator Manual Version 1 0 Volume Il Operation 5 BioTOF Window 5 7 3 Diagnostics Selections in the Tool gt Diagnostic menu enable you to compare actual system values with the indicators in the BioTOF window To display the parameters set in the ESI Source controller 1 Choose Tools gt Diagnostics gt lonization Source from the menu bar to display the Electrospray lonization Source Setting dialog box Figure 5 14 Electrospray lonization Source Setting Voltages Volts Dry Gas Temp 165 ME L min APCI Neb Temp 8 E L min Corona Needle End Plate Capillary lonization Capillary Exit ESI O nes O APCI Skimmer Polarity Oftset Positive Negative Trap Gas Heater Control Lens Dry Gas v Neb Gas RF Amplitude RF Pass Figure 5 14 Electrospray lonization Source Setting Dialog Box 2 Compare the display with the settings in the ESI Source tab 3 Click Get to refresh the display if the dialog box has been opened 4 Click OK to close the dialog box Operator Manual Version 1 0 Volume II Operation 5 21 5 BioTOF Window 5 7 3 1 Spectrometer The Parameter Settings Spectrometer dialog box Figure 5 15 displays the ion polarity voltage levels and timing parameters in the mass spectrometer as set in the controller enabling you to compare actual system values with information in the Spectromete
106. nce P_ H 3H Bradykinin_ M H Glu Fib B_ M H Glu Fib B H H Figure 9 1 The files are located in the biotof systems list directory 118 686255 195 687653 328 223632 347 167361 508 207828 556 276577 574 232996 578 258521 609 280660 997 397815 1111 545877 1296 685770 658 856023 432 899774 1046 541794 523 774535 349 518782 931 514850 466 261063 311 176468 1619 822343 810 414810 540 612299 1347 735424 674 371350 449 916659 530 787977 1570 676848 785 842026 Sample Reference File 9 2 Operator Manual Version 1 0 Volume Il Operation 9 Calibration 9 2 Calibrating the System To calibrate the BioTOF system 1 Acquire spectra from a sample with known constituents 2 Click the Calibration tab to display the table of reference masses 3 Use the pull down list at the top of the tab to select the reference file for display in the Reference in the tab 4 Select the first known sample used in acquiring the spectra Angiotensin II in Figure 9 2 A red vertical line appears in the Spectra Display at the calculated position of the selected compound on the m z scale and the cursor in the Spectrum Display changes to the Max cursor 17 Int BioT OF Spectrum 54000 Points rr 3X0 30 40 40 S80 oz Instrument Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition C Ze i Standby Operation EE Dese S Fit ed Linear Quadra
107. nt Likewise interrupting the protective conductor inside or outside the instrument or disconnecting the protective earth terminal creates a shock hazard for the operator and can damage the instrument WARNING The instrument must be disconnected from its power source before any cover is removed or the system is opened WARNING All connections of the instrument must be used in correct way The instrument should only be used with the wires and cables delivered with the system or otherwise provided by the manufacturer Noise Declaration 70 dB operator position normal operation per ISO 7779 1988 Instrument Identification Each instrument is identified by a unique serial numbers These serial number is located on a label on the lower left side near the rear of the instrument When corresponding with Bruker Daltonics Inc about your instrument be sure to include the model and the full serial number Write the serial number of the instrument here for reference Model Serial Number Part Numbers In this manual Bruker Daltonics Inc part numbers are generally listed in parentheses after the name of the part A few tools and supplies listed have no part numbers and are not available from Bruker Daltonics Inc Most of these can be obtained from laboratory supply companies Manual part number A5001 BioTOF Series Getting Started Version 1 0 Copyright Bruker Daltonics Inc Support 978 663
108. nt name and the extension ref Note Do not include any spaces in the file name 3 Delete any line that you do not want in the reference table but keep at least one line to use as a template 4 For each new constituent add a line consisting of the name and the reference mass aligning the references masses on the decimal 5 Follow these rules Use the space bar not Tab to insert spaces between the name and the reference mass value Use the underscore character instead of spaces in the compound names Do not press Enter immediately after the last reference mass Operator Manual Version 1 0 Volume Il Operation 9 5 9 Calibration 9 6 Operator Manual Version 1 0 Volume Il Operation 10 Maintenance 10 Maintenance This chapter covers routine maintenance and basic and advanced troubleshooting 10 1 Daily Maintenance Daily cleaning of the ESI Source is necessary to maintain optimum performance and sensitivity for the BioTOF system To clean the ESI Source 1 Click Standby in Instrument group to turn off the high voltage 2 Turn off Dry Gas Heat and allow the system to cool 3 Flush the transfer line from the syringe pump or LC system Generally you can flush the line with the solvent last used in operating the source It is recommended to flush with a volume of at least 1 to 2 mL of solvent Open the spray chamber and wipe it with a methanol soaked tissue 5 Remove the spray shield and wi
109. nterface is located immediately behind the ESI Source wr RF lon Guide in the ESI Source Detector Multideflector Repeller Accelerator Flight Tube Figure 3 2 Orthogonal Interface The packet of ions emerging from the ion guide in the ESI source is collimated by a lens electrode and travels into the orthogonal interface with a velocity related to its kinetic energy acquired by acceleration due to the offset voltage of the ion guide 3 2 Operator Manual Version 1 0 Volume I Theory 3 TOF Mass Spectrometer Operation 3 1 1 Accelerator After the transfer delay the ion packet reaches the extraction region of the accelerator Here a high voltage pulse applied to the Repeller Figure 3 2 changes the trajectory by 90 and pushes the ions into the mass spectrometer flight tube The pulse coincides with the start of a digital timer that measures the time of flight of the packet from the accelerator through the Reflectron and ultimately to the ion detector When the high voltage pulse is applied to the Repeller to accelerate the ions down the flight tube the voltage also prevents other ions from entering the accelerator When the packet of ions has safely entered the field free drift region of the flight tube the high voltage in the accelerator is turned off Figure 3 3 shows the Orthogonal Interface placed on end Note that the accelerator includes a series of gold plated rings separated by resistors that f
110. ntial is held high after the ions enter the flight tube Thus when the ions return from the Reflectron they are reflected back into the flight tube for another round trip After the ions have made the additional round trips in the flight tube the Repeller voltage is dropped and the ions pass through the accelerator and to the detector Figure 3 9 Orthogonal Interface Reflectron Drift Region Accelerator Detector Flight Tube ESI Source Figure 3 9 lon Path in Multipass Mode Multipass effectively creates a much longer instrument Refer to Multipass on page 8 7 for information on setting up multipass 3 12 Operator Manual Version 1 0 Volume I Theory a BRUKER Se BiolOF Series Operator Manual Volume Il Operation BioTOF BRUKER DALT ONICO Copyright Copyright 2003 Safety Information WARNING Part Numbers Bruker Daltonics Inc Safety Class All connections of the In this manual All Rights Reserved instrument must be used in Bruker Daltonics Inc The instrument is a Safety correct way The instrument part numbers are generally Reproduction adaptation Class I instrument and has should only be used with listed in parentheses after or translation without prior been designed and tested the wires and cables the name of the part written permission is in accordance with IEC delivered with the system prohibited except as Publication 1010 or otherwise provided by A few tools and supplies al
111. nual Version 1 0 Volume II Operation 5 15 5 BioTOF Window Table 5 4 Icon Function Display Crosses About TOF Control BioTOF Tool Bar Icons Continued Description Toggles between the use of crosses and dots to display data points Use the crosses to make actual data points more obvious Displays version and copyright information for the S BioTOF Control software Context Sensitive Help Launches BioTOF Help m Default Size Hides the Parameter tabs so that more of the Spectra Automatic Mode Display is visible Drag the bottom edge of the Spectra Display window up to bring the tabs into view Default Size Includes the Parameter tabs in the default BioTOF Advanced Mode display Drag the bottom edge of the Spectra Display window down to hide the tabs Start HyStar Launches the HyStar Software for Chromatography and i Hyphenated Experiments application placing the BioTOF system under the control of the HyStar application until you exit that application 5 16 Operator Manual Version 1 0 Volume II Operation 5 BioTOF Window 5 5 2 Status Bar The Status Bar displays the following information A short description of the currently selected function or field Any Spectra Display options such as Max Cursor that have been selected from the tool bar The X and Y coordinates of the cursor in the Spectra Display window To toggle the display of the status bar at the bottom of the
112. on Gas Flow T emperature ee Dry Gas Temp Et E E c ESI C nESI C APCI High Voltage Electrodes Volts Lens Volts Polarity Cylinder 2000 Capillary Exit 140 Trap 45 2 Positive Negative Skimmerl 40 lene 50 Gas Heater Control v Dry Gas End Plate 3500 Skimmer2 303 pr Amplitude 600 5 v Neb Gas Capillary 4000 Offset 153 iv DG Heat Figure 3 6 Gas Heater Group and ESI Source Tab 3 6 Getting Started Version 1 0 3 Quick Start 4 Atthe Source Control panel set the nebulizer gas pressure to approximately 30 psi 5 Set the drying gas flow so that the ball is at about 1 3 scale see Figure 3 7 ESI APCI CONTROL UNIT Nebulizer Gas Flow Meter Nebulizer Gas Control Knob Drying Gas Flow Meter Drying Gas Flow Control Knob Figure 3 7 Nebulizer and Drying Gas Controls 3 5 Prepare the Sample Samples of the following compounds are well suited for electrospray ionization Leucine Enkephalin 556 Reserpine 609 Angiotensin 1046 523 e Angiotensin Il 1296 648 Argenine Glycine Aspartic Acid 347 Leucine Enkephaline and Reserpine produce singly charged molecular ions MH The larger peptides generate doubly charged ions M 2H and triply charged M 3H ions A typical ESI solvent for these peptides is a 1 1 mixture of methanol water with 0 1 to 1 acetic acid Getting Started Version 1 0 3 7 3Q uick Start To prepare a standard ESI sample 1 2
113. or basic troubleshooting of the ESI Source The flowchart is meant for new users of the source and provides a simple step wise guide based on observations of key parameters Source Control LCD Move the cursor to End or Cap No Signal push the syringe and watch the spray current Spray current OK No Spray current but no signal Check if the HV Indicator is ON Check sample concentration Green LED on Source Control and solvent conditions Turn on the HV and push the Voltage On No Check drying gas and gas needle button Yes Check the spray voltage Check lens voltages and instrument settings Check RF box and connections Set the voltages to Too low typical values He if A Check the sample flow Check pressures No liquid flow f through sprayer Clogged or Leaking g spray Clean capillary entrance Check the transfer line and spray needle Still no current Call Service Figure 10 4 ESI Source Troubleshooting Flowchart Operator Manual Version 1 0 Volume Il Operation 10 11 10 Maintenance 10 5 Advanced Troubleshooting A more extensive guide to troubleshooting is presented in Table 10 1 Diagnostics and solutions to may of the problems depicted in these tables may require electrical tests or cleaning of the source Table 10 1 Symptom Unstable signal and current e Poor sensitivity or weak signal Diagnostic Unstable signal Unstable current Liquid droplet on the En
114. orm an homogenous field The voltages decrease evenly from the Repeller to the grid Multideflector Accelerator Repeller Dual Microchannel Plates Figure 3 3 Orthogonal Interface On End Operator Manual Version 1 0 Volume I Theory 3 3 3 TOF Mass Spectrometer Operation 3 1 2 Multideflector Before the ions enter the flight tube they pass through the multideflector which compensates for the initial orthogonal velocity of the ions and aligns the ion trajectories along the axis of the flight tube The assembly consists of a series of gates placed parallel to the intended ion flight path Figure 3 4 Multiple deflectors are used to minimize distortion Figure 3 4 illustrates the relative time difference for ions of the same m z traveling through the narrow multideflector gates compared with ions traveling through a wider single gate EL uu LLL A AAA AAA A AA AAA AAA AAA AAA t Distortion At without Multideflector E CL ES EA i Ween EE y E m A Ee gt Distortion At with Multideflector Figure 3 4 Minimized Distortion with the Multideflector 3 4 Operator Manual Version 1 0 Volume I Theory 3 TOF Mass Spectrometer Operation The multideflector uses two steering voltages Multideflector High A pulsed voltage applied across the gates steering ions as they leave the accelerator Mul
115. oss Display Da RO Show MS Spectrum Type Axes EB Annotate Masses Background Color Show m_Time Show m_TIC Show WinTips Iw Show Peak Width Title Mode v v Separate Legend Digits 0 SplitMode 0 C1 C2 C3 4 Cancel Figure 5 9 Properties Profile Display Dialog Box Cross Display Show MS Spectrum Type Annotate Masses Toggles between the use of crosses and dots to display data points Use the crosses to make actual data point more obvious Replaces the default legend Profile Spectrum Window Creates a legend showing the number of points in the mass spectrum 5 10 Operator Manual Version 1 0 Volume Il Operation 5 BioTOF Window Table 5 3 Spectrum Display Window Pop Up Menu Items Function Description Show m Time Creates a legend showing the time of the last spectra acquisition Show m TIC Creates a legend showing the total ion count Show WinTips Enables tooltips in which the value of data is displayed when you place the cursor over a data point in the display Title Mode Toggles the display of a title bar above the Spetra Display window Separate Legend Creates a separate bar above the Spectra display window for legends that would otherwise appear in the upper left corner of the window Digits Sets the number of digits to the right of the description in the display of data points in the Spectra Display window Split Mode Creates a separate legend above the MS pan
116. oubleshooting unexpected results it may be useful to turn off the pulser so that there are no ions travelling through the tube and reflected back to the detector The digitizer continues to send data to the BioTOF window allowing you to identify data points that are not a result of detecting reflected ions Operator Manual Version 1 0 Volume II Operation 7 11 7 Methods 7 8 Acquisition Tab The settings on the Acquisition tab Figure 7 10 determine display and data recording resolution Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Acquisition Window Advanced Digitizer Setting m from UH to HN Spectrum Offset 0 0350 V End of Scan Deadtime 05440 us Spectrum Size 50000 pts 20 KB Deer Hee SB Advanced Data Handing Sample Rate Digitizer Summing 20 Spectra 05 E 2 6 Mass Range Resolution Figure 7 10 Acquisition Tab Acquisition Window The m z range is set on the Mode tab Figure 7 3 on page 7 3 The m z range and the Spectrum Size set on this tab determine total size of a the spectra file The Spectrum Size is set in the number of data points per record The maximum number of data points per record is 252 000 The number of points determines the disk space required for a single spectrum as shown in kilobytes in the field to the right The Digitizer Delay is specified in us from the start of each data acquisition cycle until the digitizer begins recor
117. pe it clean with a methanol soaked tissue 6 Wipe the capillary entrance with a methanol soaked tissue and reinstall the spray shield 7 If the project has included high flow rate applications empty the high flow rate bucket behind the system and follow approved procedures for disposing the LC solvents Operator Manual Version 1 0 Volume II Operation 10 1 10 Maintenance 10 2 Monthly Maintenance This section describes maintenance that should be performed on a monthly basis You may adjust the frequency of this routine maintenance depending on usage and sample concentrations Monthly maintenance of the BioTOF system includes The cleaning specified in daily maintenance Checking the oil level in the vacuum pump Cleaning the Capillary and Skimmer 1 in the ESI Source The last task required that you shut down the vacuum pumps 10 2 1 Stopping the Vacuum System The BioTof system is normally left running in Standby mode when it is not in use so that the ESI Source and TOF Spectrometer pressure can be maintained below thresholds To shut down the vacuum system 1 At the control station stop data acquisition and bring the BioTOF system into Standby mode if you have not already done so Standby Mode turns off the ESI source and spectrometer voltages 2 Check the Enable light on the High Voltage unit and the light in the ESI High Voltage switch to ensure that the voltages are in fact off Use the manual switches on
118. pm Valine M H 118 086255 Co 482 6948 Co 482 6948 Caffeine M H 195 087653 E 120426 9651 E 224269651 Val Pro Leu M H 328 223632 a a 1 md AS M H 347 167361 Ca 0 0000 Ca 0 0000 ti Tprosine M H 508 207828 Leucine Enkephalin M 556 276577 556 2100 119 68 m 0 Methionine Enkephalin 574 232996 3 SBS 2 gt a Accept F lt X Clear Figure 7 6 Calibration Tab Operator Manual Version 1 0 Volume II Operation 7 7 7 Methods 7 5 ESI Source Tab The ESI Source tab Figure 7 7 includes groups for setting gas flow gas temperature and voltages applied to various ESI Source components The active settings on the tab depend on the button selected in the lonization group see Figure 7 2 on page 7 2 Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Gas Flow T emperature Dry Gas Temp Er 9 E 3 High Voltage Electrodes Volts Lens Volts Cylinder 2000 Capillary Exit 140 Trap s my Skimmerl 40 4 Lens s EndPlate 304 Skmma2 3031 Amplitude 602 Capil 15 lt apillary 4000 Offset Refresh Figure 7 7 ESI Source Tab Gas Flow Temperature When the Instrumentation is either ESI or nESI Dry Gas Temp is the only active field in the Gas Flow Temperature group For ESI and nESI Dry Gas Temp is typically set between 200 C and 220 C When the Instrumentation is APCI Dry Gas
119. put to the Ready switch in the external device however defined in the other system The external switches are configured and enabled from the Mode tab See Mode Tab on page 7 3 Operator Manual Version 1 0 Volume Il Operation 5 23 5 BioTOF Window 5 7 3 2 Digitizer To compare actual values in the digitizer with settings in the Acquisition tab in the BioTOF window 1 Choose Tools gt Diagnostics gt Digitizer from the menu bar to display the Parameter Settings Digitizer dialog box Figure 5 16 Parameter Setting Digitizer Acquisition Advanced Setting m z from Digitizer summing 40 Spectra to ES Spectrum Size Sensitivity mv Data Size Offset my Sampling Rate O Compression O Realtime Smooth End of Scan Deadtime 1 O Enhancer O Auto noise threshold Digitier Delay 29 000 Figure 5 16 Parameter Settings Digitizer Dialog Box 2 Make changes by typing directly in the value fields and clicking the option checkboxes 3 Click OK or in the upper right corner to close the dialog box 5 7 4 Show Servers The Show Servers selection in the Tools menu provides access to the messages generated by the various servers that make up the BioTOF application Bruker TADS Server IC1 Bruker ESI Controller ESI Con Bruker TREC Server IC1 To view server message traffic 1 Choose Tools gt Diagnostics gt Show Servers in the menu bar so that the option is checked The Windows task bar displays a
120. quires the analyte in the gas phase for proper ionization To bring the mobile phase and analyte into the gas phase APCI is typically done at vaporizer temperatures of 400 C as set on the ESI Source tab The vaporizer temperature must be carefully controlled Most compounds work best at higher temperatures while a few compounds work best at lower temperatures It may be necessary to evaluate a couple of temperatures to determine the optimal APCI vaporizer temperature Operator Manual Version 1 0 Volume I Theory 2 11 2 Source Operation 2 12 Operator Manual Version 1 0 Volume I Theory 3 TOF Mass Spectrometer Operation 3 TOF Mass Spectrometer Operation This chapter describes operation of the Time of Flight TOF Mass Spectrometer The major components of the spectrometer Figure 3 1 are Orthogonal Interface Flight Tube Drift Region and Reflectron Detector Digitizer Orthogonal Interface Accelerator Reflectron Drift Region See Figure 3 2 AL Detector Flight Tube ESI Source Figure 3 1 BioTOF Components The spectrometer directs ions on a round trip through a flight tube measures their time of flight and provides the digitized mass spectra data to the BioTOF software Operator Manual Version 1 0 Volume I Theory 3 1 3 TOF Mass Spectrometer Operation 3 1 Orthogonal Interface Figure 3 2 shows the orthogonal interface with the accelerator multideflector and detector The i
121. r Make sure that the sprayer assembly is connected to the nebulizer gas supply and turn the gas valve counterclockwise until it is fully open Close the spray chamber and secure the latch on the left side to engage the source voltage interlock Operator Manual Version 1 0 Volume II Operation 4 5 4 System Startup 4 3 Start the BioTOF Software To start the BioTOF control software 1 Turn the workstation on 2 Do one of the following when the system is initialized Double click the BioTOF desktop or tool bar icon cd Choose Start gt All Programs gt BIOTOF gt BioTOF Refer to Chapter 5 BioTOF Window for a complete description of the user interface 4 4 Turn the Gases On The next step is to set the nebulizer and drying gas controls To turn the gases on 1 Click the Dry Gas and Neb Gas checkboxes in the Gas Heater Control group Figure 4 7 if they are not already on 2 Click the DG Heat checkbox in the Gas Heater Control group to turn the heater on 3 Click the ESI Source tab and set the Dry Gas Temperature to 200 C Instrument Standby Operation Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Gas Flow T emperature MS Dry Gas Temp En E E f ESI C nESI C APCI High Voltage Electrodes Volts Lens Volts Polarity Cylinder 2000 Capillary Exit 140 Trap 45 Positive Negative Skimmerl 40 les 50 gt Gas Heater Control
122. r and Timing Sequence tabs in the BiotOF window 1 Choose Tools gt Diagnostics gt Spectrometer from the menu bar to display the Parameter Settings Spectrometer dialog box 2 Click Get to refresh the display if the dialog box has been opened 3 Click OK to close the dialog box Parameter Setting Spectrometer Polarity Timing 9 Positive O Negative Cycle Period 100 Gate Voltages Volts Repeller Transfer Repeller Reflectron Deflector off Delay 20 Reflectron Lens Aux Trigger Post Accelation Synchronization 5 Detector External Switches Multi Defl High Neg 0 External In 1 D Extemal Out 1 Multi Defl High Pos 0 External In 2 D Extemal Out 2 Figure 5 15 Parameter Settings Spectrometer Dialog Box 5 22 Operator Manual Version 1 0 Volume II Operation 5 BioTOF Window External Switches The BioTOF system supports two external devices The External Switches group includes two data fields that show the current value of the inputs and two buttons for triggering outputs External In 1 Input from the external device switches the BioTOF control software to transition from Standby to Operation This is equivalent to clicking the Operation option in the Instrument group External In2 Input from the external starts spectra acquisition This is equivalent to clicking the Start button External Out 1 Output to the Ready switch in the external device however defined in the other system External Out 2 Out
123. r particles until in the final stage de solvated sample ions emerge The electric field inside the spray chamber is shaped by potentials applied to three electrodes the cylinder the end plate and the metal coated entry of the glass capillary tube De solvation is facilitated by a heated drying gas flowing counter to the stream of droplets and ions as they are electrostatically attracted towards the capillary tube that is the inlet orifice of the vacuum system 1 2 Getting Started Version 1 0 1 BioTOF Operation lons enter the vacuum system of the mass spectrometer through the dielectric glass capillary stream through a skimmer and then enter into the RF ion guide which is biased at an appropriate offset voltage Neutral gas molecules that enter with the sample ions are pumped away in differential pumping stages at successively lower pressures lons accumulate in the RF ion guide by means of a trapping voltage applied to the guide s exit gate electrode Then the voltage on the gate electrode drops for a specified time and ions drift out of the RF ion guide Thus the ions form packets of a spatial length determined by the pulse width and velocity distribution At the end of the gate pulse the exit electrode potential is raised starting another cycle of ion accumulation in the RF ion guide Determination of the pulse width and repetition rate is explained in the next section 1 3 Time of Flight Mass Spectrometer The packe
124. range in which case the BioTOF software automatically determines digitizer delay in keeping with the other timing parameters Specifying the sampling speed and the number of data points per record The recording window is given by the time per data point multiplied by the number of points to be accumulated in each record In this case you must set the appropriate digitizer delay to assure that the mass range includes ion masses of interest 3 8 Operator Manual Version 1 0 Volume I Theory 3 TOF Mass Spectrometer Operation 3 5 Time of Flight Calculation The time of flight calculation is derived as follows All ions are accelerated by the same electrical field in the spectrometer When entering the drift region the ions have a kinetic energy equal to the electrical potential applied to the active section of the accelerator S in Figure 3 7 Accelerator Drift Region Reflection Detector So Ro gt X 0 Figure 3 7 Reference Trajectory Ekin Eg ZeUg where Ekin is kinetic energy Eq is electrical energy z is the number of charges charge state e is the elementary charge Up is the electrical potential difference E 1 2m vo where m is mass vo is drift velocity Operator Manual Version 1 0 Volume l Theory 3 9 3 TOF Mass Spectrometer Operation Therefore Zell ES SB z Time of Flight L To m o where L is the effective length of the spectrometer
125. rapid flow of gas and is dispersed into small droplets Spray Tip A A gt Nebulizing Gas Sample Liquid NebulizingGas Spray Needle v The nebulizing gas pressure is controlled through the software and with a control knob on the ESI Source Control front panel The sprayer assembly including the spray needle is electrically grounded Nebulizer Gas Assists in creating an even and steady dispersion of the sample as it is injected into the spray chamber The nebulizing gas shear forces and the strong electrostatic field at the tip of the sprayer to break the sample solution into droplets Since the sample solution is not heated when the aerosol is created ESI ionization does not thermally decompose most analytes In some low flow rate applications particularly Nanospray the nebulizer is not used as the electrical fields provide sufficient dispersion of the liquid Note The nebulizer gas pressure is set at the Source Control panel If a nebulizing gas is in use ensure that Neb Gas is checked in the Gas Heater Control Group to the left of the tabs Operator Manual Version 1 0 Volume I Theory 2 5 2 Source Operation Table 2 1 Component ESI Source Operation by Component Continued Function Cylinder The first of three electrodes that create an electrical field in the spray chamber Endplate The second of the three spray chamber electrodes A polished m
126. ration 6 19 6 Introducing Samples 6 4 3 Nanospray Settings For Nanospray only the capillary element is raised to a high voltage The magnitude of the voltage for Nanospray is lower than that needed for Electrospray applications For best sensitivity operate at the lowest possible voltage needed to maintain a stable Electrospray signal To set the chamber voltages 1 Use the arrow buttons below the Source Control Panel LCD display to position the display cursor on Cap Figure 6 15 CON edit active Remote Menu 0 D 600 Cyl End cB Capillary Electrode Figure 6 15 Select Cap for the Source Lens Current Reading 2 Setthe capillary voltage to 600 V 3 With the mass spectrometer in operating mode slowly increase the capillary voltage in 50 V increments until the current is registered on the capillary At this point the voltage applied to the capillary affects the flow rate through the needle Caution As the magnitude of the high voltages for negative ESI tends to be lower than those optimal for positive ion mode caution should be used when adjusting voltages to avoid arcing Arcing can destroy the coating of the needle tip 6 20 Operator Manual Version 1 0 Volume Il Operation 6 Introducing Samples 6 5 Configuring the APCI Spray Chamber This section describes how to convert the ESI Source assembly to use the APCI Source Figure 6 16 Figure 6 16 Source Configured with the APCI Source Note
127. red in the current session Clears records from the buffer and display resets the Spectrum Counter Operator Manual Version 1 0 Volume Il Operation 5 5 5 BioTOF Window 5 1 3 System Control Groups Figure 5 5 describes the option buttons in the four system control groups to the left of the parameter tabs Instrument C Standby Operation lonization ESI nESI APCI Polarity Positive C Negative Gas Heater Control V Dry Gas iv Neb Gas iw DG Heat Figure 5 5 System Control Groups Table 5 2 System Control Group Option Descriptions Function Description Instrument Sets the instrument mode to Standby HV off but otherwise ready to acquire data or Operation ready to start spectra acquisition lonization Identifies the source as electrospray ionization ESI nanospray nESI or atmospheric pressure chemical ionization APCI Polarity Selects positive or negative ion polarity The various high voltages in the system are automatically set to the appropriate polarity The Polarity switch on the Instrument HV panel must be in the neutral position to enable control from the BioTOF window Gas Heater Control Toggles dry gas and nebulizer gas flow and dry gas heating Flow rates are set on the Source Control Unit Dry gas temperature is set with the ESI Source tab in the BioTOF window 5 6 Operator Manual Version 1 0 Volume Il Operation 5 BioTOF Window 5 1 4 Spectra Display Window
128. rein or for incidental or damage the instrument Write the serial number consequential damages of the instrument here for in connection with the WARNING reference furnishing performance The instrument must be or use of this material disconnected from its power Model Source before any cover is removed or the system is opened Bruker Daltonics Inc Serial Number assumes no responsibility for the use or reliability of its software on equipment that is not furnished by Bruker Daltonics Inc BioTOF Series Operator Manual Version 1 0 Volume Il Operation Copyright Bruker Daltonics Inc Support 978 663 3660 Ext 1445 Manning Park FAX 978 667 5993 Billerica MA 01821 U S A Email support bdal com Internet www bruker daltonics com ii Operator Manual Version 1 0 Volume II Operation Contents GETTING STARTED GOPYRIGHT etes litio ll PHEFADE 5 deae os ae a deor VII SAFETY llblqu Vill 1 BIOTOP OPERATION 1 1 1 1 EEN ee 1 2 1 2 Eege E ee E E ES 1 2 1 3 Time of Flight Mass Gpechomeier EEN 1 3 1 4 Dat ACQUISITION EE 1 4 2 SYSTEM CONTROLS ERU ee 2 1 2 1 EIS TO aTe ON Soa cae cedida 2 1 2 2 Eront E eine Panels een 2 4 231 System Status Indica 2 5 3 X QUIGK START EE 3 1 3 1 Start the SySt EE 3 2 3 2 Check the Spray Chamber and Gpraver AAA 3 5 3 3 Start the BioTOF ein 3 6 3 4 Ji d INS Gases WEE 3 6 3 5 Prepare the Sample ege geed deer eebe de
129. rmation on using the analytical software included with the BioTOF system DataAnalysis User Manual QuantAnalysis User Manual Library Editor User Manual For technical assistance contact Bruker Daltonics customer service In the U S 1 978 663 3660 Ext 1445 In Germany 49 0 421 2205 430 In Japan 81 0 45 440 0471 Local Service Getting Started Version 1 0 vii Safety Labels These labels are placed on the instrument to indicate the respective hazard possibility SEPP Hazard Note Refer to the Operator Manual for details on this hazard Warning High Voltage A dangerous high voltage or shock hazard exists within this area Only trained service persons should perform work in this area Warning High Temperature Hot parts and surfaces are present in this area Allow the part or surface to cool before performing work in this area This may require turning off power to selected components or to the entire instrument Warning Biohazard A biohazard may exist within this instrument or specific area Handle this biohazard with appropriate caution and protection viii Getting Started Version 1 0 1 BioTOF Operation 1 BioTOF Operation This chapter provides an overview of BioTOF system operation For a more detailed description refer to the BioTOF Operator Manual Volume I Theory The BioTOF system Figure 1 1 consists of Time of flight mass spectrometer Electrospray ion ESI source
130. rward in the Nanospray tip 3 Place the filled needle in the fitting and place the fitting in the XYZ stage Use the stage adjustments and the assembly optics to position the needle on axis with the capillary with the tip about 2 0 mm from the opening in the capillary cap 5 Set the Capillary voltage in the ESI Source tab to 600 V 6 Place the system in Operation 7 Increase the Capillary voltage in 50 V increments until the current is visible in the Source Lens Current indicator Operator Manual Version 1 0 Volume Il Operation 6 15 6 Introducing Samples 6 4 E Z Nanospray The E Z Nanospray lon Source allows you to set up an off line Nanospray application without removing the Electrospray spray chamber An E Z Nanospray assembly holding a disposable needle with the analyte solution is inserted on axis into the front of the spray chamber door Figure 6 10 E Z Nanospray Assembly Figure 6 10 E Z Nanospray Assembly Installed in the Spray Chamber Door 6 16 Operator Manual Version 1 0 Volume II Operation 6 Introducing Samples 6 4 1 Preparing the Nanospray Needle Pre opened Nanospray needles such as 2 micron inner diameter or 4 micron inner diameter New Objective Tips are suggested when using the E Z Nanospray assembly To prepare a Nanospray needle for use 1 Use a micro pipetter fitted with a gel loader tip to deliver 1 to 3 uL of analyte solution through the larger diameter end of the tip into
131. s system electronics and additional electrical outlets on the power supply labeled Computer These pushbutton switches light up when they are engaged The Vacuum Gauge Controller on the top right includes an LCD display of the vacuum pump gauges Use the numbered membrane buttons on the right to select the vacuum gauges Gauges 1 and 3 are used in the basic BioTOF configuration Below the selection buttons is an array of buttons for selecting units of measure and pressure thresholds The Source Control Unit is used to set power for the ESI source and to control drying and nebulizing gas flows The unit is powered up when the System Electronics are turned on from the Main Control Panel The unit also has its own power switch in the lower right corner of the unit panel The Instrument HV Panel includes two 3 position toggles one for enabling the supply of high voltage power to the BioTOF electrodes the other for setting the ion polarity When a toggle is centered the HV or polarity is controlled from the BioTOF software Main Control Vacuum Gauge Panel Controller Source Control Unit ESI Power Switch Source HV Switch Instrument HV Panel Figure 2 3 2 4 Getting Started Version 1 0 2 System Controls 2 3 System Status Indicators Figure 2 4 shows the set of indicators located on the front of the cabinet above the ESI module These include a source lens current gauge and indicators for ion polarity high voltage vac
132. sembly in the XYZ stage Note Be careful not to bump the needle against the capillary cap 3 Use the stage adjustments and the assembly optics to position the needle on axis with the capillary with the tip about 2 0 mm from the opening in the capillary cap Connect the transfer line to the nanoLC system or a syringe Set the Capillary voltage to 600 V in the ESI Source tab Start the nanoLC system or syringe pump and place the system in operation Increase the Capillary voltage in 50 V increments until the current is visible in the Source Lens Current indicator Adjust the XYZ controls on the Nanospray assembly to optimize the spray 9 In the BioTOF window check DG Heat in the Gas Heater Control group and set the Dry Gas Temp in the ESI Source tab to 50 C 10 At the Source Control panel set the dry gas flow to one quarter of the scale 11 Adjust dry gas flow and temperature to optimize for solvent conditions ot 9 E ge 6 14 Operator Manual Version 1 0 Volume II Operation 6 Introducing Samples 6 3 3 Off Line Nanospray For Off Line Nanospray applications use Pre opened Nanospray needles such as 2 micron inner diameter or 4 micron inner diameter New Objective Tips For off line Nanospray 1 Use a micro pipetter fitted with a gel loader tip to deliver 1 to 3 uL of analyte solution through the larger diameter end of the tip into the Nanospray needle 2 Shake down the assembly to allow the liquid to flow fo
133. spectrum and recalibrate the instrument two passes Save spectrum and save the method with new parameters and new calibration Proceed to setting up a three pass method accordingly Operator Manual Version 1 0 Volume II Operation 8 9 8 Acquiring Data 8 3 3 Tips on Using Multipass Tuning Experience shows that the sensitivity of the Reflectron voltage tuning increases with the number of passes as well as the importance of fine steering of the ion beam provided by Multideflector Low voltage Eliminating un wanted ions from the mass spectrum In a complex sample ions of m z higher or lower than the accepted mass range may appear in the spectrum after the wrong number of passes Those ions are typically not in focus but may be of significant intensity particularly if their number of passes is lower that is they are higher m z ions than specified by the multipass method To eliminate higher m z ions Switch to the Timing Sequence tab and set Deflector off Delay to a low value such as 2 or 3 ps This setting turns off the steering voltage before high m z ions have passed through the multideflector into the flight tube The trajectories of those ions are not be corrected for the orthogonal drift and the ions are effectively eliminated from the spectrum Try this approach in single pass mode first and observe the affect on the mass range transmission To eliminate lower m z ions Switch to the Timing Sequence tab an
134. spheric pressure to the vacuum region of the mass spectrometer A barrier separating the atmospheric pressure region of the source from the vacuum region Made of a dielectric material that electrically insulates the spray chamber from the vacuum side Drying Gas A heated gas typically nitrogen or dry air flowing counter current to the spray of charged droplets that evaporates the solvent thus decreasing the droplet diameter and forcing the charges closer together While the drying gas assists in the de solvation process it does not thermally decompose the analytes If a drying gas is in use ensure that Dry Gas is checked in the Gas Heater Control Group to the left of the tabs The flow rate is set on the Source Control panel and the temperature is set from the ESI Source tab in the BioTOF window Capillary Exit The first of the vacuum side voltages The metallized exit end of the capillary is typically set in a range from 80 V to 180 V through the Capillary Exit voltage field on the ESI Source tab As ions exit the capillary they are electrostatically drawn towards the first skimmer which is at a lower voltage Due to the dimensions of the capillary and the pressure gradient the ions emerge from the end of the capillary embedded in a jet flow of neutral gas High capillary exit voltage can facilitate ion fragmentation Operator Manual Version 1 0 Volume Theory 2 7 2 Source Operation Table 2 1 ESI Source Operation
135. ssure is below threshold Power Indicates that system power is on Source Lens Measures current flow at one of the three spray chamber Current electrodes The electrode is selected with the Source Control unit 1 4 Operator Manual Version 1 0 Volume I Theory 1 System Components 1 2 Computer Workstation The principal means for controlling and monitoring the BioTOF system is a Windows XP based application configured on the workstation From a single multi pane window you can start the BioTOF system set system parameters acquire and display spectra and save the data for use with the DataAnalysis processing software included with the system and other optional software tools An array of hardware controls and indicators are configured on the front of the BioTOF cabinet These include On Off switches for the mass spectrometer and ESI source components Many of these controls are replicated in the BioTOF software and can be set from the BioTOF window 1 3 BiolOF Software This section gives a brief description of the main window for the BioTOF application Refer to Chapter 5 in the BioTOF Operator Manual Volume Il Operation for a detailed description of the window features including the menus tool bars and options The BioTOF window appears Figure 1 4 when you start the application Once you have started the application you can leave it running minimizing the window when you are not using the BioTOF system EB 2 07 0F
136. t of ions emerging from the ion guide is collimated by a lens electrode and travels towards the accelerator with a velocity related to its kinetic energy acquired by acceleration due to the offset voltage of the ion guide After the transfer delay the ion packet reaches the extraction region of the accelerator Here a high voltage pulse on the Repeller changes the trajectory by 90 and pushes the ions into the mass spectrometer flight tube The pulse initiates a clock that measures the time of flight of the packet from the accelerator to the Reflectron and ultimately to the ion detector When the high voltage pulse is applied to the Repeller to accelerate the ions down the flight tube the voltage also prevents other ions entering the accelerator When the first packet of ions has safely entered the field free drift region of the flight tube the high voltage in the accelerator is turned off Grid Repeller Detector lon Paths Multideflector Figure 1 3 Accelerator Getting Started Version 1 0 1 3 1 BioTOF Operation Since all ions are accelerated by the same Repeller potential in the accelerator they reach individual velocities according to their m z values All ions of a given m z ratio take approximately the same amount of time to travel through the flight tube The smaller m z ions travel faster than those with higher m z values the ion packet begins to separate The ions drift through the flight spa
137. tall the Nanospray needle for On Line operation 1 Use a union to connect the Nanospray needle and the transfer line as shown in Figure 6 8 and either of the two needle configurations in Figure 6 9 Fingertight Fitting Fused Silica Capillary Upchurch Scientific LC Packings Peek ZDV Union F 120 Large Bore Fitting Flangeless Ferrule o 280 um OD Upchurch Scientific Upchurch Scientific Upchurch Scientific 0 010 P 742 F 294X P 200Nx Eni ES Syringe Fillport 1 16 Silica SealTight Sleeve Valco 357VISF1 Upchurch Scientific Silica SealTight Sleeve 380 Um green F 242x Upchurch Scientific 280 Um blue F 240x Figure 6 8 Installing the Needle for On Line Nanospray Operator Manual Version 1 0 Volume II Operation 6 13 6 Introducing Samples Teflon Tubing LC Packings TF 250 350 EE a SealTight Ferrule Uphurch Scientific F c192x Fused Silica Capillary LC Packings 75 Um ID 280 Um OD FS 75 New Objective FS360 50 15 D Fused Silica Pico Tip Stainless Steel ZDV Union Upchurch Scientific 0 007 U 411 0 010 U 435 Silica SealTight Sleeve Upchurch Scientific 280 Um blue F 240x Fused Silica Capillary Fused Silica Pico Tip LC Packings New Objective 50 Um ID 280 Um OD FS360 50 15 D FS 50 Silica SealTight Ferrule Upchurch Scientific F 192x Figure 6 9 Typical Needle Configurations 2 Place the as
138. the Electrospray Direct low flow rate is capable of higher sensitivity as there is no dilution Low flow is often associated with small sample volumes In the high flow configuration the actual delivered flow rate is determined by the LC pump The flow from the syringe pump is tee d into the flow from the LC by means of a low dead volume fitting inserted into the flow path directly before the Electrospray union The addition of solvent from the LC system can reduce certain buffer effects The increased flow rate also makes it possible to optimally use infusion for APCI work where higher flow rates are optimum If the target application for a particular analysis is found in LC analysis then tee ing into this flow with a representative standard can be useful during method development while optimizing instrument conditions One of the most common uses of direct infusion is to provide continuous introduction of sample while optimizing the mass spectrometer for a particular analysis The most general case is the direct infusion of a standard tuning solution These solutions can be introduced either in the low flow configuration or in conjunction with the LC pump depending on whether the target analysis will occur with the LC Operator Manual Version 1 0 Volume Il Operation 6 1 6 Introducing Samples 6 1 1 Syringe Pump The syringe pump Figure 6 1 can be used to drive glass syringes from 5 uL to 5 mL The BioTOF system includes a 100 u
139. the Nanospray needle Figure 6 11 Main Nanospray Assembly Body Collet Nanospray Needle NE A m E Funnel Needle Shroud Figure 6 11 Preparing a Nanospray Needle 2 Slide the rear of the needle into the entrance of the collet and mount the collet onto the needle shroud Figure 6 11 3 Shake down the assembly to allow the liquid to flow forward in the Nanospray tip Figure 6 12 In Figure 6 12 Nanospray Assembly Operator Manual Version 1 0 Volume II Operation 6 17 6 Introducing Samples 6 4 2 E Z Nanospray Installation To insert the E Z Nanospray Assembly in the ESI Source 1 Open the spray chamber door remove the spray shield from the endplate so that the Capillary is visible and close the spray chamber door Figure 6 13 Capillary Endplate with Spray Shield Removed Figure 6 13 ESI Source with Spray Shield Removed 6 18 Operator Manual Version 1 0 Volume II Operation 6 Introducing Samples 2 Remove the louvered plastic cover and the glass cover from the port on the front of the spray chamber Figure 6 14 Figure 6 14 Port Covers Removed for E Z Nanospray Note Do not insert the E Z Nanospray assembly unless the spray chamber door is closed and the latch on the left side is fully engaged 3 Insert the E Z Nanospray assembly into the port and push the assembly until it can go no further into the spray chamber Operator Manual Version 1 0 Volume II Ope
140. ther RF amplitude is Defective capacitor Call for service above a certain value None of the above Defective RF driver Replace the RF driver Error 122 Check heater and Loose cable Tighten the cable No heat from TC cable ang gas Check the resistance of the heater Should be 6 E0 5 Check the Blown fuse Replace the fuse heater fuse f the Temperature Heater has Lower the setting setting is over insufficient power temperature or apply 300 C an extra 24 V 100 W power supply on the second heater Operator Manual Version 1 0 Volume II Operation 10 13 10 Maintenance Table 10 2 provides the test points and values for all high voltage and low voltage elements to the source When performing an electrical test use the software to set the voltage of the element to a typical value listed in Table 10 2 The voltage interlock to the source must be Active The Active state indicates that the high voltage button to the API 100 1600 is On and the spray chamber door is close Table 10 2 Electrode Test Points and Typical Values Pin to Element Electrode Pin and Connector Resistance Capillary Pin 3 of HV Connector lt 5 Ohms Endcap Pin 2 of HV Connector 5 Ohms Capillary Exit Pin 1 of lens Connector lt 5 Ohms Skimmer 1 Pin 6 of lens Connector lt 5 Ohms Skimmer 2 Pin 2 of lens Connector lt 5 Ohms Trap Extract BNC SMA lt 5 Ohms Lens BioTOF only Pin 4of lens Connector lt 5 Ohms Typical
141. thod 100 D ee SN TR ETT ig m c Cep gib Start EINE Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition E IN G i MOM HL 5 Stary Operation Acquisition Window Advanced Digitizer Setting Lose Bu m z from 147 to 874 lonization Manual Stop ESI C nes Spectrum Size pts 200 KB PreSet Spectra Spectrum Offset 0 0350 Y End of Scan Deadtime 0 5440 Polarity Digitizer Delay us soz Ze Positive C Negat E N Sample Rate Digitizer Summing 20 Spectra Gas Heater Control ol ul El GS s VW Dry Gas RH x Clear pes Range IV Neb Gas Advanced Data Handling Resolution For Help press F1 X 347 1343 Y 1845 0000 Autoscaling Checkboxes Figure 5 2 BioTOF Window and Autoscaling Checkboxes 5 2 Operator Manual Version 1 0 Volume Il Operation 5 BioTOF Window 5 1 1 Status Indicators Group The four fields in the Status Indicators group Figure 5 3 provide essential information about the current status of the BioTOF system Instrumentation MS ONLY System Automation Current method Last Good Method Figure 5 3 Status Indicators Table 5 1 Status Indicator Descriptions Function Description Instrumentation Displays one of three instrumentation modes as set in the Mode tab MS ONLY for mass spectrometry only LC MS for chromatography and mass spectrometry when the sample is supplied fro
142. tic Preset lonization Constants a ESI C nESI C APCI Name Reference Mass Current Mass Error ppm Leucine Enkephalin Dim 1111 545977 Co 475 1750 Co 476 1743 j Angiotensin M H 1296 684770 E 119436 9164 1194369194 Polarity Angiotensin M 2H 648 846023 A 5 d i Positive Negative Angiotensin M 3H 432 899774 Ca 0 0000 Ca 0 0000 Angiotensin Il M H 1046 541794 Gas Heater Control gt Angiotensin M 2H 523 774535 to n m c m Iv Dry Gas Angiotensin Il M 3H 349 518782 v e Iw Neb Gas gt ba RE jw DG Heat X Clear lr p 77 EL Lea X 523 7703 Y 31955 0000 Figure 9 2 Calibrating the BioTOF System Operator Manual Version 1 0 Volume II Operation 9 3 9 Calibration Drag the cursor immediately to the right of the mono isotopic peak of the reference compound and click the left mouse button The cursor snaps to the peak and the mass appears under the Current Mass in the reference table in the Calibration tab Use the same technique to snap the line to the next peak but keeping second cursor below the top of the first peak A minimum of two peaks is required for linear calibration If you add more than two peaks the software computes a least squares fit Click the Linear button in the Fit group to display the new calibration constants Check for obvious errors such as negative constants These errors can occur if the peaks selected are not the proper compounds or the
143. tideflector Low A constant voltage applied across the gates to provide steering as the ions return to the orthogonal interface and in a multipass application enter the flight tube again Multideflector High and Multideflector Low are set in the Spectrometer tab 3 2 Flight Tube The ions pass through the multideflector into the flight tube which consists of a drift region and the Reflectron assembly 3 2 1 Drift Region Since all ions are accelerated by the same Repeller potential in the accelerator they reach individual velocities according to their m z values All ions of a given m z ratio take approximately the same amount of time to travel through the flight tube The smaller m z ions travel faster than those with higher m z values the ion packet begins to separate The ion flight time through the tube is proportional to the square root of the ion s m z ratio Operator Manual Version 1 0 Volume I Theory 3 5 3 TOF Mass Spectrometer Operation 3 2 2 Reflectron At the end of the drift region the ions enter the Reflectron assembly at the far end of the tube The assembly Figure 3 5 consists of the Reflectron lens and a series of evenly spaced gold plated disks with a hole in the center and separated by resistors similar to the rings in the accelerator The Reflectron Lens assists in collimating the beam A potential higher than the Repeller accelerator voltage is applied to the far end of the Reflectron the ions gra
144. tion and are specific to each instrument Modifying these parameters can adversely affect system performance The BioTOF system must be re calibrated if any of the Spectrometer tab values are changed Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition High Voltage Volts Repeller Ka Post Acceleration 20002 o Detector 1600 Reflectron 3300 Multi Deflector Low 502 Reflectron Lens 100 MultiDeflectorHigh 704 Figure 7 8 Spectrometer Tab 7 10 Operator Manual Version 1 0 Volume II Operation 7 Methods 7 7 Timing Sequence Tab The Timing Sequence tab is used to set the cycle delay time and duration used in the mass spectrometer The values shown in Figure 7 9 are the default values which are appropriate for most cases Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Advanced Spectrometer Timing 25 u ms Repeller 10 E us Gate 100 us 100 us Cycle Period Transfer Deflector off Delay 25 us Figure 7 9 Timing Sequence Tab Pulse ON OFF The Pulse ON OFF button in the lower right corner of the Advanced Spectrometer Timing group toggles operation of the HV pulser that supplies a high voltage pulse to the Repeller in the accelerator This pulse changes the trajectory or the ion and sends them into the TOF flight tube The pulser must be on for the BioTOF to collect mass spectra However when tr
145. tography direct infusion has no solvent flow between samples to flush the sample path The entire sample path must be flushed with solvent before and after each sample infusion If you have enough sample flush the entire system with the new sample before acquiring data To flush the entire system 1 Fill a syringe with solvent and connect it to the sample tubing 2 Ensure that the nebulizing and drying gases are on 3 Inject one full syringe of solvent or sample through the sample path Note Push the liquid through slowly 4 Check all fittings for leaks Tighten fittings if necessary 5 Verify that the spray is straight with respect to the needle If the spray is not straight the needle may be bent or partially clogged 6 6 Operator Manual Version 1 0 Volume Il Operation 6 Introducing Samples 6 2 Liquid Chromatography LC The most common form of sample introduction is accomplished by means of an LC system There are different configurations that are possible with LC which can include use of a pump automatic sampler and column Frequently the system includes an inline UV detector for dual detection Certain analyses may not necessarily require the use of an analytical column and are referred to as flow injection analysis FIA The Electrospray system accepts solvent flows up to 1 mL min It is compatible with both standard LC columns 2 1 mm to 4 6 mm inner diameter and microcolumns 320 um to 1 0 mm inner diameter
146. tons 1 6 5 4 Computer Workstation 1 2 1 5 Configure Button 7 4 Control Buttons 7 2 Custom Reference File 9 5 Cylinder 2 6 D Daily Maintenance 10 1 Data Handling 7 13 DG Heat 4 6 6 11 DG Heat Checkbox 4 6 Diagnosing Problems 10 11 Digitizer 3 8 5 24 Setting 7 13 Drift Region 3 5 Dry Gas 4 6 Temp 6 11 Dry Gas Checkbox 4 6 Drying Gas 2 7 Controls 4 7 Index 2 Operator Manual Version 1 0 Volumes and Il E Electronics Button 4 3 Electrospray ion ESI source 1 2 lonization Source Setting Dialog Box 5 21 Needle 6 4 Enable 4 4 Endplate 2 6 Electrode 4 4 Endplate with Spray Shield 6 11 ESI 7 2 Server Window 5 25 Source 2 3 Source Cleaning 10 1 Source Configuration 2 4 Source Tab 4 6 6 11 7 8 Exit 2 8 External Out 1 7 5 Out 2 7 5 Start 7 4 Start Configuration Dialog Box 7 4 Switches 5 23 Extracted lon Chrom Option 7 7 lon Chromatagram Range 7 6 E Z Nanospray 2 10 6 16 F FIA MS 7 3 File Save Options Dialog Box 8 6 Flight Tube 3 5 Flushing the Syringe Pump 6 6 Format Menu 5 26 Front Control Panels 1 3 G Gas Controls 4 7 Diverter 6 12 Flow Temperature 7 8 Operator Manual Version 1 0 Volumes and Il Index 3 Gases 4 6 Gauges 4 3 Get Actual Values Option 5 20 H Help Menu 5 26 High Voltage 1 4 Voltage Electrodes 7 9 Injector MS 5 3 Injector MS Mode Indicator 5 4 Instrument Control 8 1 HV Panel 1 3 Modes 7 3 Introducing Sample 2 1 6 1 lon Path 3 12
147. tracted lon Chromatagram range 1 Click the Mode tab and select Chromatography MS in the Instrumentation Mode group if the Chromatography pane is not already displayed The pane includes a trace from the TIC MS range 2 Click the Chromatogram tab to display the display the Chromatagram tab 7 6 Operator Manual Version 1 0 Volume Il Operation 7 Methods 3 Use the Type pull down list to select Extracted lon Chrom Click the Add button to the right of the list 5 Change the low and high values in the Masses field by doing one of the following Type over the value on each side of the hyphen Drag the cursor across a section of the TIC MS trace in the Chromatography pane When you release the mouse button the start and end values are displayed in the Masses field 6 Use the pull down list in the Color field to select a color for the trace in the Chromatography pane and the marker bar in the Mass Spectrometer pane 7 4 Calibration Tab The Calibration tab Figure 7 6 provides access to reference tables so you can calibrate the spectra displayed in the BioTOF window to the mass of a known constituent in the sample Use of this tab is described in detail in Chapter 9 Calibration Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisition Reference List Peptides ref E Fit Linear Quadratic Preset Constants Name Reference Mass Current Mass Error p
148. trol drying and nebulizing gas flows The unit is powered up when the System Electronics are turned on from the Main Control Panel The unit also has its own power switch in the lower right corner of the unit panel The Instrument HV Panel includes two 3 position toggles one for enabling the supply of high voltage HV power to the BioTOF electrodes the other for setting the ion polarity In normal operation the toggles are centered and HV and polarity are controlled from the BioTOF software 5 Main Control Vacuum Gauge Panel Controller Source Control Unit ESI Power Switch Source HV Switch Instrument HV Panel Figure 1 2 BioTOF Control Panels Operator Manual Version 1 0 Volume I Theory 1 3 1 System Components 1 1 1 System Status Indicators Figure 1 3 shows the set of indicators located on the front of the cabinet above the ion source module These include a source lens current gauge and indicators for ion polarity high voltage vacuum and power SOURCE LENS CURRENT 7 _ POLARITY HIGH VOLTAGE T vacuum mmol Figure 1 3 System Status Indicators Polarity Indicates the ion polarity Appropriate source and spectrometer voltages are set automatically High Voltage Shows when the instrument high voltage is on green and off red Vacuum Indicates when the flight tube pressure is below threshold green or above threshold red High voltage can only be switched on when pre
149. troller 3 2 Getting Started Version 1 0 3 Quick Start 5 Press the A and w buttons on the vacuum gauge controller to cycle through the readouts for the gauges used in the BioTOF system Gauge 1 reads the flight tube pressure this gauge is initially off Gauge 3 reads the pressure in the first pumping stage of the ESI source 6 Select Gauge 1 and press the ON OFF button on the gauge controller The flight tube pressure must be below 5 1 0 mbar to engage the pressure interlock of the high voltage supplies 7 Select Gauge 3 When the inlet to the vacuum system is open the pressure should be between 1 5 and 1 9 mbar When the inlet to the vacuum system is closed the pressure should be less than 10 mbar The indicators labelled 1 through 6 in the upper right corner of the Vacuum Gauge Controller are lit when the vacuum levels are below the factory set thresholds When indicators 1 2 and 3 are on you can safely turn the BioTOF system on Indicators 4 5 and 6 are not used 8 Check the Power Switch in the lower right corner of the Source Control to make sure that the controller is turned on Figure 3 3 The Source Control unit LCD displays the default startup menu Source Lens Current Reading Source Control Power Switch lon Polarity Switch een Sno HV Enable Switch L INSTRUMENT HV Figure 3 3 Source Control Unit top and Instrument HV Panel Getting Started Version 1
150. ursor Free Cursor Display Dots Description Scrolls the Spectra Display down Places the Spectra Display in Zoom mode The display zooms in on the area you mark with the cursor To mark the area drag the mouse cursor from the top left corner of the area to the bottom right corner As you drag the cursor a dotted line defines a rectangular area of interest When you release the cursor the area is enlarged to fill the Spectra Display Zoom mode can also be invoked by right clicking anywhere in the Spectra Display and choosing Zoom Rectangle from the pop up menu Changes the cursor to a hand icon KI in the Spectra display enabling you to scroll the display by dragging the hand vertically or horizontally Changes the cursor to an arrow 4 and snaps to the next highest peak to the left with each left mouse click Changes the cursor to an arrow a The arrow rides along the trace as you move the mouse to the right or left Changes the cursor to the default cursor used by the computer Free mode allows you to place the cursor anywhere in the Spectra Display The cursor may leave the screen when you zoom in Toggles the trace line in the Spectra Display on and off When the line is off the dots representing data points are visible Use Displays Dots to distinguish between a peak consisting of multiple data points and a spike a single point that could be the result of system noise or another anomaly Operator Ma
151. ut so that the spray shield Figure 3 5 is visible Remove the spray shield remove any plug or rubber seal from the end of the capillary tube and re install the spray shield Remove the off axis sprayer assembly from the top of the spray chamber verify that the tip of the inner needle protrudes about 0 5 mm from the body of the sprayer and reinstall the sprayer Make sure that the sprayer assembly is connected to the nebulizer gas supply and turn the gas valve counterclockwise until it is fully open Close the spray chamber and secure the latch on the left side to engage the source voltage interlock Getting Started Version 1 0 3 5 3 Quick Start 3 3 Start the BioTOF Software To start the BioTOF control software 1 Turn the workstation on 2 Do one of the following when the system is initialized Double click the BioTOF desktop or tool bar icon cd Choose Start gt All Programs gt BIOTOF gt BioTOF 3 4 Turn the Gases On The next step is to set the nebulizer and drying gas controls To turn the gases on 1 Click the Dry Gas and Neb Gas checkboxes in the Gas Heater Control group Figure 3 6 if they are not already on 2 Click the DG Heat checkbox in the Gas Heater Control group to turn the heater on 3 Click the ESI Source tab and set the Dry Gas Temperature to 200 C Instrument C Standby Operation Mode Chromatogram Calibration ESI Source Spectrometer Timing Sequence Acquisiti
152. uum and power SOURCE LENS CURRENT ERN POLARITY Eh HIGH VOLTAGE Sa VACUUM h POWER Figure 2 4 System Status Indicators Polarity Indicates the ion polarity Appropriate source and spectrometer voltages are set automatically High Voltage Shows when the instrument high voltage is on green and off red Vacuum Indicates when the flight tube pressure is below threshold Power Indicates that system power is on Source Lens Measures current flow at one of the three spray chamber Current electrodes The electrode is selected with the Source Control unit Getting Started Version 1 0 2 5 2 System Controls 2 6 Getting Started Version 1 0 3 Quick Start 3 Quick Start This section lists the essential steps required to start the system and acquire spectra from a Known sample The sequence description assumes that the BioTOF system has been installed and calibrated and is configured with the syringe pump If the configuration includes an LC system the syringe pump is connected via a tee joint between the LC system and the ESI Source refer to BioTOF Operator Manual Volume Il Operation The sequence includes these activities Start the System Check the Spray Chamber and Sprayer Turn the Gases On Prepare the Sample Check BioTOF Parameters Start BioTOF Operation Acquire Spectra Check Calibration Place the System on Standby Getting Started Version 1 0 3 1 3 Quick Start 3 1 St
153. v Dry Gas End Plate 3500 Skimmer2 303 pr Amplitude 600 5 v Neb Gas Capillary 4000 Offset 153 Iw DG Heat Figure 4 7 Gas Heater Group and ESI Source Tab 4 6 Operator Manual Version 1 0 Volume Il Operation 4 System Startup 4 Atthe Source Control panel set the nebulizer gas pressure to approximately 30 psi 5 Set the drying gas flow so that the ball is at about 1 3 scale see Figure 4 8 Nebulizer Gas Flow Meter Nebulizer Gas Control Knob Drying Gas Flow Meter Drying Gas Flow Control Knob Figure 4 8 Nebulizer and Drying Gas Controls 6 Review Chapter 5 BioTOF Window for a description of the user interface and Chapter 6 Introducing Samples for a information on sample introduction to various ionization methods Operator Manual Version 1 0 Volume II Operation 4 7 4 System Startup 4 8 Operator Manual Version 1 0 Volume Il Operation 5 BioTOF Window 5 BioTOF Window This chapter gives a detailed description of the BioTOF window including the menus tool bars and options 5 1 Window Layout To view the BioTOF window 1 Turn the workstation on 2 Do one of the following when the system is initialized Double click zer Choose Start gt All Programs gt BIOTOF gt BioTOF The BioTOF window appears Figure 5 1 Once you have started the application you can leave it running minimizing the window when you are not using the BioTOF system System Status 4 i
154. window Choose View Status Bar 5 5 3 Resize Dialog The two options available from the Resize Dialog determine the initial size of the Spectra Display window Choose View gt Resize Dialog gt Automatic Mode to hide the Parameter tabs so that more of the Spectra Display is visible Drag the bottom edge of the Spectra Display window up to bring the tabs into view Choose View Resize Dialog Advanced Mode to include the Parameter tabs in the default BioTOF display Drag the bottom edge of the Spectra Display window down to hide the tabs Operator Manual Version 1 0 Volume Il Operation 5 17 5 BioTOF Window 5 6 Method Menu When the BioTOF window is opened the application loads Last Good Method that is the system settings and parameters that were in use when the system was closed The Method menu allows you to save the current method to a file and load previously saved methods 5 6 1 Load Method To load a previously saved method 1 Choose Method gt Load Method from the menu bar or click 4B in the tool bar An Open dialog box displays the methods that have been saved in Data Methods folder Figure 5 11 The files in the folder are filtered to include only those with the extension tofpar Look in Io Methods O e E3 1x neg 030503 emn 3x pos 030503 2 IntactProtein_022503 File name f1x_pos_030503 Files of type TOF tofpar Cancel Figure 5 11 Op
155. xternal device such as an LC system To configure the external Start 1 Click the Configure button to open the Configuration dialog box Figure 7 4 The dialog box configures two input and two output signals by defining whether a positive or negative signal activates the switch Configure Input Signal Setting Positive Negative Iw Enable In 2 Positive C Negative Output Signal Setting Ready Iw Enable Out 1 Positive Start Iw Enable Out 2 Positive Figure 7 4 External Start Configuration Dialog Box 7 4 Operator Manual Version 1 0 Volume II Operation 7 Methods 2 Use In 1 as a System Ready signal from an external device and In 2 for starting spectra acquisition from the connected device System Ready is equivalent to clicking the Operation option button in the Instrument group Figure 7 2 on page 7 2 while receiving a Start input is equivalent to clicking the Start button in the Command Buttons group on the left side of the BioTOF window 3 Enable Out 1 to send a ready signal to an external device when the BioTOF system transitions to a ready state and Out 2 to switch the connected system on when you click the Start button in the BioTOF window 4 Click Apply and close the dialog box to return to the Mode tab 5 Click Enable in the External Start group To read the current signals from the external device Choose Tools gt Diagnostics gt Spectrometer The current values from the device appear
156. y interface The pump flow can be set manually on the LC keypad or through the system software As a Pump and Sample Inlet This mode is essentially flow injection analysis but the LC performs all of the functions performed by the multiport valve in manual flow injection The LC serves as a pump and injector The LC column if one is present is switched out of the flow path and no separation takes place The injections can be manual or automated This mode of operation can be controlled manually on the LC keypad or through the system software As a Liquid Chromatograph In this mode the LC operates as a normal liquid chromatograph A column is installed and sample separation takes place Setting up an LC for operation with the Electrospray or APCI is mostly a matter of determining the correct solution chemistry The solvents and buffers must provide good chromatography and enhance or at least not inhibit ion formation Buffers can be added before or after separation pre or post column depending on the chemistry involved The correct choices are highly sample dependent The LC is typically controlled through the system software As an LC With a Second Detector If a second detector is used with Electrospray interface it must be upstream of the mass spectrometer because the sample is otherwise destroyed by the mass spectrometer 6 8 Operator Manual Version 1 0 Volume Il Operation 6 Introducing Samples Two configurations

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