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Agilent Technologies 500-MS IT Mass Spectrometer MS Workstation Version 6 Software Operation Manual

Contents

1. N p5canz pocans Averaged 34 wocans Capillary Voltage 80 0 volts RF Loading 100 s Use High Resolution High Resolution pS car a icang Isolation Window 20 mz Unstable lons Customize TurboDDS Triggers Tab Enter parameters for Trigger and Include Exclude Conditions Trigger conditions e Threshold The value selected on the MS2 line in the TurboDDS Parameters tab is copied to this section If you change the value on one tab the other tab updates to that value automatically lons that have an intensity that is greater than the Threshold will initiate additional scans After the most intense ion is analyzed additional ions up to the N specified are scanned e Minimum Intensity Ratio Select from 1 to 100 This is an additional parameter for setting the threshold when N is greater than 1 Use it to trigger on ions that are more intense than the threshold and the specified percentage of the most intense peak in the survey scan Increasing this value helps to remove noise spikes if they appear to be selected for data dependent actions especially near the apex of a chromatographic peak e Isotopic Equivalence Select from 1 to 10 m z Select a value to prevent more than one peak in an isotopic cluster from triggering TurboDDS scanning This is helpful when isotope peaks of one ion are more intense that the base peak of another Include Exclude Conditions Use this section to determine which i
2. Mazs 3 mz uN mi Mass 4 Mass E m z E 7 z i Start Halt The optimization plots are displayed in the 500 MS screen Return to the method editor and enter the CID excitation voltage in the MS MS Parameters in the method segment 119 Automation File Editor Overview Use the Automation File Editor for creating and editing SampleLists RecalcLists and SequenceLists outside of the Varian MS Workstation System Control application The off line Automation File Editor allows access to these files without disrupting automated runs that may be occurring in System Control NOTE SampleLists and Sequences that are active and running in System Control cannot be accessed simultaneously in the off line Automation File Editor Accessing the Automation File Editor E CA 2 Click the Automation File Editor icon on the Workstation Toolbar to display the Automation File Editor If the Workstation Toolbar is not already opened on your Varian MS Workstation you can start the Automation File Editor from the Windows Start Menu E Mozilla rwerox fm MS Workstation mm Advanced Applications T NIST 08 MS Demo and AMDIS 2 6 Documentation E Oracle Calendar 9 0 4 5 Utilities fan Picasa 3 E Aurora E SAP Front End gt z mm Snagit 9 a Batch Reporting mm Sonic fan Startup 2 Windows Services for UNIX i mm WinZip A Acrobat Distiller 8 p b b b b da
3. Creating or Editing a SampleList Choose either a New SampleList or Open an existing SampleList from the File menu or by clicking New or Open Automation File on the toolbar H Automation File Editor Edit Help Recalclist SampleList Sequence Printer Setup Exit Select a list type that corresponds to the AutoSampler Select SampleList Section Type Please select a SampleList Section Type 216 LC AutoS ampler 460 LC AutoS ampler 8200 Stand lone AutoS ampler 8400 4 5 on 3800 GC 8400 4 5 on 3900 GC 8400 445 on 431 GC 8400 4 5 on 450 GC Cancel 122 The SampleList window for the open SampleList type opens Click Add to add a default sample Click Data Files to select the location and name of Data Files ssi Automation File Editor New Sample List 460 LC smp 16 Cle Fle Edt Help eo amp MAA E New Sample List 460 LC smp 460 LC SampleList Alca Sample Name Sample Type E3l Ini Miection Notes Autotink Ini Mode PL Tray Vial WE 1 CEET ET Paria Loopil Al Insert v Delete Fill Down v v v 1 none 10 Left Add Lines Defaults Hardware a Data Files RecalcList 4i4id 4 4 4 4 4j4 4 4 4 4 pa apa a aj jo mjimja jc mm pa Drag the border of a spreadsheet to change the size Right click a column header to display formatting options When the table is scrolled to the right the Sample Name column
4. Sections to Import 212 LL Solvent Delivery System Module Uz 500 MS Mass Spec Module 03 500 M5 Standard M5 Reports Module 03 1 500 MS MS Data Handling Module 03 1 Select All Click Import to import selected sections to the target Method If the Method already has sections with the same module address and channel ID you are prompted to assign a new module address and channel number to the imported section or overwrite the existing section in the current method Deleting Method Sections Open the Method containing the sections you wish to remove From the Method Builder Toolbar click Delete Section Or on the File menu click Delete Section A dialog box displays the Method sections Click the desired sections Click one section and hold down the shift key while clicking another section to select a range Hold down the control key while clicking a section to add that selection to the list Click a selected section while holding down the control key to remove it Delete Method Sections Sections to Delete ProStar 430 Autos ampler Module 0 212 LC Solvent Delivery System Module 02 500 MS Mass Spec Module 03 500 MS Standard MS Reports Module 03 1 300 M5 MS Data Handling Module 03 1 Select All Cancel After highlighting the sections click delete to delete them You are prompted for confirmation before each section is deleted 63 Printing the Method Click the Print icon on the Toolbar al Select the desired Method
5. You can choose to create a new RecalcList append to an existing RecalcList or neither create nor update a RecalcList To select the RecalcList option that you want click the appropriate RecalcList button The RecalcList Generation dialog box opens RecalcList Generation fou can automatically create or update a RecalcList with files generated during automated injections Specify the RecalcList generation options for this SampleList below C Do not automatically create and update a RecalcList RecalcList name lcoyvariarmsk single tel Append to an existing RecalcList RecalcList name a Browse Overwrite the Recale List each time the SampleList Begins Cancel If you choose to create a new RecalcList the automatically generated RecalcList does not overwrite an existing RecalcList unless you also specify Overwrite the RecalcList each time the SampleList begins If a RecalcList with the same filename exists the newly created RecalcList has a number appended to its filename to make it unique and to prevent the older RecalcList from being overwritten Using QuickStart QuickStart is a fast way to inject a single sample without having to use System Control directly QuickStart can be customized and is ideal when setting up instruments for routine use Refer to the online help for further details QuickStart starts System Control and waits until all modules are in a ready state When ready the QuickStar
6. 20 3 Click Index select a topic and click Display Help Topics Saturn GC MS Workstation System Control da Contents Index Find 1 Type the first few letters of the word pou re looking for Acquisition Mode 2 Click the index entry pou want and then click Display 9200 Prospekt Command Table ekt Status and Control Window Adjustments After Temperature Changes Advanced BOOTP Settings Dialog Box Automix Routines Dialog Box Autosampler Carrousel Display Help Topics Saturn GC MS Workstation System Control IK Contents Index Find 1 Type the word s you want to find Find Similar Find Now EME 3 Click a topic then click Display About Running Analyses About Running Analyses Acquisition Activate Method Dialog Box ADC Board Injecting a Single Sample ADC Board Injecting a Single Sample Overview ADC Board eS a Single Sample ee a RecalcList 21 System Control Toolbar The system control toolbar contains the following functions CO MN Open Instrument 1 Status PAS Start an open RecalcList MS Start an open SampleList er Start an open SequenceList EQ i Suspend a running list 22 Control Module Window The 500 MS Control Module Window has five views Manual Control Auto Tune Diagnostics Startup Shutdown and Acquisition a 500 MS 56 Not Reade Manual Control Auto Tune Diagnostics Startup Shutdown Acquistion l E Control anc State Source ESI Edit Met
7. Scan Mode Enhanced Standard 15000 usec 50 000 mz HiRes 250 uzec 50 2000 mz Extended Enhanced Enhanced Resolution A prescan in Automatic Gain Control mode is used to determine the optimum ionization time lons are scanned at 5000 u sec Mass Range 50 2000m z Scan a wide range and determine if an ion is doubly or singly charged Standard The default Default uses a prescan in Automatic Gain Control mode to determine the optimum ionization time lons are scanned at15000 u sec Mass Range 50 2000m z NOTE Use the Standard mode for most routine applications The Standard mode has similar sensitivity to Enhanced mode and can provide more sample points across a chromatogram Enhanced mode is best to use if you need maximal resolution to observe multiply charged peaks for example NOTE High Resolution and Extended Mass Range are available only with the TurboDDS M option HiRes High Resolution Scan a range of 10 m z from 50 2000 m z around the specified mass with a scan rate of 250 u sec The spectrum is useful for determining the charge state of a know on by measuring the mass difference of the isotope peaks 71 owo EOI os os IEA os IEA SA Extended Extended Mass Range Scan from 100 to 3500 m z at 1500u sec The mass resolution is lower than enhanced or standard k NO oo iN Special Applications The Special Applications dialog box has controls for the
8. Summary of Status Green type indicates OK and red indicates a problem or a read back Display the vacuum pressure by checking the lon Gauge checkbox next to Hide Keypad Control and Status Save Data Source ESI lethod Record Data Method ESI Upload MS Method End Time 10 00 minutes Summary of Status Wacuum System OF Damping Gas OF Temperatures Wam Ice OF lon Gauge Pressur 33 1 uT or FA 1 Valid VI Needle 5000 Volts 0 1 uAmps TRAP Diving Gas 350C 15 PSI Trap Capillary 80 Volts Spray Shield 600 Volts Housing 50 Relays lial 21 431 4 Select Views 6 Port Valve ME elo Stor 28 Edit Upload Method Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Control and Status Save Data Source ES c Edit Method i Record Data Method ESI Upload MS Method End Time 10 00 minutes Click Edit Method to load the current active Method into Method Builder Click Upload MS Method to create a new Method You are prompted to save it with the current manual control settings and make it the active Method Save Data Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Control and Status Dae bata Source ESI Edit Method Checord Data _ Method ES Upload MS Method End Time 10 00 minutes Click Record Data to begin data collection using the parameters in the current active Method segment During d
9. ay ar VARIAN INC VARIAN MS Workstation System Control Version 6 9 3 Copyright 1989 2009 Service Code Installed Module Drivers 800 Interface Box ADC Board 8200 AutoS ampler 3800 GC 3300 GC Installation History MS Workstation S N Installed by Organization Installation date Validate Installed Files The Validate Installed Files uses checksums produced at the factory to test and document errors You can print a report that documents the file system tested the success or failure of the test the date printed amp tested the ID of the tester if login IDs are used on this computer The report can contain a signature line For more details about this application refer to the online help The following is an example Validation of installed file systems ga AEA Fie Options Help FILE SYSTEM VALIDATION REPORT File System Varian Workstation 6 9 3 Workstation ENG8657 Directory C ivarianws Source file c varianweN star val Test Results 209 files tested 209 files OK eee File System OK e Installation History Quit This menu item closes the Workstation Toolbar application If you choose to run the Workstation Toolbar automatically when Windows starts the Workstation Toolbar opens the next time you start Windows 11 Launching Applications from the Workstation Toolbar Using the Application Icon Buttons oa ACS Phen A l Tr Review Chrom chromatogram ae RESP SYR 500 F Ba eS
10. 20 00 rea MS Method Before an acquisition starts the active method is in Segment 1 which may be a data collection delay segment with the trap turned off for the segment duration MS Method Segment Mumber 1 of Segment Description ESI MS MS Scan Description ES Auto Full Scan Range 100 000 After a run starts the Segment Number increments after the segment start time is reached The Segment Description is the user entered description in the Segment Description field of the MS Acquisition Method in the active method The Scan Description indicates the onization and on preparation methods chosen in the Scan Type Group and lonization fields of the Method Segment field of the method Scan Range is the acquisition mass range MS Method Segment Number 2 of 2 Segment Description est me me Scan Descriptor LEMS ESI Scan Range 191 589 Operating Conditions Data File shows the name of the file currently being acquired The Scan Number lonization Time and lon Count for the current scan are displayed in their respective fields This display updates scan by scan throughout the run Operating Conditions Data File MS M5S test 41 5 Scan Number 103 lonization Time 4564 lon Count 3608 56 Spectrum and Chromatogram Windows Below the keypad select the Spectrum or the Chromatogram plot or both Hide Reppad Spectrum and Chromatogram Chromatogram Customize the appearance of the Chromatograph and
11. A interactively edit data handling parameters and calculate results view Edit Chromatograms The MS Workstation applications are icons on the Workstation toolbar When you pause the cursor on the icon the name of the application is displayed in a tooltip below the cursor and on the right of the toolbar a description is displayed Click an icon in the Workstation toolbar to launch the corresponding application Using the Data File and Method QuickLink Buttons RESP SYR SO0 de A reronuciity gt The two buttons to the right of applications are QuickLink to Data Files and Methods Data Files Display a menu of options for the most recently used Data File rt RESP SR 500 Wer pEdit M5 Chromatograrn Print Custom MS Report Print Standard MS Report view Custom MS Report View Standard MS Report Pick a different Data File Methods Click to display operations that can be performed on the most recently used Method 8 reproducibility iP View Edit Method Edit Compound Sets Print Method Pick a different Method System Control Overview This section introduces the features of System Control Later sections explain how to use these features Navigating the Instrument Window S System Control MS Not Ready File Edit Inject Automation Recalculate Instrument Windows Help aja Brar elos le 8 LR Sel el gt m1 Not Ready a 500 MS 01 Not Ready Manual Control Auto Tune
12. APC lonization Parameters Full Scan Parameters Automatic Gain Control Target TIL 489231 counts 100 z k lorization Time Fixed lonization Time lonization Time 100 seconds ale T a T Mas lon Time 250000 seconds Customize Set the Target Total lon Current TIC or value in lonization Control The Automatic Gain Control AGC algorithm uses the ion count from a prescan at fixed ion time along with this target value to calculate an ion time necessary to fill the ion trap with the target number of ions during the analytical scan The objective is to fill the trap with an optimal number of ions during each analytical scan In full scan mode an optimal Target TIC is automatically calculated and adjusted according to the set scan range in the Full Scan Parameters tab the calculated value for negative polarity is about 1 5 of that for positive polarity In MS MS AMD and MS n modes the optimal Target TIC is fixed This automatic setting can be overridden by setting the nnn field for example To double the Target TIC use 200 to halve the Target TIC use 50 The Target TIC should not be set too high or spectral distortions due to space charge loss of MS resolution and or shift in mass assignments for strong chromatographic peaks may result Typically the automatically calculated Target TIC at 100 gives the best results Click Customize to run fixed ion time experiments and or change the
13. Diagnostics Startup Shutdown Acquisition Control and Status Save Data Method Active Segment Checks and Adjustments Module Attributes Optimization Plots a Source ESI Edit Method Record Data Method Segment Method ES Upload MS Method End Time _ 10 00 minutes Y Detector On Scan Type Full y Scan Mode Standard y Defaults Summary of Status JV Manual FF Syringe Pump On Vacuum System OK Damping Gas OK Temperatures OK Source OK General Parameters lonization Control Electrospray lonization Parameters Full Scan Parameters lon Gauge Pressure 0 0 uTorr Fil 1 Valid Needle O Volts 0 0 uAmps TRAP 3413 Maximum Scan Time Muttiplier Offset 0 volts ii 1 DiyingGes 0C 10PSI a Data Rate J 0 29 Hz Capillary 0 Volts Spray Shield 0 Volts Relays lrirerers lt gt Hide Keypad Y Trap Select Views 6 Port Valve Infusion Pump r Method Segment Active Method Segment r Scan Function Channels I ton Gauge Position 1 Run Activate Number 1 Channel 4 1 rr UA real Do roion 9 rage Congo Peto lr gt A tlo el 5 My il eT TAE ls i i 0 25 5 0 0 10 0 12 5 0 500 1000 1500 2000 minutes ji Acquired Range m z lons is Instrument 1 Mar 05 10 17 06 Module 500 MS 01 4 System Control features include System Control Menu bar System Control Toolbar 500 MS Keypad Dialog Relays Chromatogram Spectrum Optimization Plot Toolbars and Chromatogr
14. On General Parameters Tab Scan Time uScans Averaged Data Rate Multiplier Offset lonization Control Tab Control the 6 port valve state for each mode of operation selected by using the Special Applications tab and is named accordingly When the 6 port valve is configured in the Manual mode this checkbox becomes inactive and the active flow path is controlled in the Manual Control section of System Control These settings are active in the Acquisition mode Use the Syringe Pump On check box in Acquisition mode to turn the syringe pump on or off Change the flow rate in Manual Control Edit the Set Points for a particular time segment There are five set points Scan Time Scans Averaged Data Rate and Multiplier Offset Count Threshold is available when the Customize button is selected Scan Time uScans Averaged and Data Rate are linked The number of scans averaged is updated when the scan time is adjusted and the reverse is true Set the scan time by first setting the mass range from the Scan Parameters tab and then changing the scans averaged to three Three scans averaged gives the best compromise between a high chromatographic data rate and good spectral averaging San Time Range 0 47 30 seconds full range uScans Averaged Range 1 to 99 Adjusts the EM voltage as much as 300V relative to the current multiplier setting in the Module Attributes tab dialog in Manual Control usually the 10 5 gain va
15. Specify the RecalcList generation options for this SampleList below Do not automatically create and update a RecalcList C Create and update a new RecalcList RecalcList name C Append to an existing RecalcList RecalcList name r If you choose to create a new RecalcList the automatically generated RecalcList does not overwrite an existing RecalcList If a RecalcList with the same file name exists the newly created RecalcList has a number appended to its file name to make it unique and to prevent the older RecalcList from being overwritten Changing Default SampleList Entries When you add a new row to a SampleList default values are used for each cell To change the default values click Defaults in the open SampleList window The following opens Enter the desired default values and click Save Set 460 1C AutoSampler SampleList Defaults ES Sample Name Sample Type Pee Inj Injection Notes AutoLink Inj Mode Gates Tray Vial Well Washes eres E are pee Multiplier Divisor prin Default Sample Analysis Partial Loopfill X 10 Left y E Cancel 125 Using More Than One Method for Injections You can do automated injections using more than one Method There are two ways to do this e Change the active Method from within the SampleList e Use a Sequence Changing the Method in the SampleList Change the Method used during injections by activating a Method ina SampleList row Automation File E
16. Spectrometry Overview MS MS is the isolation of a precursor ion formed in the API interface dissociation of the precursor ion by energetic collisions with helium atoms and trapping and scanning of the resulting product ions The ion trap MS MS process is e Filling the ion trap with the selected precursor ion e Forming the product ion spectrum to obtain qualitative structural information to obtain qualitative structure information and e Increasing the signal to noise ratio by eliminating interfering matrix ions in the product ion spectrum through isolation and dissociation the only ions in the product ion mass spectrum originate from ions that fall into the isolation window The MS MS Scan Function Eject lt m e Eject gt m A RF Voltage nr MA SA S MN lonization Isolation Dissociation Scanning MS MS Scan Function Analytical scan only 89 Product ion MS MS is a four step process 1 The sample is ionized and the on trap fills with the optimal number of ions This is similar to full scan The selected precursor ion is isolated from all the unwanted ions The range of motion of the selected ions is increased which results in energetic collisions The selected ions fragment 4 The fragment ions are ejected from the trap and scanned For MS isolation and dissociation steps are carried out serially and the resulting ions are scanned to create the spectrum While each step reduces th
17. 6 Port Valve the Syringe Pump and Relays Method Active Segment Checks and Adjustments Module Attributes Optimization Plots Instrument Configuration Acquisition Data Type TurboDDS Electrospray Centroid C Profle TF TurboDDS Method Chromatographic Time Segments oo _ es A See ee ee 0 00 10 00 ESI Auto Full 6 Port Valve The selection affects the display in the Method Segment pane Syringe Pump When the Syringe Pump is enabled in the Method Segment pane enter the Flow Rate and if appropriate click to turn the syringe pump off at the end of the run Special Applications Ed 6 Port Valve 7 Syringe Pump Manual Flow Rate 10 00 microlters min W Turn Off at Method End Diverter Relays Injector Bice Load position M required for ready state Load Position annm Inject Position Defaults Restore ox Cancel Relays There are four TLL compatible relays on the back of the 500 MS They use a 9 pin D shell connector Select Relays and click Program Relays to open the Relay Control dialog Control the relays using a time program 72 Relay Control Tine sar reia Rears eens Delete Event s Delete All Events ESI Parameters This section details how to set parameters for an electrospray ionization method In System Control click Active Segment General Parameters Method Segment 4 Detector On Scan Type Fa f Scan Mode Stan
18. Acquisition Control and Status Auto Tune Method Action Idle Select All Clear All State Idle iM Detector Setup Integrator Zero Start Auto T i Set Electron Wulkiplier Start Auto Tune melo ly Waveform Tuning and Calibration Supplemental waveform Tuning AF Full Scale Adjust Mass Calibration Trap Frequency Calibration W lon Optics Tuning lon Lenz Tuning i High Resolution Tuning and Calibration Supplemental Waveform Tuning Mass Calibration I Turn off infusion pump and source 42 AutoTune reports can be printed The following example is of a TurooDDS AutoTune report Varian 500 MS Autotune Summary Report 4 2 2007 3 06 PM Tune Parameters OK Lens Tune Positive 15 3 48 2 18 3 24 7 Lens Tune Negative 15 0 29 3 15 6 34 4 OK Calibration Parameters Calibration Enhanced Mode Calibration Standard Mode Calibration Extended Mode Calibration HiRes Mode Supplemental Waveform Enhanced Mode Supplemental Waveform Standard Mode Supplemental Waveform Extended Mode Supplemental Waveform HiRes Mode Instrument Parameters Foreline Pressure Forward Turbo Power Rear Turbo Power Foreline Pump 1 Power Foreline Pump 2 Power 43 Control and Status Start Auto Tune by clicking Start Auto Tune Click Stop Auto Tune to stop Auto Tune The Start Auto Tune window opens reminding you to check the syringe pump Click Ok Start Auto Tune Ld 1 Please check calibration mixt
19. Check Some Auto Tunes not uptodate 23 Chromatogram Toolbar There is a Chromatogram window in Acquisition and in Manual Control The Chromatogram toolbar for each is slightly different aj Slo 10 E A OB as ue Click Arrow to hide the toolbar Click in the upper left of the display to restore the toolbar aes Click Full Scale to normalize the chromatogram vertically and horizontally Double click the lower left corner to do the same Click Previous Scale to return the chromatogram display to the scaling prior to the current one Click twice to step through the ek Click Scale to switch between Auto Scale and Fixed Mode Intensity Click Multiple Plots View to select a display Al Click Spectrum Select to display spectra from the current chromatogram If not selected the spectral display is updated aire Click Chromatogram Viewer to load the data file being acquired into MS Data Review to do qualitative analysis in real time and print results Available in Acquisition only Click Clear Plot to clear the window Only available in the Chromatogram Plot window in Manual Control Click to display a plot of the Intensity of the Base Peak To display the RIC trace and the Intensity of Base Peak plot at same time enter IBP in the lons field Clicking the Base Peak icon does not affect the display of the extracted ion chromatogram 24 Spectrum Toolbar View Spectrum display when the 500 MS module is in Manual Control Au
20. Data Handling sections then the generated RecalcList will contain both sms and run files 176 Select SampleList Section Type Dialog Box Select SampleList Section Type E Please select a SampleList Section Type 8400 4 5 on 450 40 8410 4 5 on 3800 GC 8410 4 5 on 3900 GC 8410 4 5 on 431 40 28410 4 5 on 450 40 CombiPA4L Autos ampler Cancel A SampleList File may contain multiple SampleList Sections Each installed AutoSampler can contribute its own section depending on the features that correspond to its hardware Generally each installed AutoSampler has its own unique SampleList with columns for its features A SampleList File can contain a section for each type of AutoSampler similar to the way that a Method file can contain a section for each type of Instrument Module This dialog box lets you select the specific section to edit in a SampleList file that may contain more than one section em Description S SampleList section Select a section type to use in building a new type list box SampleList opens the appropriate SampleList window SampleLists may contain sections for multiple types of sample handling devices depending upon which module drivers have been installed Creates a SampleList section of the specified type Cancels SampleList section creation 177 Data File Generation Dialog Box Data File Generation Specity the names for Data Files generated by detector modules using this SampleList
21. Ea EN Method Segment Y Detector On Scan Type Full y Scan Mode Standard T Load I Syringe Pump On General Parameters lonization Control Electrospray lonization Parameters Full Scan Parameters SetPoints Full Scan 0 00 2 00 ESI Auto Full Insert Delete Defaults Restore Special pplications Add mt Defaults C Scan Time 4423 Maximum Scan Time Multiplier Offset i volts e Scans Averaged 33 Scans Data Rate 0 23 Hz Setting the MS Instrument Configuration Most of the following configuration settings are for the entire method and are not time segment variables i Instrument Configuration Electrospray Configuration Menu There are two hardware configurations Electrospray ESI and Atmospheric Pressure Chemical lonization APCI If you change the ionization configuration in a method all the previous Time Segment information is replaced with default values 65 Choosing the MS Acquisition Data Type Acquisition Data Type Centroid Profile Either Profile or Centroid data may be acquired in a method Centroid Data The standard data acquisition type is Centroid in this approach the analog signal from the detector is processed by the analog to digital converter The centroid is the center of gravity of each mass peak that is reported to one decimal place that is m z 231 2 Centroid data are required for library
22. MS Reports Blas EN Despues Ponwotd on Save Print Options f Results Format Revision History Ha Chromabogram Format No Revision Histor Compound Reports NS R Selecting the file name at the top of the Directory displays method related information including name and directory path creation and modification dates file size file attributes and the revision history 61 ES 500 MS test mth Method Hotes Method Notes Loop irgections from autosampler EY ProStar 430 AukoSampler Control Address 1 rade oa 1005018 4 Configuration FE 212 LC Solvent Delivery System Control Addre as m Selecting Method Notes displays a blank Parameters pane where you can enter notes about the method You can enter information about the method sample preparation laboratory practices and so on It is possible to copy text from a Microsoft Word or Excel document and paste into the Method Notes however you cannot copy and paste graphics or icons this way Method Notes are printed when the Method is printed Instrument Modules Highlighting low level outline branches for the 500 MS and the other LC hardware modules those that cannot be expanded displays Parameter panes with editable parameters for controlling the instrument acquiring data and post run data processing and reporting Importing Method Sections You can copy sections from one Method to another Open the target Method file This is the Method to which y
23. Not Ready File Edit alela ES Method mth a 5OO MS 56 Not Read Inject Automation Peete Instrument Windows Help View Calibration Curves Description Begin RecalcList Begins recalculation of the open RecalcList using the active Method Begin at Selected Begins the open RecalcList at the selected RecalcList line RecalcList Line This item is enabled when a RecalcList is open After selecting this a prompt for the line in the RecalcList appears It is useful when recovering from a power failure that interrupted automation in progress View Calibration Opens the Open a Method File to View Curves dialog box to Curves view calibration curves that are stored in a Method file Instrument Menu iJ System Control Varian LC MS 1 Not Ready Fie Edit Inject Automation Recalculate AE Windows Help sl E Method2 mth we 1 Varian LEMS 1 Alt 1 m 500 M5 56 Not Ready Configuration Alt c Remove Module Mames Manual Control Auto Tune Diagnostics SI Restore Disabled Warnings Status nd Control Instrument 1 Faults Run Time 0 00 minutes Setup Ethernet Communications 1 00 minutes Edit het Setup ADE Board 1 0 Ports setup COMM Ports D Not Ready The Instrument Menu commands switch the display between the configuration screen and the instrument Menu tem Description Instrument Name Displays the Instrument Status window and Status and Control wind
24. Photo Diode Array detector Use Aurora to view HPLC Photo Diode Array data in a 3D presentation Quick Start Run a sample without a Sample List Workstation Toolbar Options Moving the Workstation Toolbar The Workstation toolbar can be moved to any edge of the Windows screen Click a portion of the Workstation Toolbar that does not contain Application or QuickLink buttons and drag the toolbar to the edge of the desired screen When you release the mouse the toolbar remains on that edge The Workstation Toolbar will open to this location the next time it is started The Workstation Toolbar Options Menu 200 NG SMS ES retrial estan E Right click For options When you move the mouse over an area of the Workstation Toolbar not containing application or QuickLink buttons you may right click to display an option menu Move to Windows Task Bar Show Hide Applications on Toolbar Enable Disable Instrument Modules 2 Application Descriptions 3mall Buttons on Toolbar Run Application Help on Pick Data File For Quick Link Buttor Pick Method For Quick Link Button Help on Workstation Toolbar Product Support Web Site About Workstation Toolbar Quit Move to Windows Taskbar You can display the Workstation toolbar as a Windows taskbar icon Select Move to Windows Taskbar from the Workstation toolbar options menu Taskbar icons appear in the lower right or bottom of the Windows taskbar the bar on wh
25. Spectrum View Spectrum and Chromatograrn e Chromatogram Select Chromatogram Plot Preferences e Spectrum Select Spectrum Plot Preferences For more information on acquiring data in Acquisition mode see Injecting Samples on page 96 97 Building 500 MS Methods Creating a New Method Click Method Builder ES on the Workstation Toolbar to open the following Click Create a New Method File and the Star Assistant Wizard opens Create Open Method File Select a method file action Create a New Method File C Open an Existing Method File ee Do not display this dialog at startup Configuring method The Method Builder will guide you through adding instrument module control and any post run processing to the method Click the Next button to continue l Show this introduction next time cos Select the instrument configuration for the method either Custom or Instrument 1 You can use Custom configuration to create methods on a PC remote from the instrument 58 Select Configuration Select an existing instrument configuration for the method or select a custom configuration for the method Then click Next to continue Select a Configuration Configuration Description Custom The following screens will guide you in selecting a C Instrument 1 custom configuration for your method Select Instrument 1 to add automatically the hardware modules currently configured in System Control and click Ne
26. User Program in the box next to Cancel and click OK UserProg_1 in the above example This User Program is then saved in the SampleList and can be exported to other SampleLists Alternatively User Programs from other SampleLists can be imported to the active SampleList using Import When you are ready to build a PS 420 SampleList or a PS 430 SampleList you can use Add at the right side of the SampleList to enter sample lines efficiently Add Lines to ProStar 420 SampleList Cal Injection Inj Wash Sample Name Sample Type ni Inj Bis AutoLink Inj Mode Vaka Vial Volume Default Sample Analysis v 1 none none Partial Loopfill v 10 Al 300 E Iv Number Sample Names from 1 Add Insert Cancel Number Vials from A1 through 81 2 The Add dialog allows you to enter common information and select the number of entries to be added sequentially to the SampleList With the PS 420 you can specify the first and last vials to be added to the SampleList with the requirement that the first and last vial numbers start with the same Tray Type letter A B C or D With the PS 430 you can specify the first and last Row and Column to be added to the SampleList You can also select to have the entries added by Rows or Columns which determines if the plate is processed from left to right by Rows or from top to bottom by Columns 460 LC SampleList To create a 460 LC SampleList do the following 1 Click the Edit Auto
27. a mass isolation window of 3 m z to ensure that the ion is centered in the window For example given a precursor ion mass of 1522 m z and a mass isolation window of 3 m z ions of masses 1521 1522 and 1523 m z are isolated If there is a need to reduce the window because of interfering ions do it in a later step because the customized parameters may require optimization Some ions are easily dissociated during the isolation step Increasing the width of the isolation window reduces the ion loss by premature dissociation at the expense of selectivity Range 1 0 14 0 m z full range Select non resonant or resonant excitation The non resonant method is usually the first choice because of its simplicity Resonant excitation involves exciting the target ions by an RF field of the resonant frequency of the trapped ions Non resonant excitation involves exciting the precursor ions using an RF field not at the resonant frequency of the trapped ions The RF storage level in m z while the dissociation waveform is applied following isolation The Excitation Storage level depends on the precursor mass but the lowest product ion must be more than 2 mass units above the excitation storage level The excitation storage level for the precursor ion can be calculated using the q calculator The q calculator is accessed by right clicking on any of the fields in the MS MS parameters table NOTE Set the excitation storage level to avoid e
28. are searched according to their intensities e Low Mass High Mass Select from 50 0 to 2000 0 m z Enter the mass range to search The Low Mass must be lower than or equal to the high mass and overlaps are not permitted among listed ranges 84 e Priority As you enter the mass ranges you can assign a priority to that range When Order by Priority is selected the mass ranges are searched according to the listed priority e Order by Specifies the order the ions from the include list are used when the method is run The options are e Abundance The most intense ion in the survey scan that meets the trigger criteria is analyzed first followed by the next most intense ion until N ions are analyzed e Priority The ions are searched for according the Priority assigned in the List regardless of intensity Only those ions that meet the trigger criteria are analyzed e Use the following commands with the Include List e Delete Click the row to be deleted and then click Delete e Sort by Masses e Click to sort the mass ranges in order of increasing mass e Click again to sort the mass ranges in order of decreasing mass e Sort by Priorities e Click to sort the mass ranges in order of increasing priority e Click again to sort the mass ranges in order of decreasing priority e Comments Enter commenis that are displayed each time you use the list e Import Export Use other programs such as a spreadsheet or a text editor to
29. are shutting down DO NOT PERFORM MAINTENANCE UNTIL SHUTDOWN IS COMPLETE After the turbo pumps reach 35 speed the remaining gases turn off Close System Control and turn off the foreline pumps by turning off the 500 MS hardware from the front panel switch The system should be fully vented after 10 minutes Hide Keypad Event Messages Turning Damping Gas OFF Shutdown Turbo Pumps Heated Zones are shutting down DO NOT PERFORM MAINTENANCE UNTIL SHUTDOWN IS COMPLETE Shutdown Turbo Pumps are shut down Turning off gases Press hardware switch to turn off foreline pumps Please wait approximately ten minutes for the system to vent Starting Up the System Restart the system by turning on the 500 MS hardware from the front panel switch and then restarting the System Control a 500 M5 56 Not Ready Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Status and Control Vacuum System gt Foreline Pumps Conditions Start Up Shut Down Pump 1 Pump 2 State Starting Up Status Mormal Normal aria Speed 100 100 Curent 1667 mAmps 1762 m mp Status Not Ready Power 443 Watts 469 Watts Preumatics Tempi BE DE i Life Damping Gas On ai Dil Ste Level OK Level OK Flow Rate Setpoint 0 8 mL min Turbo Pumps Flow Rate 0 0 mLemin Rear Forward Inlet Pressure OPS Status Starting Starting Speed 54 69 2 lon Gauge Curent 1529 m mps 1562 mAnmps Pressure 0 0 Tor Power 106 Watts g
30. channels And you can specify up to eight internal standards for each of the four channels for a total of 32 internal standards NOTE If you only want to specify a single Multiplier Divisor Unidentified Peak Factor and Amount Standard to be used for all channels of all detectors Use the SampleList RecalcList or Inject Single Sample dialog If you plan to specify parameters for each channel before opening this dialog box make sure that the active method contains the data handling sections for each channel and that their peak tables contain the standard peaks if any properly named and checked The dialog box uses information from the active method to help you make the proper specifications for the type of calibration method that you are using 171 NOTE If you change the standard peak names in the peak table you must return to this dialog for each sample to update the names and amounts Data Handling Channels For Method Method1 mth Sample testi Calculation Unid Peak rr T Standard Amount Detector Channel Time Faclo Multipher Divizor Peak 1 Standard 1 3800 44 Channel Front Uncalibrated Pe ace Insert Delete Cancel Edit Calibration Setup tem Description S Detector Channel Indicates the detector channel specified by the remaining fields in the row Channels are indicated by the detector module name bus address and channel identifier It has entries for all channels with data handling sections in
31. does not scroll Defining the Fields The top row of the SampleList window has fields for information sample The various fields and buttons in this dialog are Sample Name Enter the sample name The final name of the file depends on settings accessed by clicking Data Files If only s is listed in the Data Files dialog the sample name will be as you entered it here Sample Type Use the scroll bar to see all the options Sample Type sample Type MENER Baseline Print Calib New Calib Block Cal Level If calibration is the sample type you can designate up to ten calibration levels In Enter the number of injections You may make up to nine replicate injections at each calibration level Injection Notes Enter notes 123 AutoLink Refer to the Specifying Data Handling Parameters section in this manual Injection Autominx Amount Std Unid Peak Multioli Divi HMultiChannel Volume Routines 15 HZ only Factor el WIO MultiStandard 1 OU none In the Vial Injection Volume and Injectors Used fields enter information about the sample The fields beginning with Amt Std are used only for data files acquired with standard LC detectors Add Add a new line to the end of the list Insert Insert a new line before the currently line Delete Delete the selected line Fill Down When sections of columns or entire lines are highlighted the information in the top highlighted row is repeated to the lowest
32. end of the method to divert column effluent during a column washing step In this case the divert segment is inserted after the MS MS segment shown in the following example Method Active Segment Checks and Adjustments Module Attributes Optimization Plots Instrument Configuration Electrospray 1 00 ES Auto Full 1 00 17 00 ESI Auto Full 117 Set the valve type to Divert in the Special Applications window Enter the Flow Rate of the Syringe Pump to make up for the lack of flow from the LC pumps during diverting Plumb the 500 MS valve with the following configuration In the diverter segment check that the syringe pump is enabled Special Applications 6 Port Valve Manual z Flow Rate 4 microliters min gt Injector Load position JY required for From HPLC ready state Column Diver to Waste Load s OAPI inject Defaults Restore ox Cancel MS MS Optimization Use the 500 MS Optimization Plots View and the built in syringe pump to help your MS MS development process Begin by infusing a sample in an appropriate solvent into the 500 MS making sure your drying gas pressure and temperature are set to the desired levels After you click Start the Optimization Plot screen automatically appears adjacent to the Spectrum Plot screen You can click and drag the Bar to undock the Plot move and resize it Click the Optimization Plots tab and select CID Excitation Voltage
33. regarding a task or a feature of the system Before Using Your System 1 Is the system turned on and ready If not refer to the 500 MS Hardware Operation Manual 2 Do you need to tune the mass spectrometer Refer to the 500 MS Hardware Operation Manual and the Autotune section in this manual Prepare your samples Do you have methods built for both 500 MS and chromatography modules If not refer to the 500 MS Method section of this manual and to the manuals for your chromatography devices such as LC pumps and Autosampler Perform the injection Refer to Injecting Samples on page 96 Review your data Refer the MS Workstation Software Reference Manual MS Workstation Applications Overview The Varian MS Workstation is a series of applications that are displayed on the Workstation toolbar The tool bar can be displayed as a series of large buttons Acs iat Al m as a a Ed yy iit RESP SYR 500 P reproducibility_ gt Or as a series of small buttons ie ES FY ed ay ES E dal ES ES 12 mrese sve 5002 2 fa repraciuciviity_00 gt Tooltips show the application name when the cursor rests on the Application Icon Application Descriptions give brief descriptions of the application that will be opened when the cursor rests on the Application Button Quick Link Buttons provide menu selections of operations to be performed on the listed file When activated the Workstation toolbar is like th
34. reset Trap Detector Setpoint Multipher 1335 Volts 1361 Dynode 14966 Volts ions 15000 Select Multiplier Dynode in the Monitoring tab view to display the readings for the multiplier and conversion dynode voltages The multiplier reading is negative and reflects the voltage drop between ground and the entrance of the multiplier The dynode voltage should be close to 15 000V the polarity is the opposite of the charge of ions being detected Waveform System Waveform System Entrance Endcap Trapping Field Dipole 100 Exit Endcap Trapping Field Dipole 100 Dipole Supplemental My avetorn Had a Quadrupole Supplemental Wavetonmn 444 Select RF Control and RF button in the Monitoring tab view to display the readings for the Entrance Exit End Cap Trapping Field Dipole The values should be 100 1 48 Waveform System Entrance Endcap Trapping Field Dipole 0 3 Exit Endcap Trapping Field Dipole js Dipole Supplemental waveform 101 Quadrupole Supplemental waveform 100 A kA Select RF Control and the Waveform radio button in the Monitoring tab view to display the Dipole and Quadrupole Supplemental Waveform readings The values should be near 100 Temperatures and Gases Temperatures and Gases Setpoint Dring Gas Temp 3500 350 Drying Gas Fress 10 PSI 10 0 Spray Chamb Temp 50 E KO Nebulizer Press 35 PS 35 0 Damping Gas Flow 0 8 mL min 0 8 The temperatures of all heated zones are shown with th
35. results to be deleted from a specified channel of a Data File Results deletion is logged in the Data File Revision Log This option only affects standard LC results LC MS results are deleted Disable creation of new passwords in Method files When checked new passwords cannot be added to Methods Methods already containing passwords will still require a password before changes are saved 130 Configuring HPLC Modules Overview The first time that you use the Varian MS Workstation to collect chromatographic data and generate results you must configure the communications and modules in the System Control and Configuration Window Set the communication parameters for each chromatograph modules and drag the modules into the Varian MS Workstation instrument area After this is done the configuration is stored and you only need to reconfigure System Control when you change the physical hardware configuration of your chromatograph modules The Varian MS Workstation supports the Varian HPLC modules the 500 MS CVM and the LC Combi PAL AutoSampler Refer to Data Acquisition with LC Control part number 0391473200 for details on the operation of the Varian MS Workstation with Varian HPLC modules It covers configuring LC modules in an instrument for data acquisition building a method and running samples This manual is included on the Varian MS Workstation CD ROM 131 System Control Command Reference System Control Menus File
36. section Selecting the Multiplier Dynode checkbox activates the Multiplier and Dynode parameters in the Trap data area lower Monitor Window section Selecting the RF Control checkbox activates the Waveform System data area lower Monitor Window section Underneath the RF Control checkbox there are two radio buttons RF and Waveform that indicate which elements of the Waveform System to monitor Selecting the RF radio button activates the Entrance End Cap Trapping Field Dipole percentage and Exit End Cap Trapping Field Dipole percentage Selecting the Waveform radio button activates the Dipole Supplemental Waveform percentage monitor and the Quadrupole 152 Supplemental Waveform percentage monitor Selecting the lon Gauge checkbox adds the lon Gauge to the components that are monitored and tested The user may also select which filament is used Filament 1 or Filament 2 by selecting the appropriate radio button Diagnostic Tests Tab The Diagnostic Tests tab has checkbox for Quick Verify This does a system verification check 500 M5 01 Not Ready Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Control and Status Monitoring Diagnostic Tests Start Diagnostic state Idle Start Diagnastic Select all Clear All Function Monitoring Status O Quick Verify Display Display either the Monitor Window and or Event Messages by selecting from the menu next to Hide Keypad Show Keypad Event M
37. sections and click Print Print Method Sections r Sections to Print ProStar Dynamax SystemModule 24 ProStar 410 AutoSampler Module 25 500 MS Mass Spec Module 56 500 415 Standard MS Reports Miodule 56 1 500 MS MS Data Handling Module 56 1 Password Protecting a Method Click Set Password on the Method Builder Toolbar or use the command File Set Password to open the Add Password dialog Add Password e Enter new password cence _ Enter the password and then re enter it to confirm After a Method is password protected it can be used in System Control for instrument control data acquisition and data handling It can be viewed in Method Builder as well However the selected password must be entered to save Method changes It is now locked and can be edited only when the password is known 64 500 MS Method Section Setting Up the MS Method Editor Screen In the Directory point to 500 MS Mass Spec Control and click MS Acquisition Method The Parameters pane shows the MS acquisition method s Method Builder 500 MS ESI CALIBRATION METHOD mih File Edit View Window Help aca ana Hexal el fal 4 gt i Re ssl fico 500 MS ESI CALIBRATION METHOD mth Maok X Instrument Configuration Acquisition Data Type TurboDDS Electrospray y Centroid Profile F TurboDDS Method Setpoint Chromatographic Time Segments Ey 500 M5 Mass Spec Control coh se Era
38. selected row Add Lines If you need to add several similar lines to the SampleList click Add Lines Enter the common information in the dialog box Some fields in the Add Lines dialog vary from one AutoSampler to another Only the fields that are related to the AutoSampler selected are displayed in this dialog Sample type ESL Inj Hwection Noles ot Im Mode at Tay Viel 2 Well Washes Automa Amount Sid o Multiples Divisor uli hanwy Routines 15 NX o ultiStanda nore Pasa Loco Usro Lett Tray Duang The Add Loves Sesser Number Cokmans from A Geough IH Number Plows frome 11 teougre 112 By Cimes Insert Cancel By Rows Number Sample Names free 11 For sequentially numbered Sample names check the box and enter the starting number and the number of entries to add to the SampleList The Sample Names will have these numbers appended to them Defaults Change the default entries in the table such as Injection Volume Injectors Used etc After defining new defaults click Save to activate the new defaults Data Files Change the location and name of the Data Files RecalcList Click to generate a RecalcList associated with single sample acquisitions Specifying the Data File Name and Path Data File names can be up to 255 characters long Sample ID injection date module name and injection number can be embedded in the file name making the Data File name correlate with each sample injection When you click Da
39. size in the appropriate boxes The following example uses a 1 0 mL Hamilton syringe with a flow rate of 10 L min If syringe pump control is specified in the method the flow rate is disabled Method Active Segment Checks and Adjustments Module Attributes Optimization Plots r Electron Multiples High Mass Calibration Last Set Not Applicable Result Default Values Voltage 1200 a walks Adjust high mass calibration Scan Mode OMAN Expected Mass 3500 00 H rz Observed Mass 3500 00 miz Dampina Gas Trap Damping Gas Flow jos l mL min Enter the diameter or select a brand and volume To add a syringe first enter a diameter then type in a brand and volume and click save syringe To delete a syringe select a brand and volume and click delete singe Brand Hamilton Volume 1 0 mL mL Save Syringe Diameter 4 610 j mm Delete Syringe Click Run under infusion pump and check that the green indicator comes on 98 Manual Injection To make a manual injection plumb the switching valve as follows Sample Loop Load Position Inject Position In the Manual Control tab click Edit Method then Setpoints Summary of Status Vacuum System OF Temperatures OF Source OF lon Gauge Pressure 0 0 uToer Fi 1 Wald Trap 3 WE Needle OVoks 0 0 uAmps TRAP ON OFF Instrument Configuration Electrospray TurboD DS F Tuoos Meha C sepon
40. spectrum area To reopen a closed view click the chromatogram button in Select Views to restore it Manual Control Tabs Method Active Segment Checks and Adjustments Module Attributes Optimization Plots I There are five tabs on the 500 MS Keypad NOTE In Manual Control after making a change in the Method tab view you must click Activate Changes to apply those changes to the active method Method and Active Segment Tab Edit methods created using Method Builder in Manual Control You can determine the effect of the changes on the mass spectra Save these changes to the original method or to a new method Refer to Building 500 MS Methods for more information on method parameters The set of tab depends on the ionization type ESI APCI and the scan type Full MS MS etc 32 Method Active Segment Checks and Adjustments Module Attributes Optimization Plots Instrument Configuration Acquisition Data Type TurboDDS Electrospray e Centroid Profile F TurboDDS Method Setpoints Chromatographic Time Segments A al Add Segment Description Scan Description T Insert 1 JESI MS MS men 10 00 ESI Auto Full iran OO D Delete EE EA Ep yi Special Applications In a multi segment method select the segment to edit using the Active Method Segment Number 2 2 esi ms ms In this multi segment example the Active Method Segment is 1 and the Sc
41. store or edit your lists The file name extension is txt Only the information on the spreadsheet mass range and priorities is saved e Open Save As Save Include lists or open them The files name extension is inc and all other information is included such as the Comments Order by selection and if the list is exclusive Include List Priority Mass low Mass thigh Delete x Ol pen Sort by Masses Cancel 2 2 100 0 104 0 ae ee Order by Exclusive List Abundance Priority Comments Import Open Export Save 45 85 e Exclude List Create a list of ions that are not searched Eliminate known background or contamination ions Click Edit to open Exclude List Activate the list by clicking the checkbox e Use the following commands with the Exclude List e Delete Select the row to be deleted and then click Delete e Sort by Masses e Click to sort the mass ranges in order of increasing mass range e Click again to sort the mass ranges in order of decreasing mass range Note Because these ions are always excluded the sort order is not important When the Exclude List is not used the sort order is from low to high m z e Comments Enter comments that are displayed each time you use the list e Import Export Use other programs such as a spreadsheet or a text editor to store or edit your lists The file name extension is txt Only the masses are s
42. the 6 Port Valve indicates the function The syringe pump can be controlled in each segment in this field Theses setting are active in the Acquisition mode lonization Control adjust the Automatic Gain Control AGC target that is the total number of ions the system attempts to generate within the trap up to the Maximum ionization time selected A lower target reduces space charge effects on resolution while a higher target may improve quantitation An optimum value of the target total ion current TIC was calculated and should give good results for a broad range of applications Adjust this by changing the percentage If the Fixed lonization Time box is checked the AGC adjustment of ionization time is replaced by the fixed ionization time selected 146 Method Segment 4 Detector Oni Scan Type Ful Scan Mode Standard Defaults IY Manual FP Syringe Pump On General Parameters onization Control Electrospray lonization Parameters Full Scan Parameters Automatic Gain Control Target TIC 489231 counts 100 Customize Electrospray lonization Parameters If checked the value overrides the setting in the Method Setpoints section Method Active Segment Checks and Adjustments Module Attributes Optimization Flots Method Segment W Detector On Scan Type Full Scan Mode Standard Defaults IY Manual Syringe Pump On General Parameters lonization Control Electrospray lonization Paramete
43. the Scan Type selected For example Parameters if AMD is the Scan Type selected then the tab reads AMD Parameters If Full is the selected Scan Type then Full Scan Parameters Full Scan Change the values of the Full Scan Parameters Low Mass Parameters m z and High Mass m z Also change the capillary voltage and the polarity for each segment These two parameters appear for all scan types When polarity is selected in each row the polarity switches from one scan to the next resulting in data files with two channels each corresponding to the selected polarity An optimum value of the RF storage level is predetermined however it can be customized by changing the percent value in the RF Loading field AMD MS MS Select one of these MS MS type ion preparation modes to see MS n Scan a table that has all available MS MS parameters Each MS MS Parameters isolation dissociation and scanning step follows across the table Precursor lon Enter exact fractional masses Range 50 2000 m z 160 tem Description S Isolation Window Waveform Type Excitation Storage Level m z Excitation Amplitude volts Product lon Start Mass m z Product lon End Mass m z The full mass isolation window range is 1 0 to 14 0 m z The actual range depends on the precursor ion The default value is 3 0 m z Both integral and fractional mass isolation windows are accepted When first developing a method start with
44. the precursor ion After making the change click Activate Changes to see the result Refer to Building 500 MS Methods for more information Select Views 6 Port Valve Infusion Pump Method mE 244 Groans 0 Active Method Segment Number Checks and Adjustments Tab RF Ramp is available in the Checks and Adjustments view Method Active Segment Checks and Adjustments Module Attributes Optimization Plots RF Ramp Adjustment Last Set Date 10 4 2005 2 28 PM Result OK Highest Count 769 Start Action Idle RF Ramp Adjustment The RF Ramp Adjustment determines if the RF voltage is in tune over the entire mass range of the 500 MS To begin click Start and then click Done when the adjustment is completed Open the door to access the RF Adjust on the front panel After starting the routine use a screwdriver to adjust the RF adjust screw Follow the instructions in the Adjustment Results field until the value of the highest part of the graph is at a minimum a 900 445 56 H Heady Marsa Cordial tony Ture Diget Startup Acqueiion ee ae E ten Das Metin Actret Segment Checks arai acierta pc de Altos Optimisation Pisti Saue ES Record Data EF Bamas access RF Rare chur Last Set bate Wot Applicable Mei ES Updo MR Md End Tome 1000 mentos nae Nee ddjusced 5 ot Sin Highest Count 0 Vera Spree DE Diampirg ar Di Tepoa i Source
45. the supplemental waveforms to the end cap electrodes All ions having masses up to and including the mass just below the precursor ion mass are resonantly ejected The supplemental waveforms are then turned off Product lon Formation Precursor ions are transformed into product ions by collision induced dissociation CID The precursor ions constantly collide with helium gas in the ion trap Normally these collisions involve relatively small energies but if the translational energy of the precursor ion is increased the collisions may convert the translational kinetic energy into internal vibrational energy If the precursor ion acquires enough vibrational energy one or more chemical bonds in the ion may be broken and in the case of singly charged ions forming ions of lower m z than the original precursor ion If the precursor ion is multiply 90 charged then the product ions may be observed throughout the mass range The ion distribution that results from the CID process depends on the characteristics of the precursor ion and the amount of energy that has been converted into internal vibrational energy The amount of energy converted to internal energy in the precursor ion depends on the number of collisions excitation time the relative energy of the collisions CID excitation amplitude and the rate at which internal energy is removed by collision deactivation related to the excitation method Collision induced dissociation in
46. to a stage in the scan or a name of a compound Click a Segment Description to update the Method Segment display in the lower window with the details of that segment Start min Indicates the time a particular segment begins Click to edit the Start Time The Start Time you enter becomes the End Time of the preceding segment if one exists End min Indicates the time a particular segment ends Click to edit the End Time The End Time you enter becomes the Start Time of the succeeding segment if one exists Scan Indicates the Scan Type and lonization Type Edit the Scan Description Type in the lower Method Segment window to change the scan parameters and update the Scan Description Add a chromatographic time segment to the end of the Chromatographic Time Segment Table Adding a segment copies the parameters from the last segment into the newly created segment The default duration for the newly created segment is 1 00 minute Insert Inserts a chromatographic time segment before the currently selected row of the Chromatographic Time Segment table Inserting a segment copies the parameters from the currently selected segment into the newly created segment Inserting a segment essentially splits the currently selected segment into two segments of equal duration Delete Deletes one or more selected segments from the Chromatographic Time Segment table However there must always be one segment in the method To delete multiple seg
47. to analyzing the sample are completed Step 3 involves editing the conditions specified in the method for the devices you are using Editing the various sections of the method in the Method Editor save the edits and activate the method in System Control 96 To begin click the System Control button if it is not open n 500 MS 01 Not Ready AE Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Source ESI Edit Method Record Data Method Segment x Method ES Upload MS Method End Time 10 00 minutes M Detector On Scan Type Full v Scan Mode Standard Defau Summary of Status IV Manu I Syringe Pump On Vacuum System OK Damping Gas OK Tempersuas ON SOUICS Ok General Parameters lonization Control Electrospray lonization Parameters Full Scan Parameters lon Gauge Pressure 0 0 uTorr Fil 1 Valid Trap SetPoints Needle O Volts 0 0 uAmps tao on off C Scan Time 341 Maximum Scan Time Multiplier Offset 0 volts DryingGas 0C OPSI Nebulizer 0 PSI pScansAveraged 33 Scans Data Rate 0 29 Hz detector Capillary 0 Volts Spray Shield 0 Volts Customize Housing 0 C Relays A be lt itl Hide Head Trap Select Views gt 6 Port Valve r Infusion Pump Method Segment Active Method Segment Scan Function Channels FT lonG D Position 1 Aun Active 1 4 Data Views dul 2j Defaut Position 2 o Purge nige S
48. 07 E desa BR sy pans 5003 3 i eedle Current ps Dee ar OS Eee Temperatures and Gases Capillary Voltage 78 5 Volts 80 1 Turbo Pumps Setpoint Spray Shield Voltage 610 8 Volts 599 8 Rear Forward Drying Gas Temp 351 C 350 lon Guide DC 5 0 Volts 50 Status Normal Normal Drying Gas Press 15 PSI 15 0 Exit Lens Voltage 8 7 Volts 89 Speed 100 100 Spray Chamb Temp 50 C 50 Focus Lens Voltage 35 0 Volts 35 1 Curent 417 m mps 564 m mps Nebulizer Press 35 PSI 35 0 Deflector 1 Voltage 9 7 Volts 10 2 Power 29 Watts 51 Watts Damping Gas Flow 0 8 mL min 0 8 Deflector 2 Voltage 9 8 Volts 10 2 Temp 33C 40C Trap Focus Lens 169 2 Volts 170 0 Life 145778 hours 618357 hours Trap Offset 14 2 Volts 14 2 16 01 01 Ion Gauge Test Started for Filament Number 1 16 01 02 Ion Gauge Test Passed 16 01 02 Ion Gauge Test Completed Control and Status This indicates the state of the Diagnostics system Idle or Running and the function the Diagnostics system is currently doing The normal function is Monitoring Status but various test functions cycle once the Start Diagnostics is clicked Monitoring Tab This provides you with choices for monitoring lon Trap and lon Gauge components Selecting the lon Source checkbox activates the temperature and pressure parameters of the nebulizer and drying gases as well as the needle capillary and spray shield voltages and ion optics voltages displayed in the lon Source area lower Monitor Window
49. 1 Ready File Edit jee Automation Recalculate Instrument Inject Single Sample i 3 15 6 a 500 M5 56 Ready Menu tem Description o Inject Single Opens the appropriate Inject Single Sample Dialog Box for your system Use to inject a single sample without building a SequenceList and SampleList manually Inject Single Sample M tann Cal Injection Amount Std Unid Peak y om MultiChannel Sample Name Sample Type level Ini Notes AutoLink IS NZ only Factor Multiphos Divisor MultiStandard Default Sample i 1 none none 1 0 1 1 none gt Inject the Sample using the Method C Varianw S startup1 mth Browse Defaults m Cancel Data Files RecalcList For more information on the Inject Single Sample dialog please see Injecting Samples on page 96 134 Automation Menu E System Control Varian LC MS 1 Not Ready File Edit Inject Pee Recalculate Instrument wi 500 MS 56 Manual Control E Status And Contr Run Time 0 00 Resume Automation 1 00 Stop Automation 0 NotBeady 2 Reset Modules No Fault Enable Automated Printing Menu tem Description Begin SampleList Begins executing the currently open SampleList The currently active method downloads and the system will begin to equilibrate and stabilize Begin Sequence Begins executing the currently open Sequence The method specified in the first line of
50. 420 SampleList Defaults Default Sample 4 Cancel Cal inj Injection Austad ink Inj Mode Inj Vial Wash Automix level Notes Volume Volume Routines 1 none none Partial Loopfill v 10 Al 300 none Sample Type Note that neither volume nor rack and vial number is displayed in this screen even if an AutoSampler is present Saving SampleLists for Later Use When you make changes to the open SampleList they are automatically saved to the SampleList file and are used for the automated runs that are in progress If you want to edit a SampleList other than the open SampleList use the offline Automation File Editor application described in the next section Using More Than One Method for Injections The MS Workstation allows you to perform automated injections using more than one Method There are two ways in which this can be accomplished The first is by changing the active Method from within the SampleList The second is by using a SequenceList 113 Changing the Method in the SampleList You may change the Method by activating a Method in a SampleList row Select Activate Method Click AutoLink Ei ProStar 420 SampleList 420_Demo smp SEE Cal Injection kral Inj Matas AutoLink Inj Mode Default Sample 1 none none Partial Loopfill X Default Sample 1 none fnone Partial Loopfill v Sample Name Sampi Type Activate Method B4 Enter location and name of M
51. 600 04 volts Spray Shield Voltage Negative 600 024 valts Drying Gas Temperature Use Ramp Rate Initial Hold Time mir Start Temperature C Ramp End Time mir End Temperature 300 C Ramp Rate 250 00 C min Method End Time 1 40 min Temperature at C cos 145 Active Segment The functions in the Active Segments tab for ESI Method ciiwe Segment Checks and Adjustments Module Attributes Optimization Plots Method Segment AAA ee IY Detector On Scan Type Full Scan Mode Standard Defaults he Manual PF Syringe Pump On General Parameters lonization Control Electrospray lonization Parameters Full Scan Parameters SetPoints f Scan Time f Scans Averaged Data Rate 3 41 1 pican 0 23 Maximum Scan Time Multiplier Offset i volts Hz Centroiding Parameters can Teal E Customize General Parameters Scan Time and uScans Averaged are linked with each other and with the Scan Type lonization and Scan Mode If you adjust uScans Averaged Scan Time and Data Rate change to values determined by the Scan Type lonization mode and Scan Mode Changing Scan Time changes uScans Averaged and Data Rate Select the state of the 6 Port Valve in this section as follows if the Load box is unchecked the 6 Port Valve configured in the Injector mode under the Method tab is in the Inject state otherwise it is in the Load state The name of
52. Also if the active method segment is an MS type the only masses that are plotted for optimization are those produced by the last precursor on in the list Optimization Plot Preferences You can customize the optimization plot to select what type of data to display and how it is appears Click the Plot Preferences icon on the plot toolbar Optimization Plot Bar Plot Preferences 150 2 C R 5 38 Optimization Plot Bar Optimization Plot Preferences a gt Mal Optimization Data Markers Colors Axes M Count Plot 3 30 Filtering 2 Smooth Data Filter Strength edito 200 0 Epia Volts Reset to Defaults gt 00 Save All as Defaults Reset All to Defaults Cancel This example displays raw data only Filter the raw data by checking Smooth Data and selecting a Filter Strength NOTE The maximum value Needle Voltage Capillary Voltage CID Excitation Voltage or RF Loading is only displayed when the Optimization Plot data is smoothed Optimization Plot Bar Optimization Plot Preferences E a gt Optimization Data Markers Colors Axes M Count 5787 2351 pPlot 3 15221 1 C Raw Data Only a Filtered Data Only 231 60 7 C Overlay Filtered Raw Data 30 z Filtering 2 7 V Smooth Data Filter Strength High X 1 1 50 00 50 200 paria Volts Reset to Defaults
53. Chrom Data Review Custom MS Reports B Method Builder v v v v y E mail Oo Micro GC Configuration program MS Data Review MI Polyview 2000 14 Quick Start wo Security Standard Chrom Reports Standard MS Reports System Control Internet Acrobat com Microsoft Office 2007 A Adobe Acrobat 8 Professional IS OY O Adobe LiveCycle Designer 8 0 Adobe Reader 9 Internet Explorer w msn 5 Outlook Express 172 PowerDVD Adobe Acrobat 8 Professional Configure Picasa Pha Viewer ml Workstation Toolbar Microsoft Office Exc Remote Assistance blo Windows Media Player Oracle Calendar 9 0 Disk Cleanup 8 Windows Messenger A Windows Movie Maker AG AM gt Workstation Toolbar P Windows Search Ws UltraMon m Wacom Tablet fan Powertoys for Windows XP gt All Programs 120 Creating or Editing a RecalcList Either choose a New RecalcList or open an existing RecalcList from the File menu Also you can click New or Open Automation File on the toolbar 53 Automation File Editor 26 Edit Help d RecalcList F p SsampleList Sequence Printer Setup Exit ii Automation File Editor Fil Edit Help ELE The RecalcList window for the open RecalcList opens lt contains many of the fields of in the SampleList While the SampleList contains AutoSampler and sample specific data handling in
54. Click a folder to see its contents You can also use the Save In box to see the hierarchy of folders To open the folder one level higher click the up arrow button on the toolbar Click to display a list of recently selected files When you select a file from this list its name is displayed in the File name box 167 Print Setup Dialog Box Specify the printers used by Varian MS Workstation applications both interactively and during automation Select File gt Printer Setup Star Printer Setup Default Star Printer Used by all Star Workstation applications for interactive printing MA BACHY w 3LabzLj2 on Wf3 LAB2 Ll2 Change Instrument Printers Used during automated printing by System Control Instrument 1 AABACHSWSLab2Lj2 on WS LAB2 LI2 Change Instrument 2 AABACHSWSLab2Lj2 on wS LAB2 LJ2 Change Instrument 3 M BACHY w 3LabzLj2 on Ww3 LABZ2 LJ2 Change Instrument 4 AABACHSWSLab2Lj2 on WS LAB2 Li2 Charge tem Description S Default Printer Displays the printer used when printing interactively from Varian MS Workstation applications Click Change to select a different printer Instrument Displays the printer used when printing under automation from Printers System Control Since the Varian MS Workstation is a single instrument workstation the printer specifications for instruments 2 4 are ignored 168 Edit Notes Dialog Box Access this with the menu command Edit gt E
55. F radio button is chosen underneath the RF Control checkbox 153 The percentages of the Dipole Supplemental Waveform and Quadrupole Supplemental Waveform are indicated when the Waveform radio button is chosen underneath the RF Control checkbox Temperatures and Gases This area indicates the spray chamber temperature the temperatures and pressures of the drying and nebulizer gases and the damping gas flow lon Source The lon Source Area indicates the readings and setpoint voltages of the needle capillary and all the ion optics components Startup Shutdown The Startup Shutdown tab provides status and control for the vacuum system and the damping gas pneumatics 500 MS 56 Not Ready Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Status and Control Vacuum System Foreline Pumps Conditions Analysis Pump 1 Pump 2 State Ready Status Normal Normal Speed 100 100 pe Curent 1778 m mps 1778 m mps Status Ready Power 442 Watts 449 Watts E Life 11623 hours 11607 hours Oi Levet Level OK Level OK Damping Gas On Turn Off Flow Rate Setpoint 0 8 mL min Turbo Pumps Flow Rate 0 8 mL min Rear Forward Inlet Pressure 79 PSI Status Normal Normal Speed 100 100 lon Gauge Curent 417 m mps 564 m mps Pressure 19 6 uTorr Power 29 Watts 52 Watts Filament OK Temp 33C 40C Life 145780 hours 618359 hours Foreline Pressure 808 mT on Reset Life Turbo Speed Setpoint 100 Hide Keypad Ev
56. If you know the product ion masses click the Plot these Masses button and enter the desired masses in the Mass 1 Mass 2 dialogue boxes If the masses are unknown click the Auto find masses radio button and select a range to look for the desired product ions Click Start and allow the 500 MS to generate an optimization plot of the desired mass or masses 118 Method Active Segment Checks and Adjustments Module Attributes Optimization Plots Method Segment W Data Collection On Scan Type MS MS scan Mode Standard kai Load E Defaults General Parameters lonization Control Electrospray lonization Parameters MS MS Parameters MRM Capillary RF Loadin Isolation Excitation Excitation Product Product Polarity Yoltage 3 I Window Storage Amplitude lon Start lon End Molts m z Level mz volts Mass m z Mass m z 60 0 100 3 0 57 8 0 66 58 141 ul Not Used Mot Used Mot Used Mot Used Mot Used Mot Used Mot Used Mot Used Precursor lon m z coco m jen me oo a 1 E O TAT AL Y Insert Add g Calculator A Customize Method Active Segment Checks and Adjustments Module Attributes Optimization Flots Dependent Parameter Plot CID Excitation Voltage RIC f Auto find masses with highest intensities 50 00 atl 2000 00 a Ange Mass Range cal mz a mz Plot these masses Stark 0 0 z z Mass 1 H mz 5 0 Default Era SAE Mass 2 m z Step 0 1
57. Instrument Windows Help 5 Q No File el El Instrument 1 Varian LC MS 1 Operator Suspended AutoStart Module 410 al 500 MS 24 56 Instrument 1 Parameters Available Modules The Configuration screen is divided into two sections the Instrument window and the Available Modules window Initially modules are displayed in the Available Modules window as they log in and can be added to the Instrument by clicking and dragging the module icon into the Instrument window When you open System Control the system restores the modules in the instrument window that were there when you last closed System Control Use the Auto Start Module area to designate which module starts the system in Automation e lf you are using an AutoSampler this example uses the ProStar 410 AutoSampler place it in the AutoStart Module area 137 e lf you are not using an AutoSampler or other automated injection device do not place any module in this area Enter an Instrument Name and Operator Name in the Instrument 1 Parameters window Ready File is used only by earlier versions of a Saturn GC MS and should be left blank for operation with 500 MS Max Errors is a number that sets the allowable number of non fatal errors during Automation When the limit is reached Automation halts An entry of 0 disables this feature Prompt on Automation Start checkbox allows you to view and edit information in this dialog each time Automation is start
58. Launching Applications from the Workstation Too0lbar ccoooncnccconcnncccnnnncononcnnononnnonnanonnononcnnonnnnnnnonancnnnnonnnnonnaneness 12 System CONTO esni ia 13 ON DP E E T E ose natn A gas stage aaadeass 13 Navigating the Instrument WINGOW ccccceccccsseeeeecseeeeecaeeeeeeeeeeeesaaeseeeseaeeeeseaeeeessaueeesaeeeeseaeeeeessaueeessageeesseaeeeenaass 13 Control Module WINKOW cccccseeecccecessseceeeceseeeeecseusceeeceassceeeeeausseeeeeeesseceeesaeueeceeesusceeeseauenseeeessaaaceesesaaeeeeessaeees 23 Manual Control Mode wiccccicdescaissascrvesndsaceceisdsscacccscessessednnssseuveatoeudeursesnesGusesisuyeavessiGessaieanessusanntedsadsearscineasversdepsudsenseies 26 AUO TEM leo as ae A NA 41 IAG INO SUGS Mode mesi ares esate a A A A A E A nel aes essa 45 Startup S NutdoWwn ModE naa ida AR 50 ACQUISIION MOGE as EA E N E A E T ace eee sacar 54 B NdNO S00MS Me MOS iuris 58 Creatina a New Melodia id 58 Method BONGE WINGOW a labo cio 60 BOOMS Metodo T a A a a 65 TUFDODDS M D ta ACQUISITION iscaccace iasceade cece Seco s casted Sec wads A aE Ne aaa a 79 NE PIM o q nagitasaasiaienaasaintacasayataiaaasaan a a sa aiantateadesanuaaeaetaaoiates 79 A a siasonsntesnentesasauaseemecdinadedsscotede saangvesieads a 80 Auto TUNE TOF TUrDODDS Maia A A dada 80 Memod BUIde Ior TUDOD DS Ml o ll tesa ads mn 81 USINO AP Olarra Na Ea aaa 87 N Ora a E A E 87 SIETI PERERA AAE en O A E A A E 87 APCI Typical Operating Parameters cc
59. Link Parameters dialog box to set the options for linking to a remote application during automation through System Control Opens the Activate Method Dialog Box to set the path name for activating a new method during automation of a RecalcList through System Control 0 000001 to 1 000 000 0 LC Files Sets the amount of the first internal standard Use to calibrate results for Internal Standard and Normalized Percent calculations Not used by calibration samples MS files Sets an IS Factor used for Analysis and Verification samples It is multiplied by the appropriate Compound Calibration Level Amount that is in the DH Method being used Note that internal standards in Analysis and Verification samples use the amount specified in Calibration Level 1 0 to 1 000 000 0 Sets a calibration factor for unidentified peaks Not used for calibration samples 0 000001 to 1 000 000 0 Sets a value for the multiplier Results for the sample are multiplied by this value Not used for calibration samples 0 000001 to 1 000 000 0 Sets a value for the divisor Results for the sample are divided by this value Not used for calibration samples 170 tem Description MultiChannel None multiple specific channel MultiStandard LC files Opens the Data Handling Channels dialog box to specify the calibration parameters for up to four different Detector Channels MS files Not used for MS data handling Adds a line to the end of th
60. Maximum lon Time for Automatic Gain Control Fixed ion time experiments are used as diagnostic tests and are not for qualitative or quantitative analysis 17 If the ion time in the baseline of the chromatogram is not below the maximum ion time the ion trap does not fill with ions to the level designated by the Target TIC Changing the Maximum lon Time improves the sensitivity of techniques such as MS MS This parameter can be increased to 650 000 usec doing so reduces the data rate APCI lonization Parameters General Parameters lonization Control APCI lonization Parameters Full Scan Parameters APCI lonization Parameters Corona Current Nebulizer Gas Nitrogen V Nebulizer Gas Pressure 5 Positive 5 microamps Negative microamps Y Drying Gas Pressure Spray Shield Voltage Y Vaporizer Gas Pressure Positive 600 0 volts Y Drying Gas Temperature Ma Warning Temperature changes of greater than Negative 600 04 volts 45 deg C may result in longer stabilization times These settings are for the entire method and can be used for many applications You can override and adjusted them for each segment Full Scan Parameters General Parameters lonization Control APCI lonization Parameters Full Scan Parameters Mass Range Capillary Low Mass High Mass RF Loading Polarity Yoltage Woltz mz m z 1 Positive 80 0 50 2000 100 2 Nouse
61. Menu i System Control Varian LC MS 1 Ready wie Edit Inject Automation Recalculate Instrument Windows Help Activate Method Upload Active Method From Modules Mew SampleList Open SampleList MS Method Segment Number Mer PlecalcList Open RecalcList Mew SequenceList Segment Description Open SequenceList Scan Description Print Scan Range Printer Setup w Remember Last Open Files Exit 1 Description Activate Activate a Method and download the Instrument Control Method sections to the modules Upload Active Name the Method that receives the instrument control Method from sections uploaded from all modules connected to the Modules instrument New Name a new SampleList SampleList Open Open an existing SampleList SampleList New RecalcList Name a new RecalcList Open Open an existing RecalcList RecalcList New Name a new Sequence Sequence List 132 Description Open Open an existing Sequence Sequence_List Prints the contents of the active window in System Control Printer Setup Select a printer and set options for it You can open the Print Setup dialog box from the Star Printer Setup dialog box The Varian MS Workstation only uses the printer for instrument 1 Remember Last Restore the active Method and any automation files that Open Files were open when System Control was last closed upon System Control startup if c
62. NOTICE Varian Inc was acquired by Agilent Technologies in May 2010 This document is provided as a courtesy but is no longer kept current and thus will contain historical reterences to Varian For more Varian Inc information go to www agilent com chem 2700 Mitchell Drive Walnut Creek CA 94598 1675 USA et Agilent Technologies 500 MS IT Mass Spectrometer MS Workstation Version 6 software Operation Manual c E R T l F l E D OVarian Inc 2009 Printed in U S A 03 954077 00 6 Trademark Acknowledgments Microsoft Windows Windows 98 Second Edition Windows NT 4 0 Windows 2000 Professional and Windows XP Professional are registered trademarks of Microsoft Corporation DemoShield and InstallShield are registered trademarks of InstallShield Corporation Acrobat Reader Copyright 1984 2008 Adobe Systems Incorporated All rights reserved Adobe and Acrobat are trademarks of Adobe Systems Incorporated which may be registered in certain jurisdictions Other brand and product names are trademarks or registered trademarks of their respective owners COPYRIGHT 2006 2009 All rights reserved Contents COMICS aia aa 1 INTFOGUCTION sacizeri cesarean deen 4 OVA a 4 Additional Manta Sat led at aletnadec 4 Belore Using Your SYSIOM set o E ese inc 5 MS Workstation ADDIICALIONS cuisine 6 MWO as A ected PC PEA ile ov fags tenn a aera peal ce Secs A T sane neon ae 6 Workstation Toolbar OPUONS irera as 8
63. Numbers will be appended to file names if the file already exists Do not include the file extension in the Data File name Directory for Data Files Data File names gt oc VARIAN WS gt DATA Example S TUESDAY Sample 11 Use the following symbols to enter the corresponding variable data to the file name Sample ID Injection number Date Detector Module name Injection Time Method Name Operator Hame Instrument Hame New Folder Drives SB Bae SE ay be lo r Shows the currently selected directory where Data Files are generated Allows you to specify the drive where Data Files are generated Allows you to create and name a new folder in the currently displayed directory Data File Names Allows you to enter the Data File name specification You may enter as many characters as you wish but the final file name including path must not exceed 255 characters in length You may embed the special symbols listed in the window to represent sample specific information in the Data File name As you enter the specification an example file name is shown Accepts the name specification Cancels the name specification 178 RecalcList Generation Dialog Box RecalcList Generation fou can automatically create or update a RecalcList with files generated during automated injections Specify the RecalcList generation options for this SampleLlist below Do not automatically create and updat
64. Numer Ed E pee En UN al lol S 12 my il 100 241 9 50 3 750e 6 e lt 2000 Acquired Range m z ESI Startup Shutdown Startup Click the Trap check box in the 500 MS View to turn on the gases and heaters If the method requires the infusion pump set the drying gas to 10 15 psi see the following sections If the method requires flow injection or uses the LC pumps set the drying gas to 20 psi or greater Allow a few minutes for the temperature and gas flows to stabilize before beginning analysis Data Views a S Ol A Shutdown Turn off the flow from the LC pumps and allow the LC pump pressure to drop and stabilize so the flow into the ESI stops Click the Trap check box to turn off the gasses and heaters The Analyzer turned off automatically when you turn off the trap 97 Pump Infusion and Manual Injection Infusion with Syringe Pump Use the motor driven syringe pump on the 500 MS to infuse samples into the source for purposes of tuning and optimizing electronic and physical settings Typical infusion rates are 10 20 uL min Connect the syringe to the luer end of the UCP 659 adapter and connect the other end to the PEEK nut on the PEEK line going into the MS Load a syringe with the sample at a suitable concentration approx 1 ppm in an appropriate solvent Set the syringe type in the 500 MS software by clicking the Module Attributes tab and selecting the syringe type and
65. Partial Loopfill 10 Fight gt AutoLink Parameters Command Other parameters Browse Injection Mode Select No Injection Partial Loopfill Full Loop or uL Pickup from the menu Inj Volume The 460 LC comes with a 100 uL sample loop Injections can be made with 1 uL increments Tray Create a sample list for either the Left or Right tray Vial Well Enter a combination of a row letter and a column number The available rows and column numbers depend on the type of plate configured e 96 well plate A1 through H12 e 384 well plate A1 through P24 112 Wash Volume Enter 1 to 9 washes Automix Routines Use these routines only with the 84 3 Adapter Tray Combi PAL SampleList The Combi PAL has settings for Tray selection Vial position and Injection Volume in addition to the features of the Generic SampleList All other sampling parameters are specified in the Combi PAL section of the Method EN CombiPAL SampleList GC_Pal_ Demo smp level Volume IS Sample Name Sample Type Cal Inj ee AutoLink Tray Vial niection An Default Sample Analysis Die Deere rio S are m oe Datspies Reactie Changing Default SampleList Entries When you add a new row to a SampleList default values are entered To change the default values click Defaults in the open SampleList window The following dialog box opens Enter the desired default values and click Save Set ProStar
66. S Method Operating Conditions Temperatures Run Time 0 00 minutes Start Acquisition Segment Number 1 of 1 Data File Drying Gas Setpoint End Time 10 00 minutes Edi Method Segment Descnptor Scan Number D Ready Scan Descipborn ES Auto Full lonization Time O NoFaut M Wait For Readyln Scan Range 100 2000 lon Count 0 x Hide Keypad Spectrum and Chromatogram Default View a lo 07 87 Aj E ue aj 0 S WS a TO The yellow Not Ready light turns green Ready to indicate that the 500 MS is ready for analysis If other LC modules are not ready Not Ready messages are at the top of the System Control window After all the hardware modules come to a ready state the Not Ready messages change to Ready To determine the ready states of each component go to the top pull down menu under Windows and see the states for the modules 94 Windows Help Show Module Windows Iconize Module Windows Show Automation Windows Arrange Icons Instrument 1 Status Message Lag Prosh r Dynamax 24 Ready After all components are ready you can start an analysis An analysis can be run as a single sample or through an automated sequence To run a single sample see Injecting Single Sample on page 101 To multiple samples see Injecting Multiple Samples on page 105 Status and Control Status And Control Aun Time 0 00 minutes Start Acquisition End Time 10 00 minutes Edit Met
67. Segment Checks and Adjustments Module Attributes Optimization Flots Electron Multiplier High Mase Calibration Last Set Not Applicable Adjust high mass calibration Scan Mode IOMA Expected Mags 2500 00 mz Observed Mase 9500 00 a mz Result Default aluez Voltage fi 200 volts Damping Gas Trap Damping Gas Flow 08 j mL min Infusion Pump Syringe Flow Infusion Flow Fate fi 0 00 aL rir Purge Flow Fate 200 00 UL mir Syringe Type Enter the diameter or select a brand and volume To add a syringe first enter a diameter then type in a brand and volume and click save syringe To delete a springe select a brand and volume and click delete sprInge Brand Hamilton Volume 1 0 mL mL Save Syringe Diameter 4 610 mm Delete Syrinae 149 Auto Tune Auto Tune is an automated process for optimizing the 500 MS performance over the entire mass range After performing major maintenance such as cleaning the ion trap or ion source run all Auto Tune routines If you have the TurboDDS option Auto Tune in TurboDDS mode before acquiring High Resolution or Extended Mass range data files a 500 MS 01 Not Ready Manual Control Suto Tune Diagnostics Startup Shutdown Acquisition Control and Status Auto Tune Method Action Idle Select All Clear All State Idle IM Detector Setup Integrator ero Start Auko T Set Electron Multiplier art Auto T
68. TIC Total lonization Count is predetermined for each Scan Type You may customize this parameter by changing the percent of its predetermined value This component is disabled if you choose the Fixed lon Time checkbox in the Customize area Generally used for diagnostic tests and not advised for lonization Time qualitative or quantitative analysis in the ion trap Choosing this disables both the Automatic Gain Control AND the Prescan lon Time functions and uses the Fixed Ionization time you entered Max lonization Changing the Max lonization time can improve the sensitivity of Time techniques such as MS MS If the ion time in the baseline of the chromatogram is at the maximum ion time the ion trap may not be filled with ions to the level designated by the Target TIC The number of ions in the trap can be increased by increasing the Maximum lonization Time If you take the second approach and are collecting data for quantitation make sure that enough data points are collected across the chromatographic peaks for reliable quantitation At least five but preferably seven or more data points are needed for good quantitative data The ion signal but not the number of ions in the trap can be increased by raising the multiplier voltage Range 10 650000 microseconds Electrospray Override the ionization method parameters edited under the lonization Setpoints tab for each segment Parameters lt Scan Type gt Appearance depends on
69. The restoring force of the trapping field converts the newly increased potential energy of the ion into increased translational kinetic energy A portion of this kinetic energy is then converted into internal vibrational energy upon subsequent collisions This process is repeated during each oscillation cycle of the low frequency dipole field An advantage of using non resonant excitation is that it is not critical to match the applied dipole frequency to the ion Consequently electronic drift space charge effects and sample concentration do not affect the method This results in reproducible product ion spectra This method is often useful with precursor ions that fragment by the breakage of a single weak chemical bond to form highly stable ions containing functional groups that do not undergo significant rearrangements A disadvantage of the method is that it is not selective with respect to excitation of ions in the trap Therefore the method cannot be selectively tuned to excite only ions having a particular m z This method is less useful with precursor ions in which multiple chemical bonds are broken and with precursor ions that undergo complex rearrangements following collision induced dissociation The second method for increasing the precursor ion kinetic energy involves resonant excitation This method requires application of a high frequency supplemental dipole field to the end caps of the ion trap The frequency must match the oscillati
70. a storage level of 48 m z and with 9 CID frequencies the mass range increases from 3 to 40 m z when the precursor ion mass increases from 100 to 400 m z Given a precursor ion mass of 400 m z and 9 CID frequencies the mass range decreases from 40 m z for a storage level of 48 m z to a range of 12 m z for a storage level of 143 m z Product lon Mass Scanning Following formation of the product ions by collision induced dissociation an RF ramp is used with the supplemental waveforms to scan resonantly ions from the trap to the conversion dynode and electron multiplier This process generates the product ion mass spectrum MS MS on the 500 MS The AMD scan type is a specialized scan function for Automated Methods Development It allows a scan function to be built quickly if the only difference between microscans is the CID amplitude The method parameters can be separated according to each step The ionization parameters are identical to those for full scan Some are on the ionization control tab and others are included in the MS MS parameters table Basic Parameters These are available on the MS MS tab of the method without clicking Customize Precursor lon m z The specified ion on which to perform MS MS Values range from 50 to 2000 Da Fractional masses are allowed Polarity Sets the polarity of the detected ions either positive or negative 93 Capillary Voltage Volts Effects the focusing of ions into the capillary as we
71. activate a method change Restore Click to Restore a previous method Active Method Segment Scroll to active individual method segments Full Scan Displays scan range while acquiring data Scan Function Channels Select a scan channel or cycle through them IN Intensity Normalization Click to normalize intensity By lonization Time or None Intensity Normalization By lonization Time C None If you select none the N is red and Data not normalized appears in red above the icon Data not nomalized 30 Hardware and Data Controls Hide Keppad Trap Select Views 6 Port Valve Infusion Pump lon Gauge lx sof Default gt Position 1 Run Data Views 1 E Position 2 Purge Trap Enable to turn the on trap on lon Gauge Turn on the ion gauge to monitor the trap pressure 6 Port Valve Switch the valve position Infusion Pump Start or stop the syringe pump with the flow rate set in Module Attributes Purge Turn on the pump at the Method purge flow rate Data Views Select to view Spectral and Plot screens Module Attributes Select to view Instrument Module information Gal Click to display Spectrum and RIC EE Click to display Optimization Plot Default Click to display Spectrum and RIC Chromatogram and Optimization Plot Screens Check either screen to open the plot screens adjacent to the real time spectrum plot screen The Chromatogram plot screen continuo
72. aintenance Before doing Auto Tune perform the RF Ramp Adjustment under the Checks and Adjustments tab in Manual Control mode Tune the 500 MS in ESI mode and use these tune settings in APCI mode You adjust 500 MS parameters voltages gain values etc to optimize the signal while infusing the tune solution NOTE Tuning Solution for 500 MS part number 0393210201 Tuning Solution for TurboDDS and Extended Mass range part number 0393211901 The Tuning Solution for non TurboDDS and non Extended Mass Range work contains the following singly charged ions e Tetramethyl Ammonium Bromide m z 74 1 e Tetrabutyl Ammonium Bromide m z 242 3 e Tetradecyl Ammonium Bromide m z 578 7 e Ultramark m z 1322 0 m z 1922 0 also provides ion with these m z values 1022 0 1122 0 1222 0 1422 0 1522 0 1622 0 1722 0 1822 0 The following is a typical spectrum of this tune solution a lo a id 5 m7 ul Scan Number 233 Time 7 852 min Base Peak 242 2 Base Amount 46087 SMOOTHED RIC 5 848e 5 lon Range 49 5 2000 5 m z lon 1872 us Segment 1 Channel 1 242 3 1 2000 Acquired Range m z Before doing an Auto Tune the 500 MS must have a stable infusion flow the trap should be on and the drying gas should be stabilized at its set point Use the following procedure 1 Ensure that the infusion pump syringe has at least 200 uL of tuning solution In Manual Control mode activate the method 500 MS ESI CALIBRATION METHOD
73. al standard calculations the unidentified peak factor a multiplier and a divisor can be entered directly into this table If you have more complex requirements such as multiple internal standards or multiple detectors requiring different entries click the button in MultiChannel MultiStandard column Each AutoSampler is supported by its unique SampleList A SampleList may have more than one AutoSampler section and may be used for different AutoSamplers The information entered for one AutoSampler section of the SampleList is not displayed or used in other AutoSampler sections SampleLists are the method by which an AutoSampler program is created since AutoSamplers operate on a per sample basis as opposed to the time resolved method for pumps and detectors ProStar 410 SampleList In addition to the Generic SampleList features that were described earlier the PS 410 supports a variety of other features such as different Injection Modes Vial specification and AutoMix functions 106 Use the AutoMix Routines button to access the parameters for diluting mixing and transferring aliquots from one vial to another E ProStar 410 SampleList Demo smp a Volume Routines Default Sample none Partial Loopfill 1 none Partial Loopfill Full Loop Sample Name AutoLink Inj Mode Inj Vial Washes Automix 4 Action Amount Reagent 1 time Destination Vial none Y Automix First Injection Only ok Cancel Mixtt y F fault A
74. al Control then uploaded and saved under the same name or as a new named method The important distinction is that the effects of changes made in Manual Control are seen immediately in the real time spectrum display using LC tuning solution ions as a sample Control and Status Control and Status shows a graphic of the ion trap system and status of the needle and capillary voltages drying and nebulizer gas pressures and temperatures Click trap in the graphic display to turn on off the ion source RF system and the multiplier Clicking the ion sources RF and multiplier turns these parameters on off individually Edit Method Edit Method opens Method Builder with the method currently active in System Control Upload MS Method Upload MS Method opens a Save Method dialog to allow the MS method parameters currently active in System Control to be copied to an existing method file or to anew method name Changes made in Manual Control must be activated using Activate Changes before they can be uploaded Save Data Save Data allows acquisition of data in Manual Control mode while the ion trap system is turned on by clicking the Trap checkbox The acquisition time is set by clicking the End Time tab Once the data acquisition starts by clicking the Start Acquisition tab it can be stopped by clicking the Stop Acquisition tab When the acquisition stops the Create Data File dialog opens for creating a data file name Checkboxes Cli
75. alues from 0 1 to 0 6 The software automatically enters a value based on the precursor ion m z Excitation Amplitude Determines how much energy is imparted to the ion during the CID process Higher values give the ion more kinetic energy making collisions with the helium damping gas more energetic If amplitude is set too high the ions are ejected during the excitation step and no product ions are formed The optimum value depends on the stability of the ion and the excitation storage level Allowable values are 0 to 59 79 volts for resonant excitation waveforms and 0 to 119 58 volts for non resonant excitation waveforms The software automatically enters an approximate value based on the precursor ion m z and the excitation storage level Product lon Start Mass m z Specifies the lowest mass that appears in the MS MS spectrum Values range from the excitation storage level to the precursor ion minus seven Daltons Product lon End Mass m z This value specifies the highest mass that appears in the MS MS spectrum Values range from the product ion start mass to 2000 Daltons More Ranges Click the more ranges check box to specify non contiguous mass ranges for the MS MS spectrum Possible ranges are the start and end masses described above with the additional constraint segments must have a minimum of 10 Daltons between them The following parameters displayed when customize is clicked allow for control over a wide range o
76. am Spectrum Optimization Plot Displays 13 File Menu iJ system Control Varian 500 LCMS Not Ready wie Edit Inject Automation Recalculate Activate Method Upload Active Method From Modules Instrument Windows Help Hew SampleList Open SampleList lartup S hutdowrn Acquisition Mew RecalcLisk Data Open RecalcList tord Data Positive Mew Sequence ist ad Time 10 00 minutes Open 5equenceList A Print Printer Setup Remember Last Open Files Exit Activate Method Open an existing method Upload Active Method from Modules Save conditions of an existing module to a new or existing module This method becomes the active method with the conditions of the active modules New SampleList Create a SampleList Open SampleList Open an existing SampleList New RecalcList Create a RecalcList Are RecalcList Open an existing RecalcList The RecalcList dialog box ollows NOTE Process a RecalcList either in System Control by clicking Begin or in MS Data Review In MS Data Review use Process Data to access this feature Mi RecalcList recalc rcl 0 mates sewn E EE a New Calib Block w c varianws mstutorials 1 0_ng sms ALLMIX 10NG Calibration c varianws mstutorials 20_ng sms ALLMIX 20NG Calibration c varianwes mstutorials 40_ng sms ALLMIX 40NG Calibration c warianws mstutorials 80_ng sms ALLMIX 80NG Calibrat
77. ames Remove the association between a Module Name and a Module Address You can connect a Module with a different Module Name at that address The next time the associated Module connects at that address you are prompted to select the correct name for the Module Restore Disabled Warnings Enable warning message you disabled Instrument 1 Faults See if there are faults in the modules in Instrument 1 except for the 500 MS An example follows Use the update key to check for new faults without opening dialog The 500 MS error messages are displayed in the Message Log Instrument 1 Faults Module Address Faults Module ProStar Dynamas 24 1 Fault s Module 410 25 O Fault s Module 5000 56 0 Fault s f Recent Messages and Faults C Selflest Messages and Faults Setup Ethernet Communication Set up communication between an Ethernet controlled device such as a 335 photodiode array detector and System Control 18 Setup COMM Ports Set up communication between a COMM Port controlled device such as the ProStar 430 Autosampler and System Control Windows Menu View the Windows associated with System Control see the following Instrument Show Module Windows lconize Module Windows O otto wind tartup S hubdor Arrange Icons Data Instrument 1 Status zord Data Message Log ad Time 1i 430 01 Ready 212 02 Ready w 500 M5 03 Not Ready de ma Gas OK Show Module Windows View the Module Windows configured i
78. ameters TurboDDS Trigger Trigger Conditions Include Exclude Conditions Dynamic Exclusion Threshold 8600 counts Minimum BE Intensity Ratio rx JE Isotopic Equivalence 33 m z Include List Exclude List M Use Dynamic Exclusion Use Include List T Use Exclude List Exclude After 23 scans Restore Never Restore C After 14399 mir Charge State _________ Y Exclude Charge State 1 Exclude Charge State 2 Edt e Include List You can use the Include List as an Exclusive List or a Non exclusive List Click Edit to open Include List Activate the list by clicking the checkbox e Exclusive List The listed mass ranges that exceed the threshold are the only ones searched This ensures that the target ions even if they have low abundance are selected lons not on the list are displayed only on the survey scan regardless of how intense they may be Select ions in order of abundance or priority e Non exclusive List The software searches for the listed mass ranges that exceed the threshold If no listed ions are found other ions in the survey scan are selected This ensures that in addition to possible low abundant target ions other ions from the survey scan are analyzed if the listed ions are absent Select ions in order of abundance or priority e When Exclusive List is not enabled the listed ions are searched for first and then ions that are not on the list
79. an Function Channel is 1 Notice that the spectrum has this information in the upper right corner ae Hide Keypad Hide Keypad L Trap Select Views 6 Port Valve Method Active Method Segment r Se ee e small ogra o E se ran eel A lo al 14 M ui C Scan Number 591 la 8 290 min i 00 1000 1500 Acquired Range vz In this multiple reaction monitoring MRM example segment 1 is the active segment The MRM of two ions creates two channels of spectral data Use the Scan Function Channel to select the spectrum of channel 1 Notice that the upper right corner of the spectrum has this information Click cycle to alternate the display between the channels M Trap r Select Views 6 Port Valve m Infusion Pump r Method Segment Active Method Segment Scan Function Channels IV lon Gauge A aj Defaut O Position 1 o L_Stop_ Activate Gestore Number H Channel 1 1 IN Data Views y Position 2 Purge nanges Ee 0 mm 4 tlo a i Bm ur Scan Number 20 Time 0 213 min Base Peak 438 7 Base Amount 462162 SMOOTHED RIC 6 695e 5 lon Range 161 5 589 5 m z lon 1295 us Segment 1 Channel 1 100 75 50 25 200 300 400 500 Acquired Range E z Instrument 1 Jun 19 15 37 53 Module 500 MS 56 End Auto Tune Check Some Auto Tunes not up to date 33 Data Views In this example the excitation storage level is increased for channel 1 to eliminate
80. an 1914 44 47 SampleList apci smp Activated Jan 19 14 44 47 Method 4PCL reserpine 60S Brin mth Activated Message Log entries are stamped with the date and time they occurred Automated MS Report Generation After adding Report sections to your Method automated reports can be generated from System Control after each injection after Recalculations or by using any of the Print actions in the Sequence window You can disable automated Report printing during the course of an automated sequence of injections or Recalculations Disable automated printing from the Automation menu in the Instrument window Automation Begin SampleList Begin Sequence Begin At Selected SampleList Line Bean At Selected Sequence ine Suspend Automation Resume Automation Shon Automaton Reset Modules Enable Automated Printing When this item is not checked report printing is disabled ASCII file generation is still performed if specified in the Report Method section Disabling automated printing is like to disconnecting the printer data is acquired but no reports are printed Stopping an Acquisition During automation and while the LC MS system is running you can suspend or stop data acquisition as follows Suspend Automation Suspends execution of automation after the current run has been completed Stop Automation Stops the current run resets all modules simultaneously and Suspends automation Resume Automation Resumes e
81. annel of the currently selected data file This does not appear if disabled by the MS Workstation Security Administrator NOTE only affects standard LC results MS results are not deleted Opens the selected data file using the selected channel Cancels file selection 174 SampleList Window Contains injection parameters specific for your sample introduction device This section describes the generic SampleList fields Refer to the appropriate Instrument Control help topic in the System Control Reference Help for a description of the device specific extensions to the SampleList sii Automation File Editor Untitled smp File Edit Help Cal Injection Amount Std Unid Peak y MultiChannel Sample Name Sample Type level Inj alee AutoLink IS N only Factor Multiplier Divisor MultiStandard ae Default Sample 1 none Add l l Insert Delete Fill Down Add Lines Defaults ttem Description Sample Name Up to 19 characters Sets the name of each sample in the SampleList Sample Type Baseline Analysis Calibration Verification Print Calib New Calib Block AutoLink Activate Method Sets the sample type or automation action of each line in the SampleList Cal Level 1 to 10 Sets the calibration level of each calibration or verification sample in the SampleList Inj 1 to9 Sets the number of injections to be made of the sample Injection Notes Up to 180 characters Opens the Note
82. ar J a 5200 45 0 Nat Ready Marc Coral Agi Tune Diagnosis Stata tha Acquiton Uani rad Sesh Eve Dele Method Actiee Segrer Checks and Achuctments Mods Abu Optimization Plats Source ES Fecod Date Deperdera Parets y Por P lt gt k Cipli ode mt MebodES Ueinadi MS Meta End Tima UOD menue UE Auto llr aiae athe hegheal pierra rauy d 498 A gt mDM _ _ Mata Flange pm 2d yz foe Te wr Waouum Ester DF Dismparo Gar T Fess ad Tepai DK Dana Er oik fe 1 A Fibi hene maie Maia 50 int laa Gauge Preise Outen Fl Wa End ENIE niai l Tas H mm T Mage 5000 at To Hag 9000 nk F hmi g 0 a omy Mata 5 ao Ju Mende Ove Outage mooi sfa 3 Dygant OPS a Nabulos OPS Pr Hep deigh be dro dj fem lo sm ms bibet ca AE pem a vaier EE tna eg o A aa a Pee q AERA B vauni CAB Lf a par EL Chareal 1 lq ri AA y E HL LALA i i ee Geer E ft A Splitter Bars Splitter Bars 3 wog iret ara T mari e Instrument 1 Jun 8 13 32 06 Module 50045 02 5 1 1 1 1 Control and Status The 500 MS Trap Icon A graphic picture of the trap on the left displays the status When the on trap is off most of the text is black indicating that the on trap is off The spray chamber housing temperature is always on and green 26 Manual Control Aut
83. ata collection you can switch to different Method segments and change individual Method segment parameters to observe and record the data Click End Time to increase the run time if necessary When the End Time is reached or when the Stop Acquisition is pressed the Create Data File dialog appears and you can specify a file name and a location The data file contains only raw data so no log information is stored NOTE The Record Data operation does not operate the hardware lt does not automatically turn on the trap switch the 6 Port valve start or stop the infusion pump etc You must do these operations before during or after data collection Relays In Manual Control click to select a relay Set up Relays in Special Applications in the Method Builder See the special applications section of the Method Builder for information about how to set up the Relays Relays rirerars 29 Quick Control Toolbar The Quick Control Toolbar has controls for the method hardware and data Hide Keypad W Trap Select Views 6 Port Valve Infusion Pump Method Segment Active Method Segment Scan Function Channels M lonGauoe ajf gt Detour Position 1 Stop Number Channel 8 1 Data Views a Jl Vo Position 2 2 _ Purge cat Tht Full Scan F UNI Method Controls Method q Active Method Segment y Scan Function Channels Activate Number 1 hand tl Changes Restore E che eat Activate Changes Click to
84. aved e Open Save As Save Include lists or open them The files name extension is inc The Comments and Order by entries are included Exclude List ES Delete OK Sort by Masses Cancel Comments Import Open La Export Save S 86 Using APCI Tuning Optimization Tune and mass calibrate the 500 MS mass spectrometer using the Electrospray lonization ESI chamber with the 500 MS Tune Solution part number 0393210201 Use the ESI tune file for acquiring APCI data For specifics regarding tuning refer to the Tuning section of the 500 MS manual The detector response varies for each type of analyte and depends upon the mobile phase composition and flow rate Usually analytes are introduced by flow injection or infusion Use the syringe pump at less than 100 uL min Adjust nitrogen flows and temperatures while performing flow injection or infusion of the analyte to achieve maximum signal to noise At low flow rates there can be arcing to the shield The most significant parameter is the vaporizer temperature Its optimum value is a function of the sample experimentally determined the mobile phase composition more water requires a higher temperature while a higher percentage of organic solvent requires a lower temperature and the flow rate a higher flow rate requires a higher temperature If the temperature is too low droplets are observed at the outlet of the vaporizer tube If the tempe
85. cable Method ESE Upload MS Method End Time 10 00 mates Result Not Adjusted Highest Count O Surrmnaty of Stabos g Vacuum System OK Dameing Gas OK Temperatures OK Source OK fi rar y Cancel AS Mesas lon Gauge Presse 19 5 ulcer Fill Vahd A z x Adpetmen Resuts 119 y fAdust RF coil tuning to obtain a straght ine in the Neede 5000 Vols 1 6 ulnps cua echal display and minimize the size of the bar graph r TIP l Once the rinmumis found adult the cof 1 9 of a tun f s 3 Oning Gas S0C 15PSI icourterchockwise y Nebulizer 35 PSI Er 4 gt DEJECION y BE Housing SOC Capilery 80 Vate Spray Shield 600 Volts fo Solect Views 6 Pot Vaive Intuson Pump l Active Molhod Segment Soan Function Channels f Jl gt Detout oo o Number A Channel t N Data Views X ns Q Poston 2 _ Pisge haa at Fil Scar i A 0 aj gt mA yn Scan Number 18 Time 0 072 min Base Peak 1999 9 Base Amount 9014 SMOOTHED RIC 2 8690 8 lon Range 199 6 20005 m z lon 651 us Segment t Channel 3 100 gt m peee nz 5 a es ieee 50 a eee eel 35 a q nn DS 250 si 750 1000 1250 1500 1750 2000 Acquired Range mz instrument 1 Jun 19 15 37 53 Module 500 MS 56 End Auto Tune Check Some Auto Tunes not up to date 148 Module Attributes Set the electron multiplier voltage damping gas flow the motor driven syringe attributes and infusion and purge flow rates Method Active
86. ccccccccccsesseecceeeeeceeeeseeeceeeceeeasseceeeeeeeaeeaseseeeeeessseaesceeeeeeesssaaaaeeeeeeeessuaassess 88 Comparison of Parameters for ESI and APCI cccccccccccseeeeeeeeeeeeeeeeeeeseceeeeeeeeeeeeeseeeeeeeeseseeeeesaaeeeeeeessaaeeeeeessaaees 88 MS MS Tandem Mass Spectrometry cccccessccceseecceneecceneescnseecenneesensesenseesenseesenseesensesses 89 STII aerate eR PORE OT a ET TC PRR Ear Ren ne RR 89 MS MS Ome 00M Sua ati nae E A ee een meee ee 93 IIE CUNG Samples cuca cias 96 OVEMIE Wo dd probada 96 Ol SLU SU AN adds ceo ea eames 97 FUmp IMIUSION ana Manual Meco Marica dolida 98 NECM SMS IMP ii IA DAA AAA A A A A a A AA 101 Injecting Multiple Samples iaa 105 MONO di gf 6 AUR Salsa odos lll did o cd ico 115 Automated MS Repo Gener ON uta ile IAS aL DO EE AA IAS E DOES N 116 SOP DING AM ACQUIS MO A EA AA A A TA 116 Settingra COlleCyDIVeN DO Vi A A A A AR 117 MSMS OBUIMIZA MON iii tii das decada dica iia 118 AUTOMAton Fie EQU cuicos tedutnceteccaecuteswetsunscuensedsenencndeceets 120 OI isa 120 reatingi Or Edita RecalchiS tapas lil 121 Gr eatind Or Editind a SamplellS tus ista 122 Creating OF Eding a Sequence isidro 127 SECUTITY ACMINISTAO Nu eee a a a a aa a 128 OVENI W idad Pee 128 PASS WO IS et ed 128 PAPO ATI IA NEO SI Mura idad 129 Pile REVISION SUN Senet Siete A oa ee aie 130 Configuring HPLC Module siisii ici 131 WEIEN ech etc a eal case rte id 131 System Control Command Reference cccsssc
87. ccesseeeseeceeneesenseesenseesenneeseaseesensessensesses 132 SE iGo oh aie a a Sa ite hate ll eh as 132 PIS DAY ii RN aati genta amauta A Era 140 OEM Wo ene a os 141 Manual CONO lia a 142 AUTO UN ac a ios a a lios teak eteasewauetelaets 150 DIagnos Sinai iria 152 Stanu gt NUI WN ai A Ge see ee te eee 154 ACQUIS ION iio 155 Method Builder Command Reference s cccssseccsseeccesseeeeneeseenseeenseesenseesenseesensessonnens 157 Automation File Editor Command Reference ccsssccceesecceeseecesseeseeseesenseesensessensesees 164 ALT Seem rene sm aoe ner re ree rt POR Se NOV Ra RE RC eee nO e 164 Maim TOODA Presa reer neat ett a NPR PRR Aa Sen eR SS Cae ee Neen 165 Open Automation File Dialog BOX a a eh eh i al ais 166 Save Automation Fileas Dialog DO a a os e a le eee 167 PRIME Set OD Didog DO il os 168 Edit Notes DIA OG BOX ra A E lies 169 FLCC AIC M INGO Wi a el e ie 169 Data Handing Channels Dialog Box id ici 171 SU e o ee eee eee 175 Jn a a a e e a 180 Introduction Overview Varian MS Workstation Version 6 9 controls the Varian 500 MS associated Varian chromatography modules and the LC Combi PAL AutoSampler Use the workstation to manage MS hardware and data acquisition display reduction and analysis This manual in association with the 500 MS Hardware manual the MS Software Reference Manual and the manuals for each of your LC modules and AutoSamplers provides you with the essential i
88. ck the Trap checkbox to turns on the entire ion trap system Click the lon Gauge to display the pressure inside the trap 142 Special Applications E Port Valve C Manual C Diverter i Injector Load position W required for ready state 6 Port Valve This indicates the active solvent sample flow path for each configuration of the 6 port valve Select by clicking the Special Applications tab in the Method field E Syringe Pump re ga Syringe Flow Rate 110 00 microliters Armin Filling sm Adapter i Tum Off at Method End Relays Use Relays LC Pump gt To API Low Position a Inject Position Defaults Restore ox Cancel This manual and the Hardware Operation manual have information about the 6 port valve Click the yellow button to the left of Load and Inject to activate the valve b Port Valve Load Inject The names of the two possible states depend on the selected configuration of the 6 port valve The Manual configuration names are Position1 and Position 2 The Diverter configuration names are Divert and MS The Injector configuration names are Load and Inject The 6 port valve state that is active in the Acquisition mode is controlled on a segment by segment basis by checking or un checking the Divert Load checkbox under the Active Segment Syringe Pump Use the built in motor driven syringe to infuse samples into the source while tuning and optimizing electronic an
89. ct Single Sample Menu iJ system Control Varian 500 LCMS Mot Ready File Edit tite Automation Recalculate Instrument Window Inject Single Sample t Inject Single Sample opens a one line SampleList The following dialog box appears when this command is invoked Click Inject bottom left corner to inject the sample by manual injection infusion or autosampler The 500 MS automatically switches to Acquisition mode See Injecting Samples on page 96 Inject Single Sample fx Cal Injection Amount Std Unid Peak uu ds MultiChannel sample Type level Ini Notes AutoLink IS NZ only Factor Multiplier Divisor MultiStandard Default Sample Analysis hone 4 b Inject the Sample using the Method C WarianwS startup1 mth Browse Defaults m Cancel Data Files RecaleList Automation Menu The Automation menu list iJ System Control Varian 500 LCMS Not Ready File Edit Inject Pie Recalculate Instrument vy t Begin SampleList alela E ns AAA Begin t Selected SampleList Line Manual Control A Control and Status Source ES E Stop Automation Method ESI U Reset Modules Summary of Stat Enable Automated Printing 1 ri Begin SampleList Automation starts at line 1 and ends at the last line of the active SampleList Open SampleList from the File menu to use this feature Begin Sequence Change methods and SampleLists during automat
90. ction P3 0UT 0 hex Activity Ready No Fault Temp 27 C Hide Keypad Cooling On Advance Tray Hardware A double click on a vial position will cause the Inject Single Sample dialog to be displayed 104 Using Inject Single Sample for Priority Samples Use either the graphical vial display or the Inject Single Sample dialog to set up a priority sample in conjunction with automated analyses To inject a priority sample 1 While automation is running click Suspend on the current active SampleList Automation is suspended after the current run is completed 2 Double click the vial position where you placed your high priority sample or choose inject single sample as described above and perform your injection 3 After completion of the priority run you can resume the SampleList that you were running by clicking Resume on the bottom of the SampleList Injecting Multiple Samples Your instrument should be configured and you should have a basic method for data acquisition If this has not been done please refer to the appropriate sections of this manual for further instructions Using a SampleList in System Control You can inject multiple samples from System Control by using the SampleList Edit Inject Automation Recalculate Activate Method Upload Active Method From Modules New SampleList Open Samplecist Open a New SampleList or New Recalclist n existing on an existin
91. d Count Threshold are regarded as noise and are not saved during the run of centroid spectra 163 Automation File Editor Command Reference Menus aii Automation File Editor File Edit Help alslals S ele File Menu Menu Item Description New Displays RecalcList SampleList and Sequence Select one to display Create a New Automation File Open Displays RecalcList SampleList and Sequence Select one to display the Open an Automation File dialog box to open an existing automation file of the selected type Save Saves the currently displayed automation file with the existing name Save As Displays the Save Automation File As dialog box You can save the currently displayed automation file with a new name Prints the currently displayed automation file Printer Setup Opens the Print Setup Dialog You can also open the Print Setup dialog box from the Printer Setup dialog box Exit Quits the Automation File Editor If you have not saved changes to open files you are prompted to do so Edit Menu Deletes a selection and copies it to the Clipboard Copies a selection to the Clipboard Deletes a selection but leaves the Clipboard unchanged Adds a new line in a SequenceList SampleList or RecalcList Inserts a new line in a SequenceList SampleList or RecalcList Inserts previously cut or copied information that was stored in the Clipboard into a document 164 Menu tem Description Select All Selec
92. d physical settings You can control two actions Run and Purge Method Activate Changes Restore These buttons become active only after one or more changes have been made through Manual Control Make a change part of the active method by pressing Activate Changes At any point before this changes may be reversed and the method segment restored to its previous condition by pressing Restore To make changes permanent click Activate Changes click Upload MS Method select a folder and file name and save the method You are prompted to save any un activated changes when you leave the Manual Control tab or change Segment number 143 Active Method Segment Number Use the active method segment control to select an existing segment of the active method for review or modification Scan Function Channels Some of the 500 MS methods have multiple channels The channels can be thought of as a subset of a segment where the scan conditions can differ These are most frequently used for alternating polarity or MRM work In Manual Control you can continuously scan a single channel or to rotate through them sequentially as happens in the acquisition mode Intensity Normalization This turns intensity normalization on and off When it is on the intensities are normalized by the ionization time The intensities are multiplied by the ion time ratio The ion time ratio is the maximum ion time specified divided by the actual ionization time Me
93. dard Defaults M Load I Syringe Pump On Genera Parame ters lonization Control Electrospray lonization Parameters Full Scan Parameters Scan Time a Maximum Scan Time Multiplier Offset 05 J volts 0 23 Dats Rate Scan Time uScans Averaged and Data Rate are linked When you change the Scan Time the Minimum number of Scans Averaged and the Data Rate are calculated for you If you adjust the Minimum Number of Scans Averaged the Scan Time and Data Rate are calculated for you The Customize button opens the Count Threshold Parameter There is a trade off between how rapidly spectra are collected and the reproducibility of mass spectra or peak areas The more microscans averaged the more reproducible the mass spectrum However collecting more data points across a chromatographic peak improves peak area reproducibility for replicate injections Averaging three scans provides good spectral quality and an acceptable data rate for most samples The Multiplier Offset adjusts the EM voltage as much as 300V relative to the current multiplier setting in the Module Attributes tab dialog in Manual Control 73 this is usually the 10 5 gain value from Auto Tune Sometimes better sensitivity is achieved particularly in techniques such as MS MS when the multiplier voltage is increased Note that this adjustment can be made on a segment by segment basis The Count Threshold is normally 1 cou
94. dit Notes while a RecalcList is opened It allows you to enter descriptive notes associated with an automation file These notes can be viewed when selecting the file and are included in the printout Notes C arianWS Examples SummaryBasic new rcl x These notes will be displayed in the Open File dialog box and in automation file printouts cancel Notepad Area Shows notes attached to the file The notes may describe the contents of the file its uses restrictions etc The Notepad Area is scrollable if the notes exceed the available space The notes are edited here either by typing text directly or by pasting text from the Clipboard by pressing the keys CTRL V simultaneously RecalcList Window sii Automation File Editor new rcl File Edit Help Data File c Warlanws examples summarybasic 6st Gstd c varlanws examples summarybasic 6st std Delete Add Data Files to a RecalcList by clicking a cell in the Data File column and typing the file name or by clicking Brower and selecting the file name To add one or more Data Files quickly to a RecalcList is to select the files of interest in the Explorer and drag them to the RecalcList Window When you release the mouse button the Data Files are automatically appended to the RecalcList A RecalcList may contain standard LC data files run extension and MS data files sms extension Some the RecalcList items described as follows are handled dif
95. ditor New Sample List 460 LC smp Ela lc File Edit Help Cal nj Iniection Notes A tol ink Inj Mode Inj Tray Vial W level Volume Default Sample i Partial Loopfill 10 Left Default Sample i Partial Loopfill 10 Left Default Sample3 gt i Partial Loopfill 10 Left Default Sample4 B a Partial Loopfill 10 Left Default Sample5 i Partial Loopfill 10 Left Default Sample6 i Partial Loopfill 10 Left Default Sample Partial Loopfill 10 Left Default Samples i Partial Loopfill 10 Left Default Sample9 i Partial Loopfill 10 Left Select Activate Method from the Sample Type cell Click AutoLink in the desired row The Activate Method window opens Enter the name of the Method or click Browse to select the Method Specify any number of Methods in the SampleList Activate Method Method PathN ame C Warianw S 500 MS Methods 500 M5 ESI CALIBRATION METHOD mt Browse Cancel 126 Creating or Editing a Sequence s Automation File Editor Recalclist SampleList F Printer Setup Exit Either choose a New Sequence or Open an existing Sequence from the File menu The Sequence window for opens Click Add to add lines to the SequenceList You may enter any number of Sequence lines Choose the action to be done in the selected step of the Sequence Enter the name of the Method or SampleList in the corresponding column or use Browse to select the Method or SampleList from a li
96. dy Equilibrating scan Description lonization Time VYaponzing Gas Temp No Fault Wait For Readyln Scan Range lon Count o 390 E Hide Keypad Spectrum and Chromatogram After a few moments the red light becomes green indicating that the 500 MS module is ready for analysis To determine the status ready of each component go to the top menu under windows to see the states After all components are ready you can start an analysis Start Acquisition Click to start data collection with the currently active method Edit Method Open the Method Builder application and load the currently active method Wait for Readyln If checked the state of the Readyln sync port is included in the checks the system performs to determine if the system is ready The Readyln port interfaces to an external device such as a non Varian Autosampler End Time The End Time is initially set to the end time of the active method You may change the end time to either shorten or prolong the end time of the data acquisition This does not affect the end time of the LC method 155 Status Display Spectrum Toolbar BEBE Al Click the green arrow to hide the toolbar Click in the upper left of the full screen display to restore the toolbar Click the full scale button to normalize the spectrum Double click the lower left corner of the scale to do the same a Click the previous scale to restore previous scale a Click spectrum plo
97. e RecalcList Adds a new line before the highlighted line Deletes the highlighted row in the RecalcList Fill Down Copies the contents of the top cell in a series of highlighted cells to the cells below it Use to edit all the cells in a column quickly Default Displays the Set Defaults dialog box allowing you to specify default values for each applicable field in the RecalcList Browse Opens the Open Data File dialog box to select a Data File to add to the RecalcList Shows the results report for the selected Data File Actions Displays a list of applications that can be opened with the selected data file that is Print Standard Report Begin Opens the Begin RecalcList Dialog Box to specify a method to use while recalculating or printing all RUN files in this RecalcList and to then begin recalculating or printing the files Only available in the System Control version of the RecalcList Suspend Suspends execution of this RecalcList after the current file is completed Only available in the System Control version of the RecalcList Resume Resumes execution of this RecalcList after it has been suspended Only available in the System Control version of the RecalcList Data Handling Channels Dialog Box NOTE Use this dialog box to specify standard LC data handling MS data handling uses only one detector channel You can specify the Multiplier Divisor and Unidentified Peak Factor channel by channel for up to four different detector
98. e Windows taskbar lt can be docked on any of the four sides of the display screen and other Windows programs do not cover or go behind it when they are opened in full screen mode If the Workstation toolbar is not already opened open it from the Windows Start Menu as follows ToS Cordura pro Adobe Aerobie A proessa T Adobe Lille Deyr 0 0 E idite es gt FAY Starelard Mi Repais m sien e nitro di nier Ene r aa Ph vw FRA y Dubicok ipren o E Pero eo Ar a a Winde Media Player a Wires Medidores a Winker Merde Mates A fj Winders eh Application Buttons View Edit Methods View and edit instrument operation data acquisition and data handling methods 4 Do off line editing of SampleLists RecalcLists and SequenceLists Review chromatograms and spectra perform library searches and review and process quantitation results Convert Kodiak Dat files to MS Workstation xms format Generate standard reports for a group of Data Files by dragging and drop ping them on the Batch Report Window View Edit Chromatograms Review standard GC chromatograms interactively edit data handling A parameters and recalculate results Preview standard chromatogram and results reports Compound Set Editor ACS Create or edit sets of compounds in the MS Data Handling method This Ed allows activation deactivation under automation PolyView 2000 Fl View and process PDA data from your Varian HPLC
99. e a RecalcList f Create and update a new RecalcList RecalcList name ic warianwstuntitled rol 0 Append to an existing RecalcList RecalcList name Overarite the Recalc List each time the SampleList Begins Cancel tem Description Do not automatically No RecalcList is generated or updated when you create and update a create Data Files as a result of injections RecalcList Create and update a Enter the name of a new RecalcList created when new RecalcList Data Files are generated as a result of injections If the RecalcList name that you specify already exists when the injections are performed a number is appended to the name so that it is unique Append to an existing Browse for an existing RecalcList file When Data RecalcList Files are generated as a result of injections their information is appended to this file Accepts the RecalcList specification Cancels the RecalcList specification AutoLink Parameters Dialog Box With AutoLink you can specify a program to be executed after all injections of a sample have been performed or two programs to be executed after each injection of that sample When AutoLink appears as a sample type ina SampleList or RecalcList the associated program is executed at that point in the SampleList RecalcList after the previous SampleList RecalcList line has completely finished The AutoLink can be specified as part of a SampleList line that has a Baseline Analysis Calibra
100. e automatically calculated Target TIC at 100 gives the best results 74 General Parameters lonization Control Electrospray lo Automatic Gain Control Target TIL counts 100 E lonization Time Fixed lonization Time Max lon Time 250000 useconde Use Customize to run fixed ion time experiments and or to change the Maximum lon Time for Automatic Gain Control Fixed ion time experiments are generally diagnostic tests and are not used for qualitative or quantitative analysis If the ion time in the baseline of the chromatogram is not reduced below the maximum ion time the ion trap does not fill with enough ions to match the Target TIC Changing the Maximum lon Time can improve the sensitivity of techniques such as MS MS This parameter can be increased to 650 000 usec however doing so reduces the data rate Electrospray lonization Parameters m Method Segment V Detector On Scan Type Full Scan Mode Standard Defaults M Load M Syringe Pump On General Parameters lonization Control Electrospray lonization Parameters Full Scan Parameters SetPoints Scan Time 4 42 Maximum Scan Time Multiplier Offset 0 volts Scans Averaged 34 Scans Data Rate 0 23 Hz These default settings are suitable for many low flow rate applications They can be changed for each segment 19 Full Scan Parameters General Parameters lonization Control Electrospray lonization Pa
101. e excitation storage level and the excitation amplitude are automatically computed Modulate RF If using resonant excitation checking this causes the q value at which the ion is stored to be repeatedly ramped in a small region around the specified value This effect slightly changes the resonant frequency of the ion and ensures that the ion is in resonance with the applied CID frequency under all conditions The default is for this to be checked Number of Frequencies If using resonant excitation this parameter specifies the total number of frequencies applied By increasing the number of frequencies used for excitation the user may excite a broad range of ions which is useful for dissociating all of the ions in a halogen cluster NOTE The excitation amplitude used with this feature must be increased proportionally with the number of frequencies used Values are odd numbers of frequencies from 1 to 119 CID Frequency Offset kHz If using resonant excitation this parameter allows the applied CID frequency to be different from the calculated frequency by the amount a user specifies This is useful for advanced users who operate with the RF modulation turned off Values range from 10 to 10 hertz 95 Injecting Samples This section describes instrument setup operation and data acquisition and uses System Control and Method Editor Acquiring data may involve these steps e Create or modify an existing method that includes sections f
102. e gas pressures and the damping gas flow The ion source must be on to view the drying gas and nebulizer readbacks In APCI mode the Vaporizer temperature and pressure readbacks are displayed lon Gauge lon Gauge lon Gauge Pressure 32 4 UT orr iv on Gauge Filament 1 OF gt O Sens Filament 2 Untested O Filament 2 Foreline Pressure 865 mT orr If the lon Gauge is selected the ion gauge pressure in uTorr is displayed in the lon Gauge field of the Monitor window Select one of the two filaments to produce the reading The foreline pump pressure is just below this lon Source ESI lon Source Setpoint Needle Voltage 43949 Volts S003 Needle Current o ump Capillary Voltage 28 5 Volts ol 1 Spray Shield Voltage 625 6 Volts 599 5 lon Guide DL 5 0 Volts 5 0 Exit Lens Voltage 11 0 Volts 11 2 Focus Lens Voltage 44 7 Volts 44 3 Deflector 1 Voltage 11 8 Volts J2 2 Deflector 2 Voltage 11 8 Volts J2 2 Trap Focus Lens 1 70 6 Volts 170 0 Trap Offset 22 4 Volts ee 49 After the ion source is turned on these readbacks are displayed The needle voltage set in the active method produces a readback The capillary and spray shield voltages are also set in the active method The other setpoints are from the Autotune procedure NOTE In APCI mode the Needle Voltage and Current readbacks refer to the Corona Needle In this case the Corona Needle Current set in the active method produces a Corona Needle Volta
103. e overall signal the signal from unwanted ions decreases faster than the signal from the target ion lon Formation and Matrix lon Ejection lonization of the sample and any co eluting matrix occurs in the API interface A notched waveform may be applied to the end caps of the ion trap to eject resonantly ions above and below the specified precursor ion mass This may or may not improve the performance of the instrument depending on the sample composition The waveforms are most effective when there is a large amount of matrix interference in the low mass range Removal of most of the space charge from the trap improves mass resolution in subsequent MS MS steps The ions remaining in the trap include the optimum number of precursor ions and ions that are in the coarse isolation window Precursor lon Isolation The precursor ions are then isolated from remaining matrix ions in a two step process In the first step a broadband waveform is applied to the trap to eject simultaneously all masses above the precursor ion mass Isolation occurs at elevated RF levels where mass resolution is optimal Thus it is possible to achieve unit mass isolation over the entire mass range of the ion trap This may be useful in isolating a single mass in an isotopic cluster or in separating the precursor ion from interfering matrix ions In the second step the ions with masses below the precursor ion mass are ejected by ramping the RF amplitude while applying
104. e results in an averaging of the energy coupled into the ion motion and provides a reproducible product ion spectrum even with changes in concentration An additional parameter is available with resonant excitation Increasing the number of CID frequencies permits simultaneous excitation of ions within a range of masses This may be useful when trying to excite an entire isotopic cluster at once As the number of CID frequencies increases the range of excited masses also increases For a given number of CID frequencies the range of excited masses increases with increasing ion mass and decreases with increasing excitation RF storage level Mass Range Excited 60 Excitation RF Storage Level m z 48 Q oS AA A JL l l l l E I I l l AA A AEE 4 l l l l I J HUE PU A O ioe 29 Number of CID Frequencies Mass range excited vs number of CID frequencies for an excitation RF storage level of 48 m z 92 Mass Range Excited 30 Excitation RF Storage Level m z 143 re Ss ed ii i Mi N ll N O Aia l aslo ene A LE 4 a a eer ee eii Ae 1 l 1 l 1 l i 1 l mk N N mk N oe oo oO N Number of CID Frequencies H E m z 500 m z 400 m z 300 m z 200 Mass range excited vs number of CID frequencies for an excitation RF storage level of 143 m z With
105. eList are disabled when the User Program is selected E ProStar 420 SampleList 420_Demo smp Inj Wash Automix User lt Inj Mode Volume vial Volume Routines Program Al 300 none Sample Name For the PS 430 use the Plate column to specify the plate location of the well or sample of interest You can enter a plate number between 1 and 7 This column is disabled unless the PS 430 is equipped with the Plate Feeder option The PS 430 Well Vial column specifies the well or vial from which you want to aspirate the sample for injection The location is specified as a combination of row letter and column number The available rows and column numbers depend on the type of plate that is configured in the AutoSampler Hardware You can enter A1 through F8 for the 48 standard vials A1 through H12 for the 96 well plates and A1 through P24 for the 384 well plates You can use the Well Vial column to randomly access any well or vial for example you can periodically recalibrate using a single standard at a fixed location For the PS 420 the Vial column specifies the vial of interest The location is specified as a combination of Tray type letter and Vial number The available Tray types and Vial numbers depend on the Tray Segments configured in the PS 420 Hardware You can use the Vial column to access randomly any vial for example you can periodically recalibrate using a single standard at a fixed location The Wash Volume column is
106. ectra with better quality but fewer spectra are produced Fast chromatographic peaks may not be well resolved e Capillary Voltage Enter from 0 to 300V The sign is set to match the polarity selected e RF Loading Enter from 0 to 300 Sets the trapping conditions as the ions enter the trap e For full scan applications enter 100 to let the software set the optimum value e For target analysis optimize the RF loading for each compound to increase sensitivity 81 MSn Condition Parameters n Select from 2 to 5 to determine the number of MS level scans If 3 is selected after the survey scan is done MS2 is done on ions that meet the trigger criteria MS3 is done on product ions of the MS2 scans that meet the trigger criteria Auto Mass Range When enabled the mass range used to detect product ions is determined automatically e Singly charged ions Start m z is determined by the RF storage level and the End m z is the precursor ion m z plus 20 e Doubly charged ions Start m z is determined by the RF storage level and the End m z is twice the precursor ion m z plus 20 uScans Select from 1 to 9 to determine the number of scans that are averaged for that MS level N Number of ions from the previous MS scan level that will be scanned for the current MS scan level For example if N is 3 on the MS2 line then 3 ions from the survey scan are used to generate MS2 spectra Start and End Mass If Auto Mass Range is not
107. ed Instrument 1 Parameters Instrument Varian LC MS 1 Dperator Ready File Mans Errors 0 Prompt on Automation Start Cancel Windows Menu System Control Varian LC MS 1 Not Ready Fie Edit Inject Automation Recalculate Instrument Paiste Help Gee Gee EE startup mth GGE E Iconize Module Windows a 500 M5 56 Not Ready Show Automation Windows Iconize Automation Windows Manual Control Auto Tune Diagnostics Startup S hutdow Control and Status Arrange Icons Save Data Sequence List Untitled seq Source ESI Edit Method Record Data SampleList MANUALI SMP Unload ME Method End Tj RecalcList SIMGLEL1E9 RCL Method El pAs e Henan 10 Instrument 1 Status Summary of Status Message Log Vacuum System OE Damping Gas OF ProStar Dynamax 24 Ready Temperatures Off Source OK 410 25 Ready lon Gauge Pressure D O uTorr FIA Valid w SO0 M5 56 Mot Ready Menu ttem Description Show Module Opens the Status and Control windows for all the modules Windows in the Instrument It is disabled when all the windows are open even if some are hidden under others Iconize Module Iconizes the Status and Control windows for all the modules in the Instrument It is disabled when all the windows are 138 Show Automation Windows Iconize Automation Windows Menuitem Description Displays the windows for all the automation files open in the Instrument It is disab
108. ed when you move the mouse over application buttons in the Workstation Toolbar Small Buttons on Toolbar When this menu item is checked the Workstation Toolbar is reduced in size Run Application Lists the applications on the Workstation toolbar Select an item from this list to launch the application Help on Lists all applications on the Workstation toolbar Select an item from this list to display online help Pick Data File for Quick Link Button Lists the eight most recently used Data Files in order of use Select a Data File from this list to display it in the Quick Link button Pick Method for Quick Link Button Lists the eight most recently used Methods in order of use Select a Method from this list to display it in the Quick Link button Help on Workstation Toolbar Displays the help currently in use Product Support Web Site If you have Internet access and a web browser installed on your computer this automatically opens the MS Workstation Product Support Web Site Here you will 10 find the latest software and documentation updates for the MS Workstation suite of products along with additional notes tips and answers to frequently asked questions Visit this site periodically for new information About Workstation Toolbar The About window has information about the MS Workstation software version installation history and a list of the instrument control modules installed The following is an example
109. elp aaa ala BB S 14 4Jp gt I ee sl S S El 500 M5 test mth E Method Notes Location CAWanarda 54500 M5 Methods500 M5 test mth Ey ProStar 430 AutoSampler Control Address 1 Created Wednesday October 21 2009 16 17 38 ay Configuration ia 212 LC Solvent Delivery System Control Addre Modified Wednesday October 21 2009 16 18 35 wx ES Pump Program Size 90086 bytes S00 MS Mass Spec Address 3 Method File Attributes 7 500 M5 Mass Spec Control TI MS Acquisition Method O Read only O Hidden H Channel 1 M5 Data ya E Standard MS Reports El Archive O Requires Password on Save 2 Print Opbions 2 Results Format Revision History E Chromatogram Format No Revision History Es Compound Reports es Calibration Block Report Format Summary Report Format 1 MS Data Handling Calculations Setup Compound Table Results Treatment The Method is displayed in two panes The Directory pane on the left is an outline of the Method Click an item in the Directory to change the content of the Parameters pane on the right Click and drag the splitter bar to resize the panes 60 co To hide the Directory click Show Hide Method directory Els on the Window toolbar The menus at the top of the screen are File Edit View Window and Help Click each to see its contents Below these menus there are three toolbars the Main toolbar opens saves and prints Methods the Directory toolbar tra
110. emperature Vaporizing Gas Temperature Ramp APCI only In gradient applications that start with very low aqueous mobile phase and end with very high aqueous phase you can ramp the temperature of the vaporizing gas Setting the vaporizing gas temperature ramp 1 Click Use Ramp Rate to enable it a Enter an Initial Hold Time if needed b Enter the Start Temperature c Enter the Ramp End Time d Enter the End Temperature 68 Editing Chromatographic Time Segments NOTE The following settings are not specific to the entire method Each time segment can have individual settings You can program the mass spectrometer to acquire data differently by dividing the analysis into chromatographic time segments The default method has a single time segment Up to 250 time segments may be defined for a single analysis The maximum run time is 650 minutes Instrument Configuration Acquisition Data Type Turbo DDS Electrospray Centroid Profile TuboDDS Method Setpoints Chromatographic Time Segments Add meone as ee omona Insert 1 Full Scan 0 00 2 00 ESI Auta Full Delete EA MEN Defaults E Petore Restore E EA is Special Applications Click a row number to highlight it Click Add to add a new segment with the same MS parameters of the last segment in the table Click Insert to insert a new row before the currently selected row with the same parameter values Delete deletes the c
111. enabled enter the start and end m z value see Auto Mass Range The product ions of these TurboDDS scans must be in specified scan range to be observed Threshold lons that exceed the threshold number of counts and meet the trigger criteria are scanned at the next MS level Use High Resolution When enabled a high resolution scan is done for each ion from the Survey Scan that meets the criteria In the profile mode you can determine up to 4 positive charge states by measuring the m z difference between isotopic peaks High Resolution uScans Select from 1 to 9 Enter the number of uscans used to average each High Resolution analysis Customize Click Customize and two options appear Unstable ions Select to decrease the possibility of accidentally dissociating less stable precursor ions in the isolation step Isolation Window Select from 1 to the maximum window The maximum window size is a function of the precursor mass and ranges from 3 to 14 m z The wider the isolation window more ions remain in the ion trap after the isolation step and the intensity for less stable precursor ions improves 82 General Parameters lonization Control Electrospray lonization Parameters TurboDDS Parameters TurboDDS Trigger Sn Conditions MS n 2 Auto Mass Range Star Mase End Mass Threshold m z mz counts Moe fi 34 0 2000 8600 Survey Scar Stark Mass AO miz End Mass 2000 mz Polarity Positive
112. ent Messages w Status and Control After the system is shut down Startup opens Click Startup to start the vacuum and turbomolecular pumping After the system starts ShutDown opens Click to begin an orderly shutdown of the vacuum system Vacuum System status indicating whether the Vacuum system is Ready Pneumatics Damping Gas Turn On Turn Off Use Turn On Turn Off to control the damping gas flow for diagnostic purposes The flow rate is set in the Manual Control section under the Module Attributes tab 154 Vacuum System Displays the actual conditions of the foreline and turbo pumps Display Display either Event Messages or Module Attributes e Event Messages are a log of actions and events that have occurred during the Startup or Shutdown process e Module Attributes displays the current instrument configuration temperature setpoints date and results of the most recent manual adjustments date and results of the most recent Autotune procedures and a log of tuning errors Acquisition Selecting the Acquisition tab activates the 500 MS module for analysis and loads the information for the active method a 500 MS 56 Not Ready Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Status And Control MS Method Operating Conditions Temperatures Aun Time 0 00 minutes Segment Humber Data File Drying Gas Temp End Edit Method Segment Description Scan Number O 138 C D Not Rea
113. ent modules you with to disable 2002 Micro GC 2003 Micro GC 3400 GC 3600 GC 33L GC 4900 Micro GC ProStar 330 PDA Detector ProStar 400 Autos ampler 1200 Mass Spec gt gt Disable gt 4000 Mass Spec 800 Interface Box 2200 Autos ampler 9001 Solvent Deliver System 9002 Solvent Deliver System 9010 Solvent Deliver System 9012 Solvent Deliver System 9050 UY Vis Detector 9100 Autos ampler 9200 Prospekt 9300 Autot ampler ADE Board Al 200 Autos ampler CombiP L Autos ampler Mistral Column Oven ProStar 220 SOM ProStar 230 Inert SOM ProStar 230 SOM Enable lt lt Cancel Select which MS Workstation instrument modules are available When available the module appears in System Control if the corresponding instrument is connected and turned on The left list shows the instrument modules currently installed and enabled in the MS Workstation The right list shows the instrument modules installed but not enabled To disable an instrument module select it from the left list and click Disable To enable an instrument module select it from the right list and click Enable Application Descriptions E Perform automated injections and 4 recalculation and monitor instrument status Se Application Descriptions are a brief description that appears when the cursor rests on the application button When this menu item is checked application descriptions are display
114. er standard peak name from peak table Indicates names of the multiple internal standard peaks present in the specific channel s peak table To change the standard peak names for a specific channel first click the channel s row and click Edit Standard Peak s If the peak table does not contain multiple internal standards these fields are disabled Amount Standard 2 8 0 000001 to 1 000 000 Sets the amounts of the second through eighth internal standards Use it to calibrate results for Internal Standard and Normalized Percent calculations involving multiple internal standards If the peak table does not contain multiple internal standards these fields are disabled Add Adds a row to the end of the list Adds a new row before the highlighted line Deletes the highlighted row in the list Edit Calibration Setup Opens Calibration Setup to permit inspection and editing of the calibration parameters in the method for the indicated detector channel Edit Standard Peak s Opens Peak Table to permit inspection and editing of the Standard Peak names in the method for the indicated detector channel Open the Peak Table and click OK to transfer the standard peak names and amounts from the Peak Table to the indicated detector channel row 173 Open Data File Dialog Box Open Data File Look in Examples do E C QuickEP O Summary Basic RSE File name 525570 04 Files of type Data Files Arun sme x
115. essages are a log of actions and events that have occurred during the Diagnostic and Monitoring process The messages usually indicate tests Started Passed and Completed The Monitor Window displays the status and values of the following systems Vacuum Trap Waveform Temperatures and Gases lon Gauge Foreline Pressure and lon Source Display both Monitor Window and Event Messages at the start of any diagnostics and monitoring After selecting which components to activate and diagnose click Start Diagnostic in the Control and Status Area The Monitor Window has the areas that indicate the value and status of components Vacuum System Displays the status of the Turbo and Foreline Pumps The Turbo Pump area indicates the pump status Stopped Running the pump speed the pump current in mAmps and pump power in Watts lon Gauge indicates the ion gauge pressure in uTorr and the status of the Filaments OK open broken Foreline Pressure is indicated in mTorr Trap The Trap area in the Monitor Window is active only if the Multiplier Dynode checkbox was selected in the Monitoring Tab top level The Trap Area if active indicates the Multiplier voltage and Dynode voltage in Volts Waveform System The Waveform System area is active when the user selects RF Control in the Monitoring Tab top area The percentages of the Entrance End Cap Trapping Field Dipole and Exit End Cap Trapping Field Dipole are indicated when the R
116. ethod Cancel or select from a list Specify any number of Methods in the SampleList Using the SequenceList Window The SequenceList window allows you to specify multiple Methods and SampleLists to be processed during automation AA Edit Inject Automation Recalculate Activate Method Upload Active Method From Modules Mew SampleList Open SsampleList New RecalcList Open RecaleList Open a New SequenceList or an existing SequenceList New SequenceList Open Sequence ist Print Printer Setup w Remember Last Open Files Exit a system Control Varian MS 1 Ready seal Automation File Edit Inject Automation Recalculate Instrument Windows le Click New or als c 2 420_Dynamax mtt b 114 E SequenceList test seq Choose action es Action Method Sample RecalcList _ yect c warianws 420_dynamax mth c warianws 420_demo smp Add Inject E Start the run Browse for a Method or SampleList Monitoring Run Status After making an injection monitor the status in the instrument window a 500 MS 56 Running Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Status And Control MS Method r Operating Conditions Temperatures Run Time 1 07 minutes Segment Number 2 of 24 Data File NonDDSCentroidStand Drying Gas Setpoint End Time 24 00 minutes Se
117. ethod Global parameters such as Instrument Configuration Acquisition Data Type and segment and scan description are set using the Method tab Method Segment adjust some parameters segment by segment Checks and Adjustments adjust the RF ramp Module Attributes set the electron multiplier voltage trap damping gas flow syringe type and the syringe infusion purge flow rates Optimization Plots optimize the capillary voltage CID excitation voltage RF storage level and needle voltage Auto Tune select and run tuning routines Diagnostics tab run the built in diagnostic routines of the vacuum system and electronics Startup Shutdown start or shut down the vacuum system and heated zones in an orderly manner Acquisition run samples and acquire data files with the Inject and Automation menus of the System Control window 141 Hide Show Keypad Click Hide Keypad to Hide the Control and Status window This becomes the Show Keypad button to restore the Control and Status display While the keypad is hidden the On Off controls for the trap and infusion pump are still accessible as well as the method segment channel and intensity normalization selectors Manual Control Manual Control is closely linked with the Method Builder function of the software A method may be built in Method Builder activated in System Control and then manipulated in Manual Control Conversely a method may be modified and fine tuned in Manu
118. ethod to determine the number of levels of MS scans to do and the number of ions to be scanned The survey scan which is a full scan is the first scan Set threshold parameters and include exclude rules to determine which ions from the survey scan will be precursor ions for the subsequent MS scan levels Up to 5 levels of scans can be done High resolution scans can be done on the ions that meet the triggering requirements Enter the parameters using the TurboDDS Parameters tab and the TurboDDS Trigger tab in the Method Builder e TurboDDS parameters determine the number of ions to select and the number of MS scan levels done and the thresholds for each scan level e TurboDDS Trigger parameters determine e How close the m z values of ions can be before they are considered the same ion isotopic peaks e How many times an ion is analyzed before going to the next most intense ion in the survey scan e How specific ions in the survey scan are selected for subsequent MS scan levels Acquire a TurboDDS data file and open it in MS Data Review MS Data Review has tools that simplify examining spectra The Software Reference Manual has information about reviewing TurboDDS data 79 TurboDDS Hints Focus the survey scan in the mass range of interest More scans are acquired across the chromatographic peak when the mass range is smaller Optimize the number of micro scans e Using fewer micro scans with narrow peaks allows more data poi
119. f parameters 94 Isolation Waveform Check this to turn on an isolation waveform to use as the ion trap is loaded Use this to reduce the intensity of low mass matrix ions which reduce the storage capacity for the precursor ion NOTE this waveform often reduces the intensity of the precursor ion Low Offset m z This is specified in Daltons and is used to reduce the size of the isolation window on the low m z side Values range from 0 7 to 0 7 Da The usual value is O Da High Offset m z This is specified in Daltons and is used to reduce the size of the isolation window on the high m z side The values range from 1 0 to 1 0 Da The usual value is O Da High Mass Ejection Factor The high mass ejection factor specifies the relative amplitude of the isolation waveform that removes ions at a higher m z than the precursor ion The values range from 0 to 200 Typically this value is 100 unless the precursor ion prematurely dissociated in the isolation step Waveform Type The CID excitation waveform is either resonant or non resonant For a description of these waveforms please see the section of this manual entitled MSMS Tandem Mass spectrometry Excitation Time msec This is the time the excitation waveform is applied Generally longer times require smaller excitation amplitudes so optimize these two parameters together Values range from 0 to 650 msec The algorithm uses the default value of 10 msec when th
120. ferently for the two data file tyoes When the list is processed the appropriate data handling is used automatically for each data file Cal Recalc E Amount Std Unid Peak ae site MultiChannel Sample Name ievel i Notes Autolink 15 Nx only Factor Multiplier Divisor WultiStandard Add Insert Fill Down Defaults Browse Report Actions 169 Data File Sample Name Sample Type Cal Level Recalc Notes AutoLink Amount Standard Unid Peak Factor Multiplier Divisor Shows the name and path of the Data File for each sample Cannot be edited Shows the name of each sample in the RecalcList Baseline Analysis Calibration Verification Print Calib New Calib Block AutoLink Activate Method Sets the sample type or automation action of each line in the RecalcList 1 to 10 Sets the calibration level of each calibration or verification sample in the RecalcList Cannot be edited Shows the number of injections made of each sample in the RecalcList Up to 180 characters Opens the Notes window for the selected record to edit or create a note about the sample This note is stored separately from the original injection notes There are two AutoLink commands when Sample Type is Baseline Analysis Calibration or Verification One AutoLink command when Sample Type is AutoLink One Activate Method Command when Sample Type is Activate Method Opens the Auto
121. formation the RecalcList contains the Data File name and data file specific data handling information The fields that are common to both the RecalcList and the SampleList are described in the sections after the creation of the SampleList sisi Automation File Editor tylosin_tutorial rcl it SEE File Edit Help als co SS Bala Drag the border to size the column EN tylosin_tutorial rcl RecalcList Right click column headers for Seles options hecalc Notes AutoLir Add Insert Delete Fill Down Defaults Browse Report Actions Sample Type hat Inj Data File Sampl Name Calibration Calibration Calibration Calibration c documents and settings 1 0_0 c documents and settings 5U0_0 Data File and Sample Names are i visible as the columns are scrolled to the right 121 Recall Notes Enter notes about the Recalculation AutoLink Enter post calculation operations to be done Report Click Report to generate a report for the currently selected data file Note that if a data file is not highlighted in the Data File column no report is generated Actions If a report was generated using the Report button clicking Actions shows actions that may be done DENE View Edit MS Chromatogram Print Custom MS Report Print Standard MS Report View Custom MS Report View Standard MS Report
122. g one Open RecalcList New Sequence List Open Sequence ist Print Printer Setup w Remember Last Open Files Exit a system Control Yarian M 1 Ready Click the New or Open File Edit Inject Automation Recalculate Instrument Automation File on the toolbar eer ES 420_Dynaman mtt 3 En en dal The SampleList window for the open SampleList opens It contains fields that are specific to the sampling device configured in the instrument 105 Generic SampleList If there is no sampling device configured in the Instrument a generic SampleList is used which does not contain columns for selecting vial positions sampling options or hardware configuration options Size spreadsheet columns by drag ging borders fe ab out Move columns by dragging them using the right mouse button Right dick column headers for formatting options Enter sample information Click to inject samples Enter postrun operations MM Generic SampleList DemoList smp Sle Sample Name Sample Type ioe Inj ee AutoLink Add Fill Down 6 y Add Lines Defaults 8 v v gt Eggin Data Files RecalcList Select Data Files Specify RecalcList options When the table is scrolled to the right the Sample Name column does not scroll so you can easily tell for which sample you are entering additional parameters for commonly used data handling parameters the amount for single intern
123. ge readback Event Messages Display Displays messages when diagnostic tests are performed Startup Shutdown Mode Use the Startup Shutdown tab to properly shutdown the 500 MS for maintenance and to start it up again Monitor the vacuum system and damping gas pneumatics during Startup and Shutdown in Manual Control a 500 MS 56 Not Ready Manual Control Auto Tune Diagnostics Startup hutdown Acquisition Vacuum System Foreline Pumps Status and Control 50 Conditions Analysis H5652 H5652 State Ready Status Mormal Normal E Speed 100 100 Curent 1879 m mpe 1611 m mpz Status Ready Power 465 Watts dd atte alos Life 17035 hours 22099 hours Ol Level Level OF Level OF Damping Gas On Turn Off Flow Rate Setpoint 0 8 mL min Turbo Pumps Flow Rate 0 8 mL mir Rear Forward Inlet Pressure E4 PSI Status Hormal Normal Speed 100 100 lon Gauge Curent 417 m mpe FO m mps Pressure 25 uTor Power 30 Watts A atte Filament OF Temp 37C AEC Life F355 hours 13760 hours Foreline Pressure Jar mT or BE Turbo Speed Setpoint 100 Status and Control Status and Control Conditions Analysis Shut Dorr State Ready Vacuum System Status Ready Pneumatics Damping Gas On Turm Off Flow Rate Setpoint 0 8 mL min Flow Rate 0 8 mL mir Inlet Pressure 79 PSI lon Gauge Pressure 49 5 uT orr Filament UF Foreline Pressure 1000 mT orr The Conditions field displays either Analysis
124. gment Description MS MS 3MRM Pos Scan Number 197 350C Running Scan Description APC Auto MS MS lonization Time 705 Vaporizing Gas Setpoint NoFaut 7 Read Survey Scan Range 467 1632 lon Count 13238396 350C Spectrum and Chromatogram 7 Default View x a joj 11 m Scan Number 199 Time 1 082 min Base Peak 1290 0 Base Amount 1134607 RIC 9 117246 lon Range 396 5 1432 5 m z lon 932 us Segment 2 Channel 3 1421 9 373187 500 lol 1 8 Al mue MCount 400 Acquired Range m z RIC NonDDSCentroidStandardEnhancedMix_APCI 4 3 2007 2 30 39 PM XMS is 15 20 A minutes Nons y Instrument 1 Apr 03 14 30 27 Results will not update any RecalcList The module status is shown in the status and control windows and on the Toolbar Automation actions and errors are logged in the Message Log the most recent line of this log is at the bottom of the screen When you double click the status bar at the bottom of the instrument window the entire Message Log window opens 115 Jan 19144323 Automation Began Jan 1914 43 23 MessageLog Name Ciara SMSGLOGSMSGL0G1_1 19 2005_ 14 43 23 MLG Jan 1914 43 23 MS Workstation 6 4 1 MSDR5 BB Version 6 41 Jan 19 14 43 23 Workstation METG 7583 Jan 19144323 Instrument 1 Varian MS H Jan 1914 43 23 ProStar Dynamas at address 24 Jan 1914 43 23 CombiP4L at address 36 Auto S tart Jan 19144323 1200 at address 42 Jan 19144323 Operator icheng J
125. gt i Filt d daah Save All as Defaults Reset All to Defaults tered raw data nas x axis maximum cmai 39 Overlay raw data with the filtered plot Optimization Plot Bar Optimization Plot Preferences a 3 Ez Aj Optimization Data Markers Colors Axes M Count 3 C Baw Data Only C Filtered Data Only jon 30 Yh erate gt n A A MV Smooth Data Filter Strength High T 1 T 1q Iy E dd vor 200 panilary Volts Reset to Defaults iH Save All as Defaults Reset All to Defaults Canoa Use the Markers tab to display the individual data point Optimization Plot Bar Optimization Plot Preferences Optimization Data Markers Colors Axes Data Point Markers F Show Markers and Lines ca Maximum Markers Per Centimeter a 578 70 1522 1 o a 23P60 P gt Integration Area Markers Edit color and Fant Background Correction Markers Show Backaround Correction Markers System Event Markers fiz Show System Event Markers Annotation Markers W Name on Own Line ao Approximate Number of Characters per Line En capillary volts Help Reset to Defaults gt Save ll as Defaults Reset All to Defaults Cares cob ee ba BD ee be bb D ay 40 Auto Tune Mode Auto Tune automatically optimizes the 500 MS performance over the entire mass range Perform Auto Tune when the instrument is first set up and after doing m
126. he Varian MS Workstation Product Support Web Site The latest software and documentation updates for the Varian MS Workstation suite of products along with additional notes tips and answers to frequently asked questions are found here Visit this site periodically to see if new information is available Displays the About Box for the System Control application The About Box contains information about the software version installation information and a list of the instrument control modules You can also validate the installed Workstation files from this dialog 139 Display Display the 500 MS status and control window in the System Control Status window by selecting the Windows menu item and clicking the 500 MS xx menu item or by expanding the 500 MS xx icon that appears at the lower left of the System Control window E System Control Varian LC MS 1 Ready AE El E ry unions Show Module Windows Iconize Module Windows Show Automation Windows Iconize Automation Windows Arrange Icons w Instrument 1 Status Message Log ProStar Dynamax 24 Ready 410 25 Ready 500 M5 56 Ready ProSta TO 410 25 7 0 x 500M E O x E Status Hel Instrument 1 Now 29 09 28 28 Ins 1 SPEYT_COMPLETED 140 Overview of Tabs a 500 MS 56 Not Ready Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Control and Status Savo Data Source ESI Edit Method Record Data Met
127. hecked Close System Control If System Control is in the process of performing a critical operation you may be prompted before the application is closed Edit Menu E System Control Varian LC MS Automation Recalculat Inject Fill Down Edit Module Info Deletes a selection and copies it to the Clipboard Use to remove or move a selected part of a soreadsheet or entered text Copies a selection to the Clipboard Use to duplicate a selection and place the duplicate in a new place using Paste Inserts previously cut or copied information stored in the Clipboard into a spreadsheet Deletes a selection but leaves the Clipboard unchanged Adds a new line in a SequenceList SampleList or RecalcList Inserts a new line in a SequenceList SampleList or RecalcList 133 Description Select All Selects all lines in a SequenceList SampleList or RecalcList Fill Down Copies the contents of the top cell in a series of highlighted cells to the cells below it Use to edit all the cells in a column quickly Edit Notes Opens the Edit Notes Dialog Box to permit editing of the notes associated with the SequenceList SampleList RecalcList or Method file displayed in the active window Edit Module Info Opens the Module Information Editor Dialog Box to permit editing of the Module Information associated with the module displayed in the active window Inject Menu B system Control Varian LC MS
128. hod de Ready gt No Fault Wait For Readyln This is how the Status and Control field looks before an acquisition starts The Run Time is 0 00 minutes because the run has not started The End Time is the run time specified in the active method The Ready and No Fault lights are green You can use the Start Acquisition button to override automation and start a run automatically even before the system is Ready The file name of this type of run is named using the last data file naming convention specified in the last SampleList used in Acquisition mode Click Edit Method to open the Method Builder and modify the method You are prompted to re activate the method after you save changes and return to System Control Wait For Readyln This feature is used with external devices that are not networked in to the system such as fraction collectors If Wait For Readyln is selected the system waits for an external Ready signal before starting an acquisition Status 4nd Control Aun Time 10 96 minutes Stop Acquisition 15 00 minutes Edit Method a Running gt No Fault O fait For Feadin After an acquisition begins the run time increases in 0 01 minute increments If you need to increase the end time for the run click End Time and select the 99 desired time The Stop Acquisition button terminates the MS run but not other LC hardware runs You stop the runs of those modules individually Endtime Please enter a new endtime
129. hod Method ESI Upload M5 Method ave Lata Record Data End Time 10 00 minutes For more information see the appropriate section 500 MS Keypad Click Hide Keypad to expand the plot area The button name becomes Show Keypad Click it to restore the keypad section Keypad F Trap Select Views E Porte Inbusion Pump Method Segment Acie Method Beren Exa Funcion Charrelh as y L Peaitige 1 sl a moei O al rere roe aie eel Espot ES EE Seer eri Hoan Miumnber 28 Time 0 807 min Base Peak 5 55 Base Amount SSS SMOOTHED AC 2675048 lon Range 995 2000 5 mz lon 330 us Segment 1 Charmed 1 100 SpA 2i 1522 4 e Expand plot 2129046 A 1322 3 1725 Gee 1 445045 are J PE c 1000 1500 Acquined RERE miz barua Cortrol Aito Tune Diagros cs Sistup Shutdonn Acaiston Sekei Views 5 Pot yave iefunon Puro Method Segnert Arte Method Segment Scan Functor Channels lon Gauge sf Daaaj 1 Peso 0 i Number Danan 37 al gt j Doras 2 Fuge re Ful Som F BED 8 1 opi Scan Number 50 Tama 1 464 min Base Peak 5788 Base Amount 2735255 SMOOTHED RIC BGe 5 lon Range 995 2000 5 mz fon 330 us Segment 1 Channel 1 5788 100 277406 242 3 2 118e 6 75 r t 1522 4 1 744036 1422 3 16225 1 9006 1 475046 4 50 1222 2 832631 19225 25 2020 Acquired Range m z Instrument 1 Jun 19 15 13 04 Module 500 M5 56 End Auto Tune
130. hod ESI Upload MS Method End Time 10 00 minutes Summary of Status Vacuum System OK Damping Gas OK Temperatures OK Source OK lon Gauge Pressure 0 0 uTorr Fill Valid Trap Needle 5000 Volts 1 2 udmps Drying Gas 350C 15 PSI Nebulizer 25 PSI LC Misc LA DA BE Housing 50 C Capillary 80 Volts Spray Shield 600 Volts Relays Pair 231 4 a toe Select Views 6 Port Valve uge D Load Data Views y Du 2j Dela O Inject Aj lol 9 1 4 E m unl Scan Number 31 Time 0 624 min RIC 4 538e 7 lon Range 49 55 2000 50 m z 576 21 142646 Method Active Segment Checks and Adjustments Module Attributes Optimization Plots Instrument Configuration Acquisition Data Type TurboDDS C Centroid Profile TurboDDS Method Setpoints Chromatographic Time Segments Add Segment Description E Si Scan Description ta Insert q fFulScan aaa 200 ES Auto Full Sf RE HA EA TEE y Special Applications Infusion Pump Method Active Method Segment Scan Function Channels Sr Case e EE Base Peak 142085 Base Amount 301575 Smoothedi7 a keak H B x le lon 2540 us Segment 1 Channel 1 1820 45 152193 146527 1121 15 1920 61 dead 54654 0 25 050 075 100 125 150 175 1500 2000 minutes Acquired Range m z B Control and Status Window Five tabbed sections provide access to 500 MS operations Manual Control adjust all parameters of the currently active m
131. ich the Start button appears Click the Workstation Toolbar icon to display the options menu Show Hide Applications on Toolbar Show and Hide Applications on Toolbar Applications not shown on the Toolbar Select applications you wish to add to the Toolbar and click on the Add button Applications shown on Toolbar Select applications pou wish to remove from the Toolbar and click on the Remove button Aurora Dat to sms conversion Batch Reporting Compound Set Editor Custom MS Reports Edit Automation Files Pole 2000 Quick Start Review Process M5 Data Security Administration SMS MMS File Conversion Standard Chrom Reports Standard MS Reports System Control Automatior Mew Edit Chromatograms View Edit Methods gt gt Remover gt Add lt lt Cancel Select which MS Workstation applications are represented by icons on the Workstation toolbar The list on the left shows the applications that are currently displayed The list on the right shows applications that are installed but not displayed in the toolbar To remove an icon from the toolbar select it from the left list and click Remove To add an icon to the toolbar select it from the right list and click Add Enable Disable Instrument Modules Enable Disable Instrument Modules Instrument Modules curently disabled Select instrument modules you wish to enable Instrument Modules currently enabled Select instrum
132. ie Nba Cancel hikin Mew los Goose Pue 15 ot Fi Vad A E Une CAE sed rr do phas a a baa a Bae A heeds 5000 als 1 6 wienps TRAP al dol aed mos e pe al thee ba ah yA ca BLE noe the mineras le hour ahii he ood 1 2 el aium 1 Dir ar HOC 15 PS High ace ay thule PS l a BE e Me j m 7 ioa Es Adjust to minimum value Hou 500 20 Volta Coen Shoplet EDO led Hic eros rc ad bh rcs Actora Method anuncio Channel lb al Dewal _ Pisa h me ef City ieee 2 J tte Bonn as d j Al ce Peak 1538 Base Amount 2014 SMUOTHED en ES ys Segment 1 Channel 3 2 0 5 Be uh Scan Humber 16 Time 0007 mei RF Ramp Value RIC 2 8690 8 lon Range 139 5 20005 mz E instrument 1 Jun 19 15 37 53 Module 500 45 56 End Auto Tune Check Some Awto Tunes mol apto date 34 Module Attributes Tab Method Active Segment Checks and Adjustments Module Attributes Optimization Flots Electron Multiplier High Mass Calibration Last Set Not Applicable V Adjust high mass calibration Result Default Values Voltage fi 200 volts Damping Gas Trap Damping Gas Flow jae mL min Infusion Pump Syringe Flow Infusion Flow Rate f 0 00 UL rie Purge Flow Pate 200 00 ulmin Scan Mode Extended Expected Mass 2500 00 mz Observed Mass 3500 00 raz Syringe Type Enter the diameter or select a brand and volume To add a syringe first enter a dia
133. ion 11 none none none none none none none none c varianws mstutorials120_ng sms ALLMIX 120NG Calibration cwananwsimstutorials 160_ng sms ALLMIX 160NG Calibration c varianws mstutorials 200_ng sms ALLMIX 200NG Calibration c varianws mstutorials 80_ng sms ALLMIX 80NG Analysis 44 4 4 44 4 444 Suspend Resume New SequenceList Create a SequenceList 14 Open SequenceList Open an existing SequenceList The SequenceList dialog box follows MM SequenceList sequence seq Inject co waranweest fulll mth cc WwaranwWs amples smp Recalc cara esfull2 mth cc Wwaranws amples rel Insert Delete El Begin Suspend Resume Edit Menu Click Edit and select Edit Module Info B System Control Varian 500 LC Inject Automation Recalculate Ctrl S5hiFt Cel Chrl f Ctri Ins Chrl f Shift Ins Insert Ins Select All Ckrl 4 Ctrl Enter Fill Down Edit Motes Edit Module Info Each hardware module in your system 212 LC 500 MS Combi PAL AutoSampler etc contains its own message log and documentation screen Use the Module Information Editor to keep a record of performance maintenance hours used or other comments MA Module Information Editor C VarianWS MODULE56 MDF Description lon Trap LEMS Lab 5 Serial Number 0106 Date Installed Nov 19 2005 Date Last Serviced Jan 23 2006 Use Add Edit Help and Delete to assist you Click Done when done 15 Inje
134. ion A Sequence must be open from the File menu to use this feature Begin At Starts automation at a particular Sequence or SampleList line and runs until the last line is completed A SampleList or SequenceList must be open to use this feature Suspend Automation Stops automation at the last completed sample Resume Automation Starts automation after it has been suspended Automation starts from where it was suspended 16 Stop Automation Ends automation immediately and resets all modules Reset Modules Brings the various modules to their ready states Enable Automated Printing Print reports during automation See Injecting Samples on page 96 for more information Recalculate Menu ey System Control Varian 500 LCMS Mot Ready File Edit Inject Automation ARRE Instrument Windows Help ale a E ES 500 ESI Full 500 ESI Full Begin Begin RecalcList Begin Ab Selected RecalcList Line View Calibration Curves Begin RecalcList Start an automated recalculation A RecalcList must already be prepared and opened under the File menu or a new RecalcList will need to be made Begin at Selected RecalcList Line Process a RecalcList starting with a selected line and proceeding with the rest of the list View Calibration Curves View any curve for the calibrated compounds in the active method An example of the calibration curve screen follows For a complete explanation of this screen go to the Quantitati
135. ion File dialog box to open an existing automation file of the selected type a Save Automation File Saves the currently displayed automation file with the existing name 5 Save Automation File As Displays the Save Automation File As dialog box allowing you to save the currently displayed automation file with a new name amp Print Automation File Prints the currently displayed automation file ES Cut Deletes a selection and copies it to the Clipboard En Copy Copies a selection to the Clipboard 165 B Paste Inserts previously cut or copied information that was stored in the Clipboard into a spreadsheet El Fill Down Causes the contents of the top cell in a series of highlighted cells to be copied to the cells below it Use to edit all the cells in a column quickly Open Automation File Dialog Box Specify or open an automation file The Open Automation File dialog box appears with several titles depending upon your selection RecalcList SampleList or Sequence lt has the following fields in forms appropriate for the type of file you are about to open s Automation File Editor EA Edit Help New tal y e eel SampleList Sequence T Printer Setup Exit Look in Lists the available folders and files To see how the current folder fits in the hierarchy on your computer click the down arrow Click a folder to see its contents The box below shows the folders and files in the selec
136. ion On e lf Detector On is checked ions are detected for data acquisition e lf Detector On is not checked the multiplier is OFF during the segment and ions are not detected Scan Type Scan Type Ful E lonization eee HE n The scan type is either full scan or a special ion preparation technique The default is Full Full Full scan data for the selected mass range AMD Automated Method Development of MS MS conditions Up to ten voltages for collision assisted dissociation of the MS MS precursor ion can be tested on a single chromatographic peak using this feature After the best dissociation condition has been identified it can be selected for MS MS MRM or MS n MS MS MS MS or tandem mass spectrometry is the isolation of a precursor ion formed during ionization dissociation of the precursor ion by applying energy trapping and scanning the resulting product ions Multiple Reaction Monitoring MRM can be performed for multiple precursor ions This is useful when target analytes co elute with deuterium labeled internal standards of the same compound It is also useful when target analytes elute too closely for different time segments to be constructed MS n Multistage MS MS a product ion from one tandem MS MS experiment becomes the precursor ion for the next stage MS 3 MS 4 up to MS 10 70 lonization lonization ES The available ionization mode depends on the attached device ESI or APCI Scan Mode
137. jection of the lowest mass product ion If for example a precursor ion of 403 m z dissociated to product ions with m z values of 350 200 and 131 do not use an excitation storage level above 126 m z Non resonant excitation amplitude range 0 119 58 volts default 20 Resonant range 0 59 79 volts default 0 2 volts Set the Product lon Start and End Masses or Low and High masses to cover the mass range required You can have multiple Product lon Start and End Masses ina single segment Click More Ranges to display the Product lon Mass Ranges Dialog box with a Mass Ranges table and buttons to Add Insert and Delete Product lon Start and End Masses Adds a line to the end of the Mass Ranges Table Insert Inserts a line after the currently selected row of the Mass Ranges table Deletes one or more selected rows from the Mass Ranges table 161 tem Description S q Calculator In scientific literature storage RF values are often reported in terms of the Mathieu q parameter The value of the q parameter determines the stability of the precursor ion trajectory More stable trajectories allow higher excitation voltages to be applied before ions are ejected from the trap It has been empirically observed that a q value of 0 4 provides an optimum yield of product ions This calculator determines the corresponding CID storage RF value m z following user entry of an ion mass m z and desired q val
138. l Clear All Quick Verity The Quick Verify test checks the entire system to see if the 500 MS hardware is operating properly Results of the test are displayed in the event messages window at the bottom of the screen If a failure is reported go to the relevant section of the Troubleshooting Guide in the 500 MS Hardware Operation Manual Configuring the Monitor and Event Messages Display You can display the Monitor window and Event Messages in a larger area by clicking Hide Keypad or recover the Keypad by clicking Show Keypad From the menu you can select to display the Monitor Window or Event Messages alone or in combination Hide Keypad Monitor Window and Event Messages 7 Monitor Window Only Vacuum Syste Event Messages Onl Forelne PASAR es 47 Vacuum System Vacuum System Foreline Pumps H5652 H5652 Status Normal Normal Speed 100 100 4 Curent 1879 m mps 1637 m mps Power 486 Watts 426 Watts Life 17035 hours 22899 hours Oil Level Level OK Level OF Turbo Pumps Rear Forward Status Normal Normal Speed 100 100 Curent 417 mArmpes 570 m mpe Power 30 Watts 51 Watts Temp 37C 46C Life 7355 hours 13760 hours The foreline pump and turbomolecular pump current and power readings during routine operation are shown You can check the oil level in the foreline pumps The running lifetime in hours of both turbo pumps and foreline pumps is displayed When a pump is replaced the lifetime hours counter is
139. led when all the automation windows are open even if some are hidden under others Iconizes the windows for all the automation files in the Instrument It is disabled when all the windows are iconized Arrange Icons Arranges the icons if any in a row at the bottom of the System Control window SequenceList SampleList RecalcList Instrument Status Message Log Configuration Log only if Configuration window Is open Module Window name varies depending on module Help Menu Opens the window for the selected file or moves it to the front Use to display the open SequenceList SampleList or RecalcList Displays the corresponding window for the instrument or moves it to the front Use to see the instrument status Message Log or Configuration Log Displays the Status and Control window for the chosen module or brings it to the front El system Control Varian LC MS 1 Not Ready File Edit i Inject aa aa Automation Recalculate startup mth a 500 MS 56 Not Ready Manual Control Auto Ture Diagnostics Startup Shutdown Acquistion Description Instrument Windows Help Topics Product Support Web Site About System Control Help Topics Displays the Help Topics window for System Control Product Support Web Site About System Control If you have Internet access and a web browser installed on your computer this option automatically opens t
140. ll as the energy imparted to them as they exit the capillary and enter the skimmer Higher masses tend to require higher voltages For a given ion higher voltages usually cause more fragmentation Values range from 0 to 300 volts as appropriate for the selected polarity RF Loading Changing this value optimizes the amount of energy that an ion acquires after injection into the ion trap Lower values may minimize fragmentation for a given ion Higher values provide a deeper potential well for trapping which also imparts more energy to the ions to overcome the deeper potential well Values range from 0 to 300 Typical values are 50 to 150 A value of 100 works well for wide scan ranges Isolation Window m z As the parent ion is isolated in the ion trap this parameter specifies how many masses are stored For example a window of three stores the precursor ion an ion one Dalton higher in m z and an ion one Dalton lower in m z The values depend on the precursor ion selected For low m z ions this total isolation window is from 1 to 3 Daltons For high m z tons this total isolation window is from 1 to 14 Daltons Excitation Storage Level m z Determines the q value for the ion during the dissociation step The higher the g values the more energy is imparted to ions This also indicates the lowest m z product ion that can be stored and subsequently scanned in the MS MS spectrum The value depends on the precursor ion m z for q v
141. ll append to new RecalcList SINGLE 1011 ACL Nov 161853505 Module 500 M15 56 Acquisition Mode waiting for tempe to stabilize before gaing into Ready State 19 430 01 Display the 430 Autosampler module control screen 212 02 Display the 212 LC module control screen 500 MS 03 Display the 500 MS module control screen Help Menu 1 Click Help on the System Control menu bar E System Control Varian 500 LCMS Not Ready File Edit Inject Automation Recalculate Instrument Windows al h Help Topics 500 ESI Full mth d a E Product Support Web Site a 500 M5 56 Not Ready About System Control 2 Click Help Topics to display the following Click an item in the Contents box Help Topics Saturn GC MS Workstation System Control FIR Contents Index Find Click a book and then chick Open Or click another tab such as Index System Control Menus gt System Control Toolbar gt System Control Windows and Dialog boxes gt Saturn 2000 GCMS Module Control gt Saturn 2000 MS Instrument Window gt Saturn 2000 MS Instrument Control Command Reference 4000 instrument Window 500 143 instrument Control 3400 3600 Gc System Control Window 3400 3600 GC Injecting a Single Sample 2400 3600 GE injecting Multiple Samples gt 2400 3600 GC System Control Command Reference 3300 GC System Control Window 3800 GC Injecting a Single Sample gt 3800 GC Injecting Multiple Samples
142. lue from Auto Tune Sometimes better sensitivity is achieved particularly in techniques such as MS MS when the multiplier voltage is increased This adjustment can be made for each segment Use the same multiplier gain for determining the calibration curve and analyzing unknown samples Range 300 to 300 volts Has either 1 or 2 editable fields depending on the Scan Type selected The Automatic Gain Control Target TIC Total lonization Count always appears with differing default values depending on the Scan Type When either AMD or MS MS is selected you can set the Prescan lon Time in microseconds for that particular time segment Also you may click Customize which displays additional options to configure Fixed lonization time Max lonization Time and the Prescan lon Time if applicable 159 tem Description Automatic Gain Uses the ion count from a prescan at a fixed ion time along Control Target with this target value to calculate an ion time necessary to fill TIC the ion trap with the target number of ions during the analytical scan The objective is to fill the trap with an optimal number of ions during each analytical scan Usually the Target TIC is not set below 10000 for full scan acquisitions but it should also not be set too high or spectral distortions due to space charge loss of MS resolution and or shift in mass assignments for strong chromatographic peaks may result An optimum value of the AGC Target
143. ma b Cancel Aun Information Result Information File LA Examples STO Sample 925 91D Recalo Date NO RESULTS Inject Date 6 6 2002 11 06 AM Run Time 9 026 29 615 min Peak Measurement Calculation Type Workstation Detected Peaks Instrument M A Rejected Peaks Module Identified Peaks Aun Mode tem Description S Lists the available folders and files To see how the current folder fits in the hierarchy on your computer click the down arrow Click a folder to see the contents The box above shows the folders and files in the selected location You can also double click a folder or file in that box to open it To open the folder one level higher click the up arrow button on the toolbar File list Lists the folders and files in the selected location Double click a folder to see the contents You can also use the Look In box to see the hierarchy of folders To open the folder one level higher click the up arrow button on the toolbar Shows the currently selected file Files of type Restricts the list of files to only those that match the selected Run Information Shows information about the currently selected file Result Shows information about the results if any calculated from Information the current data file Channel Specifies the detector channel that will have Result Information displayed Note that this applies only to standard LC results Delete Results Delete results from currently select ch
144. mation Files button to open the Automation File Editor 2 Click the New Automation File button and click new SampleList 3 Enter a file name and click Save 2 Automation File Editor a Sis S aaa Create a new SampleList File Save in CQ VarianwS 0 e Ed 5 0200sys E ChromExamples methods Release 325 M5 Methods data 5 MSGLOG DsSatSys 3 500 MS Methods jdata_xyz gt MSTutorials Service 40005Service Debug O QuadService SYSLOG E 40005ys Examples E quadsys System S Autotune Logs Library O QuadTest O TutorialsQua lt gt File name Tesi smp Save Save as type SampleLists smp Cancel Recent Files gt 110 4 Select the 460 LC AutoSampler and click OK 8200 Stand lone AutoS ampler 8400 4 5 on 3800 GC 8400 4 5 on 3900 GC 8400 4 5 on 431 GC 8400 4 5 on 450 GC Cancel 5 An empty SampleList with your file name opens e Click Add to add one sample i Automation File Editor Test smp File Edit Help Sample Name Sample Type hay Inj Injection Motes AutoLink Inj Mode Inj Tray Vial Well Washes Volume Ea Default Sample Analysis Partial Loopfill 10 Left Add Lines to 460 LC AutoSampler SampleList Sample Type Cal Inj enon nase AutoLink Inj Mode Inj Automix Amount Std Unid Peak E MultiChann level Volume Tray Vial Z Well Washes Routines IS NZ only Factor Multiplier Divisor _ MultiStanda Default Sample Analysis none Par
145. ments select the entire row for each segment Defaults Sets the selected segment to default values for the selected instrument configuration Restore Restores a chromatographic time segment values to those from the original method file or from the last time the file was saved within the current editing session Restore is only valid for saved or existing method files For a new method file use the Defaults button to set the segment values to default values Special Select the mode for the 6 port valve and control the syringe Applications pump and relays 158 tem Description Method Segment Detector On Scan Type Control which MS scan function is performed Full scan AMD MS MS or MS n For each Scan Segment selected in the upper Chromatographic Time Segments table you see menus and tabs that provide the options to create the method View all the available options if the Detector On button is selected If the Detector On is not enabled the ions are deflected off the trap by the ion optics components and the RF voltage and multiplier are OFF during the segment NOTE For all data acquisition segments the Detector On box must be enabled There are four Scan Types Full AMD MS MS and MS n Select a scan type to load its default values into the Parameters and Control tabs in the lower section Scan Mode The four options are Standard Enhanced HiRes and Extended Manual Divert Load Syringe Pump
146. meter then type in a brand and volume and click save syringe To delete a syringe select a brand and volume and click delete syringe Brand Hamilton Yolume 1 0 mL mL Save Syringe Diameter 4 610 mm Delete Syringe Electron Multiplier You can override the Electron Multiplier voltage determined in Auto Tune NOTE The Electron Multiplier voltage set by Autotune determines the voltage needed for multiplier gain of approximately 10 Although this value can be overridden changing this voltage setting affects the optimal performance of the ion trap To adjust the multiplier voltage on a single chromatographic time segment enter the value in the General Parameters tab of the Active Segment Damping Gas Set the Damping Gas flow NOTE The helium damping gas maintains mass spectral resolution and improves the trapping of ions The 500 MS was optimized for a flow rate of 0 8 mL min Although this value can be overridden changing this flow rate setting affects the optimal trapping efficiency of the ion trap It can also cause the instrument to lose mass calibration Infusion Pump Set the purge and infusion flow rates for the syringe pump Alternatively the infusion flow rate may be specified in Special Applications The infusion pump motor speed is determined from the syringe volume and diameter There are two preset brands of syringes Hamilton and SGE These syringes have different volumes and diameters To add a cu
147. mount 2015578 SMOOTHED j Ric 2 787 9 lon Range 995 2000 S mir p lor FH ua Seg 1 Channel 1 147505 Fill 1000 1500 AO Acquired Range mz In this example the RF Loading is varied on a MS MS segment Automatically the five largest product masses of the precursor ion 1422 0 m z are found and the RF Loading effect on these masses is plotted Method Active Segment Checks and Adjustments Module Attributes Optimization Plots Dependent Parameter Plot RF Loading y Auto find masses with highest intensities End 300 Detaults 37 Hide Keypad lv Trap 6 Port Valve Infusion Pump Active Method Segment Scan Function Channels Intensity Normalization I lonGauge Load QUE ite re Number 2 H Channel 24 By lonization Time Data Views y T l O Inject _ Purge 40802 esi ms ms l Cycle C None 2 slo 8 Ly 3 als ai Base Peak 1290 1 Base Amount 23217 SMOOTHED a xc la EZRM RIC 537233 lon lon 2608 us Segment 2 Channel 2 M Counts 12900 3 100 1290 1 Pepe 1090 1 3 1078 1 89 27 4 978 1 75 84 9 50 25 0 i ES ie A A EO 50 100 150 200 50 500 750 1000 1 E Storage pct PPE rango miz EN fl If the active MS MS method segment is to monitor multiple precursor ions MRM the scan function channel must be set to the precursor ion The Cycle checkbox must be unchecked Active Method Seg Number 2 esl meme
148. mth in the VarianWS 500 MS Methods directory a From the Module Attributes tab set the Infusion Flow Rate to 10 uL min b Turn on the trap and allow the drying gas temperature to stabilize c Start the infusion pump by pressing Run or by pressing the front panel button If you have just loaded the syringe you can click the Purge button for a few seconds to move the solution to the needle 41 d Monitor the real time spectrum As the tune solution infuses into the trap the ionization time and ion amplitude if enabled readback values fluctuate The spectrum should look like the previous one some of the m z values might be shifted Wait for the ionization to stabilize ionization time varies by less than 15 2 Leave the trap on and the infusion pump running and go to the Auto Tune tab After major maintenance is performed such as cleaning the ion trap or changing the multiplier or a change to the Trap Damping Gas Flow is made run all Auto Tune routines Specific routines may be run periodically as part of a system check The Auto Tune window has three major sections the Control and Status display and the Auto Tune Method display and the Spectrum and Event Message windows The Auto Tune Method and Control and Status sections are displayed NOTE The High Resolution option is only available if you have purchased the TurboDDS option a 500 M5 56 Not Ready Manual Control Suto Tune Diagnostics Startup Shutdown
149. n APCI E Method Builder 500 MS ESI CALIBRATION METHOD mth EJE File Edit Yiew Window Help aca ABB sexa S el Nal 4 gt dtl fea ssl Sel 500 MS ESI CALIBRATION METHOD mth E3 Method Notes B8 ProStar 420 AutoSampler Control Address 2 4 Configuration Y 500 M5 Mass Spec Address 56 tr S500 MS Mass Spec Control y MS Acquisition Method A Instrument Configuration Acquisition Data Type TurboDDS Electrospray y C Centroid Profile F TurboDDS Method Setpoints Chromatographic Time Segments Add Te ee Insert Full Scan 0 00 2 00 ESI Auto Full Delete Defaults Pete Restore Ea Ea sl Special Applications v Method Segment A Y Detector On Scan Type Full Scan Mode Standard Defaults T Load Syringe Pump On General Parameters Ionization Control Electrospray lonization Parameters Full Scan Parameters Electrospray lonization Parameters Needle Voltage Nebulizer Gas Nitrogen Positive 5000 volts r v Nebulizer Gas Pressure 60 0 psi Negative 50004 volts Y Drying Gas Pressure 20 0 psi Spray Shield Voltage Y Drying Gas Temperature 350 E Positive 600 0 volts Waming Temperature changes of greater than 45 deg C may result in longer stabilization times Negative 600 04 volts System setting Enable or disable available modules If you have this option you can create TurboDDS methods Acquisition Ei
150. n DN Click to change polarity Click or enter a value to add a second scan channel Select the mass range for data acquisition for a segment The default value for RF Loading is 100 An optimum RF storage level is calculated automatically for the mass range that you select However you may customize the RF storage level by entering a percentage of this predetermined level The polarity of the acquired ions can also be selected for each segment Polarity switching can be accomplished by adding a second scan channel to the segment by clicking Not Used General Parameters Ionization Control APCI Ionization Parameters Full Scan Parameters Mass Range Capillary PA E Polarity Voltage aa res oa ptas RF rl olts 1 Positive 60 0 50 2000 100 2 Negative 80 0 50 2000 100 78 TurboDDS Data Acquisition Overview NOTE TurboDDS and High Resolution are only available if you have the TurboDDS chip installed Use Turbo Data Dependent Scanning TurboDDS to do automated MS scans on peaks as they elute from the column You do not need to know the retention times or m z values of the ions before making the injection In a typical TurboDDS run a survey scan is done followed by a high resolution scan and then multiple MS sequences are done Please review the material in the preceding sections if you are not familiar with auto tuning creating and using methods Set parameters in the m
151. n the instrument In this example the Modules available are the 212 LC pump 430 Autosampler and the 500 MS Iconize Module Windows Change the individual Module Windows to icons at the bottom of your screen Show Automation Windows View the automation screens currently open These could be the SampleList RecalcList and SequenceList Iconize Automation Windows Change any open automation window into an icon Arrange Icons Arrange the icons in a row at the bottom of the System Control window SequenceList Display the active SequenceList SampleList Display the active SampleList RecalcList Display the active RecalcList Instrument 1 Status View the status of the modules connected to the system Message Log View the event log for the MS module A sample log display is shown The error messages of the 500 MS Module are displayed MM Message Log MSGLOG1_11 16 2005_15_33_01 MLG Nov 16 15 33 01 Single Sample Automation Began Nov 16 15 33 07 MeszageLog Name CW arian SSM SGLOGSMS5GLOG1_11 16 2005_75 33_07 MLG Nov 16 15 33 07 MS Workstation Demo RC1 Version 6 6 Noy 16 75 33 01 Workstation 0013420187 Nov 16 15 33 07 Instrument 1 Yarian 500 LEMS Nov 16 18 amp 3301 ProStarDynamas at address 24 Noy 16153301 410 at address 25 Nov 16 15 33 01 500 M5 at address 56 Nov 16185 3301 Operator 4 Chemist Noy 16 153306 Activating Single Sample ex Noy 16 153306 Method 500 ESI Full nth Activated Noy 16153306 Results wi
152. nformation you need As you start up your system and begin acquiring data with it you interact with a window that simplifies your tasks for controlling the 500 MS system As you build methods edit and analyze acquired data you will notice that the interface has the versatility and power needed for complex mass spectrum and chromatography data handling This Operation Manual is organized to help you understand your instrument quickly After the introduction applications that compose the Varian MS Workstation are outline Read this section to get an overview of the Workstation structure and operation The sections following the Workstation Overview outline how to do manual injections and automated injections If you plan to move your system or change its configuration read the sections on installing the software and configuring your system The manual for qualitative and quantitative analysis is MS Workstation Software Reference Manual part number 0391496300 Explore many features and advantages of this software Additional Manuals The documentation for the 500 MS includes the following manuals Before using the system refer to the following manuals on the Varian MS Workstation CD Read all instructions thoroughly 500 MS Hardware Operation Manual This provides information for installing maintaining using and repairing your 500 MS System All of the information included in this manual is also available in the Online Help section of
153. nge a miz Taj me z 7 C Plot these masses Start Mazz 1 End 16144 Defaults Step 61 Linear step through the range Halt Plats disabled in TurboDOS mode j Mase 2 Mass 3 E mz En E Mass 4 es Ele a Mass 5 NOTE Before starting be sure the trap is on the drying gas temperature is stabilized and the syringe pump is running and has a stable flow In this example in order to optimize the Capillary Voltage of two MS MS precursor ions the voltage varies from 0 300 in 3 volt steps while plotting the effect on the two precursor ions The active method must be full scan to monitor both ions simultaneously 36 After clicking Start the Optimization Plot screen automatically appears adjacent to the Spectrum Plot screen You can click and drag the Bar to undock the Plot move and resize it J a it a Stim lib Tare DO rr Sada roe SS Baro Arar 4414 ore 1 Mb ca A EY Li a 1177 1 A i Larra ye i m ib EA Pi apa pa T UUN ira Esque Aja A Click the Toolbar to overlay stack or normalize the plots Double click the Title Bar to dock the window to its default location LH Li Ceti Piat Das al eo 11 gt al Mounties a Fa fi 00 iii m i A e ia gt i LEI A Takni Veen di AA Geen Finn DE Data Vier Gia E Lael Bonu Y he rans orice J BES Eterm Scan Number 45 Timo 1 44 min Base Peak SAS Base A
154. nt a value of 2 3 counts reduces the number of low level ions reported in the mass spectrum This approach may improve library searches and reduce data file size at the cost of somewhat less detailed information in the mass spectra The count threshold is shown only if the Customize button is selected lonization Control General Parameters Ionization Control Electrospray lor Automatic Gain Control Target TIL counts 100 E Customize The Target Total lon Current or TIC is adjusted in lonization Control view The Automatic Gain Control AGC algorithm uses the ion count from a prescan at fixed ion time with the target value to calculate the ion time necessary to fill the ion trap with the target number of ions The objective is to fill the trap with an optimal number of ions during each analytical scan In full scan mode an optimal Target TIC is automatically calculated and adjusted depending upon the set scan range in the Full Scan Parameters tab the calculated value for negative polarity is about 1 5 of positive polarity In MS MS AMD and MS n modes the optimal Target TIC is fixed This automatic setting can be overridden by entering the nnn field For example To double the Target TIC use 200 to halve the Target TIC use 50 Do not set the Target TIC too high or spectral distortions due to space charge loss of MS resolution and or shift in mass assignments for strong chromatographic peaks may result Typically th
155. nt masses below the precursor ion Initially adjust in 0 2 m z increments Range 1 0 to 1 0 m z Click Customize to view The High Mass Ejection is the amplitude of the broadband waveform used to eject masses above the isolated precursor ion An optimum value has been predetermined but you can enter a percent of it Range 0 200 Click Customize to view The Excitation Time is the time allotted for collision induced dissociation CID by ion excitation The default excitation time is 10 msec for both resonant and non resonant excitation Range 0 650 msec Click Customize to view This parameter checked true specifies whether the RF storage field is modulated during resonant CID This modulation greatly reduces the effect of changing secular frequency with concentration 162 tem Description S Number of Frequencies Defaults Count Threshold Click Customize to view This is the number of frequencies at which the CID excitation waveform is applied The default value is 1 frequency applied at the end caps A value of 3 generates three frequencies the resonance frequency and frequencies 500 Hz above and below that frequency Range 1 119 frequencies odd numbers only Sets the parameters of a method segment to their default values Generally any user modified mass values are preserved Appears in the General Parameters Customize option of all Scan Types Any ions with intensity less than the selecte
156. nts to be acquired across the peak e Using more micro scans with wide peaks produces spectra with a higher S N Use dynamic exclusion to ignore ions after they have been analyzed for several scans MSn scans will be done on the ions of lesser intensity If an ion may also be present later in the chromatogram use dynamic exclusion to restore it after the set time Occasionally ions of similar heights may not be dynamically excluded as expected Dynamic exclusion specifies that an ion will be excluded after a certain number of consecutive scans If the most intense ion varies from scan to scan the dynamic exclusion list may not be updated If an ion is on the exclude list it will always be excluded from TurboDDS The exclude list takes precedence over the include list Auto Tune for TurboDDS Use the TurboDDS Tuning solution part number 393211901 Auto Tune for TurboDDS is similar to Auto Tune for non TurboDDS applications The Auto Tune Method section has e Parameters in common with non TurboDDS Detector Setup Waveform Tuning and Calibration lon Optics Tuning Turn off infusion pump and source e Parameters unique to TurboDDS High Resolution Tuning and Calibration S System Control 500MS EP 3 Not Ready File Edit Inject Automation Recalculate Instrument Windows Help al 6_9 ESI calibration extended enhanced 1u gt ws EJE El a 500 MS 56 Not Ready Manual Control Auto Tune Diagnostics S
157. o Tune Diagnostics Startup Shutdown Acquisition Control and Status Source ESI Edit Method Method ESI Upload MS Method 10 00 minutes Summary of Status Vacuum System OF Damping Gas UF Save Data Temperatures UF SOLIce OE lon Gauge Pressure 0 0 uTorr Fil Valid Needle O Volts 0 0 u mps DningGas 0C OPSI Nebulizer O PSI Housing OC Capillary 0 Volts Spray Shield 0 Volts Relays Arann The ion trap turns off when there is an instrument fault Clicking the underlined text turns on that part of the ion trap system Off underlined text is black and lower case On underlined text is green and in capitals Trap Needle 5 Volts 0 1 wimps trap on off Drying Gas 163C OO PSI Nebulizer 0 FSI Capillary 0 Volts Spray Shield 25 Yolts Housing 50 C Click trap on off to turn on the entire trapping system 27 Click ion source Nebulizer amp Drying gases and needle voltage rf and detector multiplier text to turn on those functions The appropriate fields turn green when ready This indicates that the ion trap is on as displayed next Summary of Status Vacuum System LIF Damping Gas UF Temperatures Varn SOUrCE OK lon Gauge Pressure DD uTorr FA Valid Reni hale Trap are in red Needle 5000 Volts 1 8 w amp mps TRAP ON OFF Drying Gas 290 C 15 PSI Nebulizer 25 PSI Capillary 20 yolks Spray Shield 600 Volts Housing 50 C
158. on frequency of the trapped ion The resonant frequency of the trapped ion depends on ion mass space charge within the ion trap RF trapping field amplitude and other instrumental factors Thus it is difficult to calculate precisely its value The amplitude of the RF trapping field is therefore modulated over a fixed range Because the resonant frequency of the trapped ion depends 91 on the magnitude of the RF field modulating the RF field amplitude modulates the resonant frequency of the ion Modulating this frequency causes the frequency of the ion to match periodically that of the applied supplemental dipole field Thus the energy coupled to ion motion is maximized and the effects of shifts in the ion resonant frequency are minimized The resonant excitation method is selective for the mass range that is excited It permits coupling of energy to the motion of an ion having a particular m z value in a very controlled way Consequently at the correct excitation amplitude the rate that the amplitude of the ion motion increases is balanced by the rate that energy is removed by collisions The ion is not ejected and energy can be deposited as internal energy by increasing the number of collisions Increasing the excitation time increases the number of collisions Thus it is possible to fragment precursor ions that require the breakage of multiple chemical bonds or that undergo significant rearrangements Periodic modulation through ion resonanc
159. on section NOTE Recalculation may be performed in System Control or in MS Data Review from the Process Data icon W Calibration Curve Propyl Paraben Print Print All Export Overlay Point Info Coefficients paraben_ ca 200ul Ub mth 1200 42 Propyl Paraben External Standard Analysis Curve Fit Linear Origin Ignore Weight None Resp Fact RSO 17 43 Coeff Detir2 0 999636 t3 lel eee fie oe eb ee i Feplicates 4 Amount pob Peak Name Ea E Exact View Curve Fit Type Linear vw Curve Only Flot Type Origin Point Ignore XS Cursor Linear Rever Regression None TETE C Log Cancel 17 Instrument Menu The Instrument menu follows ic system Control Varian 900 MS Not Ready File Edit Inject Automation Recalculate Bian gs Windows Help 2 55 ES startup mth e 1 Varian 300 MS Alt 1 500 MS 01 Not Ready Manual Control Auto Tune Diagnostics St Control and Status Savel Source ES Edit Method Rec setup Ethernet Communications i Setup ADC Board 1 0 Ports Method ES Upload MS Method En Setup COMM Ports Varian 500 MS The configured instrument Configuration Alt c Remove Module Names Restore Disabled Warnings Instrument 1 Faults Configuration Add new modules to the existing instrument This is necessary if additional hardware is added such as a Combi PAL AutoSampler Remove Module N
160. onitor Window and Event Messages v Detector x lon Gauge eee a Setpoint Pressure 0 0 uTor Multiplier Filament 1 OK HS652 HS652 Dunode ynode Filament 2 Untested Status Normal Normal Waveform System Foreline Pressure 956 mT on Speed 100 100 lan Soles Current 1879 m mps 1637 m mps Setpoint Power 484 Watts 426 Watts Life 17035 hous 22899 hours rene o 5 Dil Levet Level OK Level OK Noodie Curent do Temperatures and Gases Capillary Voltage 0 2 Volts 0 0 Turbo Pumps Setpoint Spray Shield Voltage 13 9 Volts 0 0 Rear Forward Drying Gas Temp 68C 0 lon Guide DC 0 1 Volts 0 0 Status Normal Normal Drying Gas Press 2PSI 0 0 Exit Lens Voltage 0 1 Volts 0 0 Speed 100 100 Z Spray Chamb Temp 50 C 50 Focus Lens Voltage 1 8 Volts 0 0 Current 417 m mps 570 m mps Nebulizer Press OPSI 0 0 Deflector 1 Voltage 0 1 Volts 0 0 Power 30Watts 51 Watts Damping Gas Flow 0 8 mL min 0 8 Deflector 2 Voltage 0 1 Volts 0 0 Temp 37C 46C Trap Focus Lens 1 2 Volts 0 0 Life 7355 hours 13760 hours Trap Offset 0 1 Volts 0 0 Keypad Field The upper portion of the screen is divided into three sections Control and Status Monitoring and Diagnostics Tests Control and Status The State field indicates Idle when diagnostic tests are not running or Running if tests are in progress The Function field indicates Monitoring Status unless a diagnostic test is running in which case the name of the current test is displayed Start Diagnos
161. ons are subjected to data dependent scanning The sections are Charge State Dynamic Exclusion Include List and Exclude List e Charge State The options are e Exclude Charge State 1 lons with a charge state of 1 are excluded This is useful for applications such as peptide digests e Exclude Charge State 2 lons with a charge state of 2 are excluded This is useful for applications such as drug metabolites e Dynamic Exclusion After an ion has been analyzed you can eliminate it from further data dependent actions either for a set time 83 or for the rest of the run Excluding these ions allows for the analysis of less intense ions e Use Dynamic Exclusion Check Box Select to apply e Exclude After n scans Select from 1 to 9 scans After an ion is observed for the specified number of consecutive scans it is added to the dynamic exclusion list No further High Resolution or MSn experiments are done on it e Restore The options are e Never Restore Keep the ions on the dynamic exclusion list for the rest of the run e After n min Remove the ions from the dynamic exclusion list after the specified time This is useful for excluding ions from a chromatographic peak and searching for them in other peaks Method Segment Y Detector On Scan Type TurboDDS Scan Mode Standard Defaults Y Manual F Syringe Pump On General Parameters Ionization Control Electrospray lonization Parameters TurboDDS Par
162. or Shut Down The State displays Ready Shutting Down or Starting Up Conditions Shut Down State Shutting Down Status and Control Conditions Start Up State Starting Up Vacuum System Vacuum system displays either Not Ready or Ready The speed of the turbomolecular pumps must be 100 for Ready to be displayed Pneumatics Displayed if auxiliary helium damping gas is flowing to the ion trap Damping gas flow is required for adequate mass spectral resolution and sensitivity It is set at 0 8 mL min which is the optimum flow rate for most applications This flow rate is set in the Module Attributes tab in Manual Control The damping gas automatically turns off during Shutdown and automatically turns on when Startup is complete If necessary the damping gas flow can be turned off for diagnostic purposes When off the flow remains off in all modes except for Acquisition mode where it automatically turns on 51 lon Gauge Monitor the internal pressure here The ion gauge filament is automatically turned on when you open the Startup Shutdown window It turns off automatically after 20 minutes Vacuum System Monitor the status of the foreline pumps and turbomolecular pumps during Shutdown and Startup The turbomolecular pumps speed is 100 2 during normal operation The rate at which the turbomolecular pumps start up is proportional to how long they have been shut down After short shutdowns the time to reach 100
163. or your 500 MS HPLC modules and AutoSampler It may also include sections for data processing e Setup and plumb the valve for manual injection e Setup a SampleList that may include standards and unknown analytes e Setup RecalcLists where appropriate for analysis of data e Prepare and load the samples into the AutoSampler The following sections are a general overview of these steps and the steps required for optimization of the 500 MS system Overview When making a single injection with the 500 MS LC MS system use the Inject Single Sample button It defines the sample and method that it used in the analysis When you programming multiple injections through automation use a SampleList to name each sample and specify the injection Making injections with your 500 MS system requires that these three steps be completed prior to starting your analyses 1 The 500 MS and the necessary chromatography modules were powered on and have reached their operational setpoints The sample s are prepared and ready to be analyzed A method with the desired analytical conditions exists Step 1 involves turning on the various components if they are not already on making sure there is enough solvent in HPLC solvent reservoirs paper in printers the MS is pumped down tuned and any set temperatures have been reached and are stable Step 2 requires that the samples be in solution diluted and any other sample specific preparations necessary
164. ou want to Import one or more sections Use the File gt Import Section command to open the Import Method File dialog Method Builder 500 MS test mth ESAS Edit view Window Help Pes Open Close Save Save As Sek Password Print Print Preview Print Setup 4dd Module Control Delete Section Import Section 1 500 ESI M5 M5 mth 2 500 ESI Full rmth 3 LEMS test mth 4 500 M5 test mth Exit Prompt For Action ak Startup Ctrl M Cirio Ctrl 5 Import Method File Ctrl F i ESso00 600fast mth F500 APCI AMD mth E5 500 ESI M5 M5 2 mth ES 500 APCI Full mth ES 500 ESI MS M45 mth ES500 APCI MS MS mth ES 500 M5 test mth ES 500 ESI AMD mth ES 5000 ESI Full mth Add Post run Processing E4250 segs mth E5 500 ESI Full 2 mth ES Autotune mth MT gt File name FOO ES Full rath Files of type Methods mth Cancel Recent Files gt Select the source Method from which you will import Click the sections that you wish to import If you click one section and then hold down the shift key while clicking on another section all of the sections in between are selected Hold the control key down while clicking on a section to add it to the selected files 62 Clicking a highlighted section while holding down the control key removes that section from the list of selected files Import Method Sections
165. ows for the named Instrument which is Varian LC MS 1 for this example Enter or edit the Instrument name in the System Control Configuration window Configuration Displays the System Control Configuration window Remove Module Brings up a dialog listing any associations you have set up Names You may select items from this list to remove individual associations in preparation for renaming LC modules Instrument Displays the Instrument Faults dialog box with recent and Faults self test messages and faults The list may be printed from 136 Description this dialog box Setup Ethernet Available if an optional module driver is installed that uses Communications Ethernet to communicate with the device See the Operation Manual for your module driver for a detailed description if applicable Allows set up of the communication between the 3800 GC and System Control Setup ADC Opens the Setup ADC Board I O Ports Dialog Box to Board I O Ports configure the ADC Board base address and determine the Switch S1 settings for each ADC Board Applicable only if the ADC Board module driver is installed Setup COMM Available if an optional module driver is installed that uses Ports Serial I O to communicate with the device See the Operation Manual for your module driver such as the ProStar Dynamax pump for a detailed description if applicable Configuration Window El System Control Configuration File Automation
166. pted for a new password if no previous password exists for the Method for the old and new password if the Method already contains a password NOTE If creation of new passwords is disabled in the File Revision tab you will not be able to add a new password to a Method that does not already contain one Method passwords can also be added and changed in the Method Builder and Interactive Graphics applications Remove Method Passwords Remove Method Passwords using Security Administration without entering the existing password This is useful if the password for a Method has been lost Click Select Method select the desired Method and the password is removed Application Locking Passwords Application Locking File Revision Settings Unlocked Applications Select applications you wish to lock and click on the Lock buttar CW arianw S Program Manager Star Toolbar rock System Control Yaran GC MS 1 Not A Locked Applications Select applications you wish to unlock and click on the Unlock Button Unlock Unlocked Applications Shows the top level windows currently running that are not locked Select any number of them click Lock and they move to the Locked Applications list box and their windows are disabled they will not respond to mouse or keyboard input 129 Note that applications that are locked by the Security Administration application remain locked even after the Security Admini
167. r conserving the tuning solution When this box is checked the infusion pump and source turn off at the completion of Auto Tune Auto Tune routines are performed in the order listed beginning with Integrator Zero All of the auto tune methods are selected if you click Select All or cleared if you click Clear All To do Auto Tune click the desired check boxes and click Start Auto Tune Stop Auto Tune at any time by clicking Stop Auto Tune For a complete description of all the auto tune routines see the Auto Tune section presented earlier in this manual 151 Diagnostics Control and status for monitoring and diagnosing the Mass Spectrometer a 500 MS 56 Not Ready Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Control and Status Monitoring Diagnostic Tests State Idle Trap On Off lon Gauge Function Monitoring Status lon Source Vv Jon Gauge Filament 1 C Filament 2 N Multiplier Dynode MV RF Control RF Waveform Hide Keypad Monitor Window and Event Messages y Detector lon Gauge pisar Sta Sree Setpoint Pressure 20 3 uTorr Multiplier 1451 Volts 1475 Filament 1 OK Pump 1 Pump 2 Dynode 15029 Volts ions 15000 Fiamert 2 Untested Status Normal Normal Waveform System Foreline Pressure 807 mTorr Speed 100 100 Entrance Endcap Trapping Field Dipole 102 as i se ati a mAmps Exit Endcap Trapping Field Dipole 101 Setpoint wer 441 Watts 448 Watts the Thom 116
168. r watts Filament Untested Temp 32C WC Life Foreline Pressure 977 mTorr boss Mia Hide Keypad Event Messages Mow 18 05 52 04 Startup Pumps starting up 53 During start up watch Pump Spin Speeds increase The software locks in Startup Shutdown and Diagnostics modes until the pump speeds reach 100 You can also monitor the startup from the Diagnostics tab The Damping Gas flow automatically turns on after the Pump Spin Speed reaches 100 and Quick Verify runs Quick Verify checks the entire system to determine if everything is operating properly Hide Keypad Event Messages Mow 16 O8 52 04 Startup Pumps starting up Mow 18 08 53 37 Startup Pump Startup Complete Mow 15 08 53 37 Guick Verify Started Mow 15 08 53 37 GuickVerify Completed NOTE If any hardware problems were detected upon startup or from the Quick Verify test the system automatically loads the Diagnostics tab where you can determine the problem Acquisition Mode About Running Analyses Selecting the Acquisition tab activates the 500 MS module If you start an analysis while the instrument is in another mode the software automatically shifts the MS module into Acquisition mode T System Control MS Ready File Edt Inject Automation Recalculate Instrument Windows Help SOFE HERMED a 500 MS 01 Ready p ale Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Status And Control M
169. rameters lonization Control APC lonization Parameters Full Scan Parameters setPaints f Scan Time 341 Maximum Scan Time Multiplier Offset 03 volts pican Averaged 3 uScans Data Rate 0 29 Hz r Centroiding Parameters Count Threshold 1 Customize 76 There is a trade off between how rapidly spectra are collected and the reproducibility of mass spectra or peak areas AS more microscans are averaged the relative intensities of ions in the mass spectrum become more reproducible However collecting more data points across a chromatographic peak improves peak area reproducibility for replicate injections Averaging three scans provides good spectral quality and an acceptable data rate The Multiplier Offset adjusts the EM voltage as much as 300V relative to the current multiplier setting in the Module Attributes tab dialog in Manual Control this is usually the 10 gain value from Auto Tune Sometimes better sensitivity is achieved particularly in techniques such as MS MS when the multiplier voltage is increased Note that this adjustment can be different for each segment The Count Threshold is normally 1 count a value of 2 3 counts reduces the number of low level ions reported in the mass spectrum This approach reduces data file size at the cost of less information The count threshold is shown only if Customize is selected lonization Control General Parameters lontzation Control
170. rameters Full Scan Parameters Mass Range Capillary Polarity Voltage a rl ae HF fa Molts 1 Positive 80 0 50 2000 _2 NotUsed Click to change polarity Select the mass range for data acquisition for a segment The default value for RF Loading is 100 An optimum RF storage level is calculated automatically for the mass range that you select However you may customize the RF storage level by entering a percentage of this predetermined level The polarity of the acquired ions can be selected here for each segment To do polarity switching for each segment add a second scan channel to the segment by clicking Not Used Enter the parameters for that scan and click Not Used until Negative is displayed General Parameters lonization Control Electrospray lonization Parameters Full Scan Parameters Mass Range Capillary Low Mass High Hass RF Loading Polarity Yoltage Volts m z m z 1 Positive 80 0 100 2000 100 a0 0 100 2000 _2 Negative APCI Parameters This section explains how to set parameters for an atmospheric pressure chemical ionization method General Parameters Scan Time uScans Averaged and Data Rate are linked When you change the Scan Time the Minimum number of Scans Averaged and the Data Rate are calculated for you If you adjust the Minimum Number of Scans Averaged the Scan Time and Data Rate are calculated for you General Pa
171. rature is too high the sample could thermally degrade If the gas flows is too low the baseline may be unstable If the gas flows is too high the size of the peaks may be reduced The vaporizer can be moved back in the spray chamber to lessen the likelihood of fouling the vacuum capillary when the matrix contains a large amount of nonvolatile material but at some expense in signal intensity To adjust the position of the vaporizer relative to the capillary entrance loosen the four mounting screws on top of the spray chamber and slide the vaporizer assembly forward or backward A numbered scale on the right side of the vaporizer assembly helps to reset the position Be sure to tighten all four screws snugly when you are finished to avoid heat damage to the vaporizer It must be firmly attached to the spray chamber for proper heat sinking The following lists typical APCI operating parameters 87 APCI Typical Operating Parameters WARNING BURN HAZARD The APCI Source may become hot during operation Take appropriate precautions Drying Gas 20 psi at 150 C Nebulizing Gas 60 psi Nitrogen in positive mode Air in negative mode automatically switched Auxiliary Gas 40 psi as hot as possible without decomposing sample maximum temperature setting is 550 C Corona Current 5 10 yA Housing Temperature 65 C Comparison of Parameters for ESI and APCI PARAMETER ESI APCI Auxiliary Gas N A 88 MS MS Tandem Mass
172. rs Full Scan Parameters Electrospray lonization Parameters Needle Voltage Nebulizer Gas Nitrogen Positive 5000 volts Nebulizer Gas Pressure 35 0 pal Negative 5000 vols F Dring Gas Pressure 10 0 pal spray Shield Voltage Drying Gas Temperature 350 C Positive eo0 0 volts Negative 600 0 wolts 147 Full Scan Parameters Select the mass range over which data is acquired An optimum RF storage level is calculated automatically for the mass range selected However you may customize the RF storage level by entering a percentage of this predetermined level Also select the polarity of the acquired ions for each segment General Parameters lonization Control Electrospray lonization Parameters Full Scan Parameters Mass Range Capillary 7 Voltage Low Mass High Hass HF See Molts Af Posie 100 Checks and Adjustments Adjust the RF ramp RF voltage ramp is a check of linearity of the RF voltage over the operating mass range of the system The display ramp should be linear and with minimum amplitude The RF adjust screw is on the front panel of the 500 MS a 500 M5 56 Not Ready TB Manual Control Auto Tune Diagnostics Stertup Shutdown Acquistion Control and Status Save Data Method Active Segment Checks andAdustments Module Atirbutes Optimization Piots Sowce ESI Edit Method Record Dats RF Ramp Adjustment Last Set Date Not Appli
173. s are selected if you click Select All They are cleared if you click Clear All Auto Tune Method Select All Clear All i Detector Setup Integrator Zero Set Electron Multipher i Waveform Tuning and Calibration Supplemental Waveform Turing RF Full Scale Adjust Mass Calibration Trap Frequency Calibration lf lon Optics Tuning lon Lens Tuning ie High Resolution Tuning and Calibration Supplemental Waveform Tuning Mass Calibration i Turn off infusion pump and source To perform Auto Tune click the check boxes and click Start Auto Tune Each of the routines you selected will be executed Stop the auto tune by clicking Stop Auto Tune NOTE Run all available routines during an Autotune However any of the three specific routines may be run periodically as part of a system check Diagnostics Mode In diagnostics mode monitor the status of the vacuum system applied voltages gases and temperatures in real time lt also offers diagnostic tests The Diagnostics Window is divided into two panes the Keypad above and the Module Window and Event Messages display below 45 a 500 MS 56 Not Ready Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Control and Status Monitoring Diagnostic Tests State Idle Trap On Off lon Gauge Function Monitoring Status lon Source lon Gauge Filament 1 Filament 2 F Muliplier Dynode l RF Control RF C Waveform Hide Keypad M
174. s Temperature TF Use Ramp Rate Initial Hold Time 0 20 min Start Temperature 350 C Ramp End Time 0 20 mir End Temperature 350 E E Ramp Rate 0 00 C min lo Time 10 00 min VETER NOTE An explanation of the Drying Gas Temperature ramp rate follows the APCI Setpoints screen capture 67 APCI lonization Setpoints Spray Chamber Temperature al Corona Current Positive 5 microamps 5 Nebulizer Gas Pressure US Corona Current Negative microamp e Drying Gas Pressure d i Spray Shield Yoltage Positive 600 0 volts a r T Vaporizer Gas Pressure US i Spray Shield Yoltage Negative 600 0 volts Drying Gas Temperature TF Use Ramp Rate Initial Hold Time 0204 min Start Temperature Ramp End Time 0 20 mir End Temperature Ramp Rate 0 00 C min Method End Time 10 00 min Ea Vapornzing Gas Temperature FP Use Ramp Rate Initial Hold Time Start Temperature 0 20 min Ramp End Time 0 20 min End Temperature Ramp Rate 0 00 C min Method End Time 10 00 min and Drying Gas Temperature Ramp ESI and APCI In gradient applications that start with very low aqueous mobile phase and end with very high aqueous phase you can ramp the temperature of the drying gas Setting the drying gas temperature ramp 1 Click Use Ramp Rate to enable it a Enter an Initial Hold Time if needed b Enter the Start Temperature c Enter the Ramp End Time d Enter the End T
175. s generated by detector modules using this SampleList Numbers will be appended to file names if the file already exists Do not include the file extension in the Data File name Directory for Data Files Data File names ES ca Zd _ s VARIAN WS Z 6 9 EVA Example 8 5 2003 Sample 1 Use the following symbols to enter the comesponding variable data to the file name Hew Folder AE eae Injection number Date Detector Module name Injection Time i Drives Er canos The left side of the Data File Generation dialog box allows you to select the drive letter and subdirectory path where the data files are to be stored The default directory is the data subdirectory of your VarianWS directory The right side of this dialog box allows you to create a filename specification You can combine text entry with the variable symbols shown to specify filenames that contain sample injection specific information An example of the filename is dynamically updated as you type in the filename specification This makes it easy to see how a data file created with this filename specification would appear 102 Specifying a RecalcList The files created by the execution of the SampleList can be stored ina RecalcList which keeps track of the names of the created files and other parameters The RecalcList can be reprocessed later for example to reprint all the reports or to process all the data with another method
176. s window for the selected sample to edit or create a note about the sample AutoLink Two AutoLink commands when Sample Type is Baseline Analysis Calibration or Verification One AutoLink command when Sample Type is AutoLink One Activate Method Command when Sample Type is Activate Method Opens the AutoLink Parameters dialog box to set the options for linking to a remote application during automation through System Control Opens the Activate Method dialog box to set the path name for activating a new method during automation of a SampleList through System Control Unid Peak Factor 0 to 1 000 000 0 Sets a calibration factor for unidentified peaks Not used for calibration samples 175 ttem Description S Multiplier 0 000001 to 1 000 000 0 Sets a value for the multiplier Results for the sample are multiplied by this value Not used for calibration samples Divisor 0 000001 to 1 000 000 0 Sets a value for the divisor Results for the sample are divided by this value Not used for calibration samples Amount Standard 0 000001 to 1 000 000 0 LC Files Sets the amount of the first internal standard Use to calibrate results for Internal Standard and Normalized Percent calculations Not used for calibration samples MS files Sets an IS Factor used by Analysis and Verification samples It is multiplied by the appropriate Compound Calibration Level Amount in the Data Handling Method used Note that internal s
177. searches and quantitation Profile Data Profile data is raw data The data files are much larger than centroid because ten data points are collected at each m z Profile data can be used to diagnose difficulties with mass resolution or mass assignment Profile Data is used to determine molecular weight of voltage charged molecules The following shows centroid data in the top and profile data in the bottom Profile data can be quantitated E Spectra noe spectrum 14 bed 10 967 min Scan 995 Channel 1 lon 1385 us RIG 220526 AC 145 Centroid Data ee eee ee Spectrum 18 PROFILE 10 945 min Scan 1034 Channel 1 lon 1255 us RIC 374881 143 Profile Data 141 142 143 144 145 146 147 148 a Range mlz 66 Setting Method Setpoints Instrument Configuration Acquisition Data Type Electrospray Centroid Profile EEES A 10 00 ESI Auto Full Chromatographic Time Segments O menos O 2 Add Insert NOTE All the set point parameters can be changed per segment except for the temperature ramp rates for Drying Gas and Vaporizing Gas APCI They are adjusted in the lonization Parameters tab Electrospray lonization Setpoints Spray Chamber Temperature AL Needle Voltage Positive 5000 walts Nebulizer Gas Pressure i Needle Voltage Negative 500 wolts Drying Gas Pressure Spray Shield Voltage Positive 600 0 volts Spray Shield Voltage Negative 600 0 valts Drying Ga
178. ses Profile files are also approximately 10 times larger but can be converted to centroid after acquisition 144 Chromatographic Time Segments Use the Chromatographic Time Segments to time program analysis conditions to get the best results for each segment Time segments up to 1440 minutes in length can be added inserted or deleted using the Add Insert and Delete tabs You can acquire a mass spectrum in full scan with a single analysis segment Or you can tailor variables such as acquired mass range ionization parameters insert MS MS segments for individual analytes Double click the required field to edit the Segment Description Start and End times The Scan Description information is automatically entered based on the choice of Scan Type and lonization mode made in the Method Segment Use Special Applications to set the configuration of the 6 Port Valve Setpoints The parameters which can be modified at the method level by clicking Setpoints are shown for Electrospray lonization These settings can be overridden for each segment under the Active Segment section The actual values of these parameters are displayed in the schematic picture on the left in the 500 MS module window Electrospray lonization Setpoints Spray Chamber Temperature L Needle Voltage Positive 5000 volts a Nebulizer Gas Pressure ai Needle Voltage Negative 6000 44 volts 2 Drying Gas Pressure Spray Shield Yoltage Positive
179. speed should be less than 2 minutes After extended shutdowns the pump speed increases in stages related to power consumption This slower startup improves pump lifetime NOTE Failure to reach 100 pump speed in a reasonable time indicates a vacuum leak and corrective action should be taken Go to the Troubleshooting section in the 500 MS Hardware Operation Manual Shutting Down the System To shut down the 500 MS click Shut Down in the upper left corner of the screen The damping gas and heaters turn off and the speed of the turbo pumps gradually reduces to 35 Cancel the shutdown procedure by checking Start Up Seles a 500 M5 56 Not Ready Manual Contral Auto Tune Diagnostics Startup Shutdown Acquisition Vacuum System Status and Control Foreline Pumps Conditions Shut Down Start Up HSER HEES per phe Status Mormal Normal Vacuum System Speed 100 100 Chats Not Ready Curent 1879 m mpe 1637 m mpe Power 466 atks 425 Watts Preumatics Life 17035 hours 22099 hours Damping Gas Off i Dll Level Level OK Level OF Flow Rate Setpoint OFF p Turbo Pumps Flow Rate 0 1 mL min Rear Forward inlet Pressure 20 PS Status Mormal Normal Speed 100 TOO lon Gauge Current 417 m mps 50 m rmnpe Pressure 0 0 uTorr Power 0 Watts 51 watts Filament UF Temp 3C 460 Lite 2355 hours 1360 hours Forelin Pressure 96 mTorr A DRE ect 7 16 49 37 Shutdown Oct 7 16 49 37 52 Pump Heated Zones
180. st of files si Automation File Editor sequencelist seq File Edit Help as ao S aa E sequencelist seq Sequencel ist Action Method Sample RecalcList t c varianws 500 ms methods500 ms 127 Security Administration Overview Passwords Use the Security Administration features to set passwords limit access to changing methods require comments when methods changes are saved and time and date stamp data files This window has three tabs e Passwords e Application Locking e File Revision Setting Passwords Application Locking File Revision Settings Password for this application You may set change or remove the password required to enter this application Change Method Passwords Tou may set or change passwords required to sawe changes to Methods Click on the button below to select the Method and then modif the password Select Method Remove Method Passwords ou may remove passwords required to save changes to Methods Click on the button below to select the Method Select Method 128 Password for this application Specify a password for the Security Administration application NOTE Document the password in a secure location because it cannot be recovered Change Method Passwords Method passwords can be added changed and removed from the Security Administration application Click Select Method to browse for and select the desired Method You are either prom
181. stom syringe follow the directions listed under the Syringe Type header After making changes click Activate Changes to apply them 35 High Mass Calibration NOTE Available only if you have purchased the TurboDDS option Adding a High Mass Calibration Point To improve the accuracy of mass assignments add a mass calibration point greater that the highest values in the Autotune table 2622 1 Infuse or inject a compound between 2622 and 3500 m z Note the observed m z 2 Select Adjust High Mass Calibration enter the observed m z and the expected exact m z 3 Click Activate Changes and the new calibration point becomes part of the mass calibration table Do a high mass calibration each time you do an AutoTune If you leave the Module Attributes tab and then return Adjust High Mass Calibration will unavailable but the expected mass value will contain the one you entered Optimization Plots Tab NOTE Not available in the TurboDDS option Do MS MS method development with the built in syringe pump using the Optimization Plots tab view Select a dependent parameter such as Capillary Voltage and then vary the parameter setting while monitoring and plotting the effect on one or more masses Method Active Segment Checks and Adjustments Module Attributes Optimization Plots Dependent Parameter Plot Needle Voltage RIC f Auto find masses with highest intensities 50 00 2000 00 EE Mass Ra
182. stration application is closed Locked Applications Shows the top level windows that are currently running and are locked Select any number of them Click Unlock and they move to the Unlocked Applications list box and their windows are enabled they respond to mouse or keyboard input File Revision Settings Passwords Application Locking File Revision Settings Require Revision Log entry when changes to Methods are saved IW Update Revision Log in Data Files with recalculation date time and Method name Enable results deletion from Open Data File dialog boxes M Disable creation of new passwords in Method files Require Revision Log entry when changes to Methods are saved When checked the user is prompted for a description of changes when a Method file is altered and saved The Revision Log appears in the Notes portion of the Open Method File dialog box is listed in the Method Builder application window when the Method is open and is included in the Method printout under Revision History Update Revision Log in Data Files with Recalculation date time and Method name When checked Data Files are updated with a time stamp and Method name when they are recalculated either from System Control or from Interactive Graphics The Data File Revision Log can be included in printed reports Enable results deletion from Open Data File dialog boxes When checked a button appears in the Open Data File dialog box allowing
183. t to displays a window previewing the spectrum JE Click scale to select Auto Scale Intensity or Fixed Scale Intensity krm _ Click mass range to choose Acquired Mass Range Fixed Mass Range or Maximum Me 22 Click to choose graphics or a text display of ion intensity and status information Chromatogram Toolbar alo B A GB 2s a A Hide the toolbar Click in the upper left of the full screen display to restore the toolbar l Restores the chromatogram to full scale Double click in the lower left corner of the scale does the same i Restore previous settings pa Select Auto Scale Intensity or Fixed Scale Intensity A Select display of multiple chromatograms generated by specific ions entered in the lons field Stacked Overlaid or Normalized ea Clear and restart the chromatogram display EF ai Display a plot of the Intensity of the Base Peak To display the RIC trace and the Intensity of Base Peak plot at same time enter IBP in the lons field Clicking the Base Peak icon does not affect the display of the extracted ion chromatogram lons Display chromatograms by entering the m z values 156 Method Builder Command Reference The available commands depend on the Instrument Configuration and if TurboDDS is enabled In the following graphic the Instrument Configuration is Electrospray ESI The other available Instrument Configuration is Atmospheric Pressure Chemical lonizatio
184. t window opens E E il DAT JAN Es Click to open QuickStart 103 Enter the name of the folder you wish to use for storing the Run files Press Start to begin MS QuickStart 1 File Screen Options Instrument Number Instrument Name Operator Name Sample Name Sample Description 1 Sample Description 2 Primary Method Instrument H o perator D efault Sample ON E ICMWarianw S istartupl mth Folder for Data File Storage Waran S EN Sample Type C Baseline C Calibration Analysis C Yerification AutoLink Vial Al H injects i Wash Yol oo Yolume 1 Amount i Factor Hultipher a Divisor CS hn Inj Mode Partial Loopfill Hardware Enter information about the sample Enter the name of the Method you wish to use Enter sampling information This information is specific to the type of sampling device installed Selecting a Vial from the AutoSampler Display Each supported AutoSampler has a graphic representation of the carousel System Control You may do an individual injection of any vial by double clicking the appropriate vial in the display The Inject Single Sample dialog appears and after you enter the information the injection is done 420 26 Ready AutoS ampler Operation Runt ime 0 00 min EndTime 0 60 min Active Sample Status a Name Location Vial Al pez AUX 1 2 3 4 Method nie
185. ta File the Data Files Generation dialog box opens Select the path and the file name specification for the data file The left side of the Data File Generation dialog box allows you to select the drive letter and subdirectory path to store the data files Error Objects cannot be created from editing field codes Use the right side of this dialog box to create a file name specification Combine text entry with the variable symbols to specify file names that contain sample 124 Injection information An example of the file name is updated as you enter the file name specification This feature makes it easy to see how a Data File created with this file name appears Specifying a RecalcList In System Control click Inject Single Sample and OK Inject Single Sample E Cal Injection Amount Std Unid Peak E MultiChannel Sample Type level Inj Notes AutoLink IS NZ only Factor Multiplier Divisor MultiStandard Default Sample Analysis X 1 none none 1 0 1 1 none 4 gt Inject the Sample using the Method C Vanan w S startup1 mth Browse Defaults r Cancel DataFiies RecalcList Create a new RecalcList append an existing RecalcList or neither create nor update a RecalcList To select the RecalcList option that you want click RecalcList The RecalcList Generation dialog box opens RecalcList Generation You can automatically create or update a RecalcList with fles generated during automated injections
186. tandards in Analysis and Verification samples always use the amount that is specified in Calibration Level 1 MultiChannel None multiple specific channel MultiStandard LC files Opens the Data Handling Channels dialog box to specify the calibration parameters for up to four different Detector Channels MS files Not used by MS data handling dd Adds a line to the end of the SampleList Adds a line before the highlighted line Deletes the highlighted line in the SampleList Fill Down Copies the contents of the top cell in a series of highlighted cells to the cells below it Use to edit all the cells in a column quickly Add Lines Displays the Add Lines dialog box allowing you to specify the number of lines to either insert or append to the spreadsheet along with the values to use for each applicable field Certain fields such as Sample ID and vial number can be automatically incremented Defaults Displays the Set Defaults dialog box allowing you to specify default values for each applicable field in the SampleList Data Files Opens Data File Generation where you specify the naming scheme used for Data Files generated from injections If the method contains both MS and standard LC DH method sections then both sms and run files are generated RecalcList Opens the RecalcList Generation dialog box to specify the options for generating or updating RecalcLists after injections Note that if the method used both MS and standard LC
187. tartup Shutdown Acquisition Control and Status Action Idle State Idle Start Auto Tune Auto Tune Method Clear Al V Detector Setup Integrator Zero Set Electron Multiplier iV Waveform Tuning and Calibration Supplemental Waveform Tuning RF Full Scale Adjust Mass Calibration Trap Frequency Calibration iV lon Optics Tuning lon Lens Tuning iV High Resolution Tuning and Calibration Supplemental Waveform Tuning Mass Calibration iV Turn off infusion pump and source Method Builder for TurboDDSTM Enabling TurboDDS In the Method Builder window click TurboDDS Method to enable it The Method Editor dialog box appears Click OK and all segments are converted to TurboDDS TurboDDS data can be acquired in either Centroid or Profile mode e Use High resolution data in profile mode to determine the charge state e Use centroid data to export protein data to external databases urboDDS Y TurboDDS Method Acquisition Data Type FC Centroid Profile Setpoints e Use multiple TurboDDS segments to change Trigger or TurboDDS parameters during the acquisition e Use either Standard or Enhanced Scan Mode TurboDDS Parameters Tab Enter parameters for the Survey Scan and MSn Conditions Survey Scan Parameters e Start and End Mass Range 50 2000 m z e Polarity Select either Positive or Negative for all scans e uScans Averaged Select from 1 to 9 Average more to generate sp
188. ted location You can also double click a folder or file in that box to open it To open the folder one level higher click the up arrow button on the toolbar File list Lists the folders and files in the selected location Double click a folder to see its contents You can also use the Look In box to see the hierarchy of folders To open the folder one level higher click the up arrow button on the toolbar Restricts the list of files to only those matching the selected type Click this to display a list of recently selected files When you select a file from this list its name opens in the File name box 166 Save Automation File as Dialog Box Name an automation file Access the Save Automation File As dialog box with the menu command File gt Save As Save Automation File As Save in gt SummaryB asic File name L ntithed Save as type RecalcLists rel Cancel Recent Files gt Save in Lists the available folders and files To see how the current folder fits in the hierarchy on your computer click the down arrow Click a folder to see its contents The box below shows the folders and files in the selected location You can also double click a folder or file in the box to open it To open the folder one level higher click the up arrow button on the toolbar To open a new folder in the current directory click the Create New Folder button File list Lists the folders and files in the selected location
189. the Inject Single Sample icon on the System Control menu bar Alternatively you can display the Inject Single Sample dialog by selecting it from the Inject menu a System Control Varian MS 1 Ready File Edit Inject Automation Recalculate Instrument Windows Help Inject Single Sample E Click Inject Single Sample to display the following dialog box The fields in the table are specific to the device configured in the instrument 101 Number of replicates Enter sample name Inject Single S ample Cal Injection Inj Wash Automix User Sample Name Sample Type level Ini Notes AutoLink Inj Mode Volume Vial Volume Routines Program Default Sample Analysis v 1 none none Partial Loopfill y 10 A 300 none En 4 Inject the Sample using the Method Vi al D osition C Warianw S methods 420_Dynamax mth Browse Defaults r Add a note Cancel Data Files RecalcList Sato or apena Reca name of the Data Files Specifying the Data File Name and Path Data File names can be up to 255 characters long Sample ID injection date module name and injection number can be embedded in the file name making the Data File name correlate with each sample injection When you click the Data Files the Data Files Generation dialog box opens This dialog box allows you to select the path and the filename specification for the data file Data File Generation El Specify the names for Data File
190. the active method If the desired channel is not visible make sure that the correct method is active and that it contains data handling sections for the desired channel Calculation Type Internal Std External Std Normalized No Calibration Indicates the calibration method specified for the detector channel in the active method To change the calibration method for a specific channel click the channel s row and then click Edit Calibration Setup If the Calculation Type is not Internal Std or Normalized the Edit Standard Peak s push button is disabled Unid Peak Factor O to 1 000 000 Set a calibration factor for unidentified peaks Multiplier 0 000001 to 1 000 000 Set a value for the multiplier Results for the sample are multiplied by this value Divisor 0 000001 to 1 000 000 Set a value for the divisor Results for the sample are divided by this value Standard Peak 1 40 character standard peak name from peak table Indicates the name of the first Internal Standard Peak The Amount Standard 1 is applied to the Internal Standard Peak having the same name To change the standard peak name for a specific channel first click the channel s row and then click Edit Standard Peak s Amount Standard 1 0 000001 to 1 000 000 Sets the amount of the first internal standard Use to calibrate results for Internal Standard and Normalized Percent calculations 172 tem Desoription S Standard Peak 2 8 40 charact
191. the ion trap is always in competition with ion ejection If the CID excitation amplitude selected is too large the precursor ion is ejected to the trap electrodes before it can collide with background helium atoms If the CID excitation amplitude selected is too small the energy of the precursor ion will not exceed the internal energy threshold required to break the chemical bonds and form product ions This is because energy is constantly being removed by low energy collision deactivation Therefore CID excitation amplitude and excitation time are used to optimize the CID process in conjunction with an appropriate choice of excitation method When a precursor ion is selected in the CID method editor default times and amplitudes are automatically filled in The exact optimum for a specific compound can be found by using the optimization plots page The translational kinetic energy of the precursor ion can be increased using one of two methods Each method requires application of a waveform to the ion trap The waveform amplitude is the CID excitation amplitude and the length of time the waveform is applied is the excitation time The first method involves non resonant excitation In this approach a low frequency supplemental dipole field is applied to the end caps of the trap resulting in an instantaneous change in the potential energy of the ion in the trapping field The dipole field is an electric field oriented along the axis of the trap
192. the sequence downloads and the corresponding SampleList is activated Begin at Selected Begins the open SampleList at a selected SampleList line SampleList This item is enabled when a SampleList is open Upon Line selecting this menu item a prompt for the line in the SampleList appears This is useful when recovering from a power failure that interrupted automation in progress Begin at Selected Begins the open Sequence at the selected Sequence line Sequence Line This is enabled when a Sequence is open After selecting this a prompt for the line in the Sequence appears It is useful when recovering from a power failure that interrupted automation in progress Suspend Suspends execution of automation after the current run Automation completes Resume Resumes execution of automation after it has been Automation suspended Stop Automation Stops the current run resets all modules simultaneously and suspends automation Reset Modules Stops the current run resets all modules simultaneously Automation proceeds to the next injection after all the modules go to the Ready state Enable Turns on or off printing of reports automatically generated Automated during any automation action This is useful when you need Printing to disable printing if the printer is out of paper for example without having to modify the Print Options portion of the Report Method 135 Recalculate Menu E System Control Varian LC MS 1
193. the software part number 0395407600 MS Workstation Software Reference Manual This manual provides a practical way to learn quickly how to perform basic tasks using the MS Workstation Software These operations include displaying and reviewing chromatographic and mass spectral data library searching and quantitative analysis part number 0391496300 MS Data Handling Users Guide This manual contains post acquisition Data Handling procedures and tasks This manual can help you develop and select the best type of Data Handling for your method part number 0395403800 MS Workstation Tutorial Manual This manual demonstrates the MS Workstation capabilities in tutorial formats While these exercises use data files from the Saturn 2000 ion trap they illustrate the principles of the Varian WS software and can be adapted to the Varian 500 MS part number 0391498800 Data Acquisition with LC Control This manual describes the operation of the MS Workstation with Varian HPLC modules It covers configuring different modules for data acquisition building a method and running samples part number 0391473200 Other Manuals Please refer to the manual s of your other instruments Valuable information on operation as well as cautions and notes are contained within these manuals The primary interface between you and your 500 MS is through the PC hosting your Varian MS Workstation Please refer to these manuals first when you need information
194. ther Centroid or Profile Data Type Centroid is the default acquisition data type as data handling quantitation library searching and spectral comparison can be done from this type of data Profile data can be used for TurboDDS data multiply charged species quantitation and for diagnostic purposes Profile files are also approximately 10 times larger 157 tem Description Setpoints Set the ionization source parameters such as temperature and pressure for the nebulizer and drying gases and voltages applied to the components of the ionization source These parameters are specific for each ionization configuration ESI or APCI Chromato Time program analysis conditions for the best segment result graphic Time Up to 250 time segments can be created for runs up to 1440 Segments minutes in length You can tailor variables such as acquired mass range insert MS MS segments for individual analytes and set up the instrument to acquire the best data for each analyte The time segments program the states of the 6 port valve which can introduce the sample in to the Atmospheric Pressure lonization API source Double click the required field to edit the Segment Description Start and End times The Scan Description information is automatically set based on the choice of Scan Type and lonization mode in the Method Segment field below Segment Describes the nature of the particular segment and usually Description pertains
195. thod The parameters that you can modify under the Method tab are illustrated in the following for Electrospray lonization The Instrument configuration ESI or APCI cannot be changed in System Control These selections must be made through Method Builder Manual Control Auto Tune Diagnostics Startup Shutdown Acquisition Control and Status Source ESI Edit Method Record Data Method ESI Upload MS Method End Time 10 00 minutes Summary of Status Vacuum System OK Damping Gas OK Temperatures OK Source OK lon Gauge Pressure 0 0 uTorr Fil 1 Valid Method Active Segment Checks and Adjustments Module Attributes Optimization Plots Instrument Configuration Acquisition Data Type TurboDDS Electrospray Centroid Profile 7 TurboDDS Method Setpoints Chromatographic Time Segments ae a Add Segment Description Scan Description E Insert i 0 00 1 00 ESI Auto Full Delet 1 00 1100 ESI Auto Full Delete ASS Save Data Needle OVolts 0 0 uAmps trap on off Drying Gas 0C OPSI Nebulizer 0 PSI Special Applications t amp Acquisition Data Type Centroid data is the most common acquisition type as data handling library searching quantitation and spectral comparison can be done from this type of data Profile data is typically used to determine the charge state of multiply charge ions such as proteins and peptides quantitation and for diagnostic purpo
196. tial Loopfill 10 Left v AT 4 A l 7 a Using Left Tray During This Add Lines Session Number Columns from fa through H Iv Number Sample Names from f Number Rows from h through 12 By Columns Add Insert Cancel C By Rows Click Hardware to specify the tray type for each position sample loop volume and indicate if the Syringe for Prep Mode is installed 460 LC AutoSampler Hardware Prep Mode 2500 uL Syringe Cancel 8443 Adapter Tray Comcel Left Tray Type ES High ells Right Tray Type 396 High Wells Mot Installed K Loop Wolume J6 Low wells 96 High Wells 304 Low Wells do Vials 12 Vials 111 Sample Name Enter the root name for all samples Sample Type Select Analysis Calibration Verification or Baseline from the menu Sample Type Analysis Analysis Calibration Vernticaton Baseline Cal Level This numerical field is used with Calibration and Verification Sample Types Inj Enter the number of injections in this numerical field Injection Notes Enter a note about the sample or the injections in this text field SampleList Cal Injection level Inj Nois AutoLink Inj v 6 I none none Partial L Notes Test method Tr Revert Cancel Injection Hotes WN Test method 24 AutoLink Add a Command or Other parameters for post run event action Notes AutoLink Inj Mode Inj Tray Vial Volume stmethod none
197. tic is grayed out unless a test is selected in the Diagnostic Tests tab dialog Control and Status State Idle Function Monitoring Status After you select a diagnostic test and click Start Diagnostic the test runs and the results are displayed in the Event Messages at the bottom of the screen The Start Diagnostic button becomes Stop Diagnostic Manual Control Auta Tune Temperatures Diagnostics Control and Status State Running stop Diagnostic Function System Yertication Test 46 Monitoring Tab Dialog Monitoring Diagnostic Tests Trap On Off lon Gauge i fon Source lon Gauge f Filament 1 C Filament 2 W Multiplier Dynaode i RF Control le RF C waveform The Trap On Off section is where you select the ion trap features to monitor If you select lon Source the lon Source section of the Monitor window becomes active If you select Multiplier Dynode the voltages of the electron multiplier and conversion dynode are displayed in the Trap field of the Monitor window If you select RF Control with RF selected you see the Entrance and Exit Trapping Field Dipole values If the Waveform radio button is selected with RF Control the Dipole and Quadrupole Supplemental Waveform values are displayed If the lon Gauge option is selected the lon Gauge pressure in utorr is displayed in the lon Gauge field of the Monitor window Diagnostic Tests Tab Dialog Monitoring Diagnostic Tests Select All Al
198. tion or Verification sample type In this case the two AutoLink programs are executed after each injection of the sample AutoLink Parameters Command Other parameters Browse Cancel 179 Command DOS command lines that open DDE applications Sets the command line used when AutoLink is invoked Other Parameters Provides information to the remote application Browse Opens the Select an AutoLink Program dialog box to select the name of an AutoLink program and enter it into the Command field SequenceList Window si Automation File Editor Untitled seg Action Method SampleRecalcList Inject e cvaranws vest low flowy mth co Wwarlarniws single smp Add Inject c vaiansssdatasesi highflow am mi c varianwes test sample_list smp Insert r Delete r Browse Shows the action chosen for each line in the Sequence Method Shows the Method chosen for each line in the Sequence Deletes the highlighted line in the Sequence Browse Opens the Select a Method File or Select a Sample RecalcList window to browse through directories and find a Method or Sample RecalcList file to add to the Sequence 180
199. tivate y Jore Number i Data Views O Position 2 Purge Changes A 100 Click the Acquisition tab and set the end time for data collection by clicking End Time and entering a value Click Start Acquisition and the 500 MS will begin to acquire data with the method specified Status And Control Run Time 0 00 minutes Start Acquisition End Time 10 00 minutes Edit Method i Ready No Fault Wait For Readyln Check that the valve is in the load position overfill the 5 uL loop of the switching valve by a factor of 2 or 3 Push the yellow button on the front of the instrument to inject the sample Allow at least 1 minute after a peak eludes before making another injection As the acquisition runs its course several seconds before the end of acquisition the 500 MS will ask if additional time is required for the current acquisition Follow the instructions in the dialog to either add more time or end the acquisition Do you wish to terminate acquisition at the specified End Time Chek Yes to terminate as specified Click No to continue acquinng data for another End Time block Yes No Cancel Injecting Single Sample This example assumes you have an autosampler such as the Varian 300 400 series or HTS PAL configured with your 500 MS LC MS Refer to the appropriate section of this manual or the hardware manual to properly configure and plumb the autosampler To perform one injection click
200. to Tune or Acquisition modes The Spectrum Toolbar is slightly different for Manual Control Click Arrow to hide the toolbar Click the upper left of the display to restore the toolbar Click Full Scale to normalize the spectrum vertically and horizontally Double click the lower left corner to do the same aj elo af te m Click Previous Scale to return the display to the prior scaling Click twice to step through the previous displays Click Printer to preview the spectrum report before printing Click Scale to switch between Auto Scale and Fixed Mode Intensity Click Mass Range to select Acquired Fixed or Maximum Mass Range Click Display Type to select between graphical and text displays of mass spectra or display additional information Click Average Spectra to average every 10 spectra and apply this average to the real time chromatogram display This is not used when recording data Not available in Acquisition Mode 25 Manual Control Mode The Manual Control Window is divided into two panes the 500 MS Keypad above and the Data Views Module Attributes display below If the screen resolution is not set properly not all of the features may be displayed Configuring the Display Slide the Scroll Bar on the right of the Keypad to view the lower section of the Keypad or slide the Splitter Bar to expand the viewing area of the Keypad Click and drag a window pane to expand and move it Scroll Bar Scroll B
201. ts all lines in a SequenceList SampleList or RecalcList Fill Down Copies the contents of the top cell in a series of added or existing highlighted cells to the cells below it Use to edit all the cells in a column quickly Edit Notes Opens the Edit Notes dialog box to permit editing of the notes associated with the currently open automation file Help Menu A aooo Help Topics Topics Displays help selected topic Product If you have Internet access and a web browser installed on your Support computer this automatically opens the Varian MS Workstation Web Site Product Support Web Site This has the latest software and documentation updates for the Varian MS Workstation suite of products along with additional notes tips and answers to frequently asked questions About Displays the About Box for the Automation File Editor application Automation The About Box contains information about the software version File Editor installation information and a list of the instrument control modules that you have installed You can validate the installed workstation files from this dialog Main Toolbar als Bs S paja a New Automation File Select RecalcList SampleList or SequenceList Select one to display the Create a New Automation File dialog box for naming a new file of the selected type 3 153 Open Automation File Select RecalcList SampleList or SequenceList Selecting one displays the Open an Automat
202. ue Customize Displays parameters to customize user settings for set points ionization parameters or scan function parameters Isolation Applied while the trap fills and ejects unwanted ions Waveform RF Loading Low Offset Low Edge Offset High Offset High Edge Offset High Mass Ejection Factor Excitation Time msec Modulate RF This is the m z value at which the RF is maintained during ionization and the coarse isolation step An optimum value is predetermined for each precursor ion Customize this value by setting a percent of the predetermined value Range 0 300 The Low Edge Offset is the mass offset to optimize the ejection of the mass just below the precursor ion mass The default value is O Increasing the mass offset increases the isolation window on the low mass side of the precursor ion Decreasing the offset decreases the window on the low mass side Adjust the offset to minimize the amplitude of the adjacent masses below the precursor ion Initially adjust in 0 2 m z increments Range 0 7 to 0 5 m z Click Customize to view The High Edge Offset is the mass offset to optimize the ejection of the mass just above the precursor ion mass The default value is 0 Increasing the mass offset increases the isolation window on the high mass side of the precursor ion Decreasing the offset decreases the window on the high mass side Adjust the offset to minimize the amplitude of the adjace
203. une a e Waveform Tuning and Calibration Supplemental waveform Tuning AF Full Scale Adjust Mass Calibration Trap Frequency Calibration I lon Optics Tuning lon Lens Tuning IM Turn off infusion pump and source 4415 a a a The three major sections of the Auto Tune window are Control and Status display Spectrum and Event Message windows and Auto Tune Method display Control and Status Click Start Auto Tune to start the routines Click Stop Auto Tune to stop them The Action field shows the tuning routine in progress The State field displays if the 500 MS is currently executing an auto tune routine or is idle 150 Control and Status Acton Mass Calibration State Calibrating Spectrum and Event Message Window Display the AutoTune tests Hide Keypad causes the upper portion of the screen to disappear and the lower portion to expand Trap Hide Reypad lan Bauge Module Attributes The current Module Attributes has information stored from the last auto tune When you are finished performing the auto tune view this message which is stored in an auto tune log M3 Workstation Demo V6 9 1 03 31 08 Version 6 9 1 Module 500 MS 1 Configuration System Type LE Ionization Source Electrospray Auto Tune Methods Three auto tune checks automatically set up tune and calibrate the mass spectrometer The last routine is Turn off Infusion Pump and Source This option is useful fo
204. ure Fluid level in syringe pump In the Control and Status section the Action field displays the tuning routine in progress and the State field displays whether the 500 MS is currently executing an auto tune routine or is idle Control and Status Actor Integrator ero State Adjusting Stop 4uto Tune Spectrum and Event Message Window The following shows the left side of the auto tune screen You select information to display during the auto tune tests Hide Keypad causes the upper portion of the screen to disappear and the lower portion to expand Hide Keypad Spectrum Spectra Spectrum and Event Messages Event Messages Module Attributes Summar Report The Module Attributes displays the information stored from the last auto tune When you are finished performing the auto tune you can view this information which is stored in an auto tune log Hide Keypad Module Attributes ms MS Workstation Demo 6 9 1 03 51 08 Version 6 9 1 Module 500 MS 1 Configuration System Type LE Ionization Source Electrospray Option Keys None 44 Auto Tune Methods The auto tune routines automatically set up tune and calibrate the mass spectrometer In addition you can select to turn off the infusion pump and source after tuning completes Auto Tune routines are generally performed in the order listed beginning with Detector Setup and ending with lon Optics Tuning All of the auto tune method
205. urrently selected row s Default inserts system default parameters to the current segment Restore restores current segment parameters that were present when the method was first opened or last saved You can enter a user selected segment description of up to 60 characters into the Segment Description field Click the fields for the Start and End times and enter the desired values in minutes The Scan Description field is entered automatically based on the selection of Scan Type and lonization mode in the Method Segment fields Chromatogram Time Gaps in Multi Segment Methods Depending upon the instrument configuration you can acquire data using Scan Type ion preparation techniques After multiple segments are acquired MS Data Review displays each segment independently in the Plots View chromatogram display There is no connection between the last point in one segment and the first point of the next segment There is a small time gap between segments This gap is less than one second 69 Programming Individual Time Segments Selections across the top determine the available parameters Method Segment W Data Collection On Scan Type Ful Scan Mode Standard ka Load E Defaults General Parameters lonization Control Electrospray lonization Parameters Full Scan Parameters Set Points f Scan Time Maximum Scan Time Multiplier Offset 0 volts uScans Averaged 3 uScans Data Bate 0 23 Hz Data Collect
206. used to specify a wash volume in the range of 300 to 9999 uL or specify 0 for no wash The Automix Routines column allows you to define a specific automixing program to be done before injecting the sample When performing an Automix routine you can program the AutoSampler to aspirate a specified amount of liquid from a 108 Well Vial or a Reagent Vial and then transfer that liquid to the sample Well or Vial from which an injection is to be made You can mix the sample by aspirating and dispensing specified amounts of the mixture and you can program the AutoSampler to wait for a reaction to take place In the Automix Steps dialog you can select Action Position Amount Speed of the syringe and Height of the needle The following is an example of an automix routine Automix Steps Position Amount Speed Height Comment 3 normal 5 mm 3 normal 5 mm 3 normal 5 mm 3 normal v Emm Destination Vial none Reagent Vial none Reagent C Vial none Reagent B Vial none Reagent D Vial none V Automix First Injection Only Cancel AutoMi 1 y Up to 240 steps can be programmed in one automix routine After entering all the steps required for handling the sample enter a name for this Automix Program in the box next to Cancel and click OK The program is saved in the SampleList and can be exported to other SampleLists Alternatively Automix Programs from other SampleLists can be imported to the acti
207. usly plots the RIC from the real time spectrum plot screen The Optimization Plot screen shows special optimization routines for specified method parameters These plots are generated from the Optimization Plots tab and are discussed in the Optimization Plots Tab section They can be undocked moved and resized p i E h Trap Smeal ea hPa a r biiain Pug Ar ln y ca Fu E ton Gs Poon 1 5 g m Pinta a taa reta as pa I a oe aj 10 8 IL m ail Bara Peak 5 96 Basa Amount Se SMOOTHED nn 31 ue Gaomank 1 E al Bil 2 4S0e48 lan lon 37 ur a E1 fi An SE RI IH p P A i RANNA SN Yass 25 80 FS iii siii FS 110 Morano w Acgured Ranga an Splitter Bar 31 a S00 MS 56 Mol Ready odie Ambae Dplimizabior Plot FIL Fede hod mares wih leg eel teres Mace Flange m aft fe Trap Sebel Wir G Post Wahre intaga Purg Actes Method Depreni Gean Funcbon Chanai pare pn biua o 5 naaa ga a E Munbec 7 Channel af H Chaba Visai i Position 2 Purge l Fl Scan F Al ele a 1 A ul Stan Number 46 Time 1 344 min Bate Peak 509 Base Amount 015671 RIC 22787048 lon Range 25 5 200005 mz E o lor 364 we Segmoni 100 2a 3 y z E ee 21138048 re Bat a 13223 1 4750 6 1725 ofr S00 1000 1500 Ag quand To restore the plot windows to their default location e Double click the title bar of the window e Drag the window to the
208. utom To add several similar lines to the SampleList click Add Lines You can then enter the common information in the dialog box Add Lines to ProStar 410 SampleList Cal Injection Inj Automix Amount Std Unid F Sample Name Sample Type level Inj Notes AutoLink Inj Mode Volume Vial Washes Funds Prep Steps IS NZ only Fac Default Sample Analysis y 1 none none Partialloopfil v 10 1 1 noe 1 0 gt Number of Lines to Add 10 1 Number Sample Nalhes from 1 Y Number Vials from 1 For sequentially numbered Sample Names enter the starting number and the number of entries to add to the SampleList The Sample Names will have these numbers appended to them When you click Begin on the SampleList you are prompted for the Method to use You can use the active Method that is pre selected or you can Browse for another Method After you click OK the run will begin 107 Begin Sample List Browse Data Files will be sawed in cM Warlaniws data Cancel ProStar 420 and 430 SampleList The PS 420 and PS 430 SampleLists are similar to the generic System Control SampleList with additional columns to support operations specific to each AutoSampler Use the Injection Mode column to select one of the three methods of injection Partial Loopfill Full Loop and uL Pickup The User Program can also be selected from this column Several columns of the Sampl
209. ve SampleList using Import The User Program column is enabled if you select the User Program in the Injection Mode column for this sample The User Program programs all possible actions required for a sample handling and injection that can be executed by the AutoSampler in separate steps When the User Program is selected as the Injection Mode the following SampleList columns are disabled Injection Volume Wash Volume and Automix Routines The User Program steps are used instead of the parameters in the Method If you use the User Program the 10 mL syringe must be selected in the Hardware Configuration The following figure shows a list of the User Program Actions that can be specified For each specified action there could be several available options such as Position Amounts Speed Setting and Height User Program Steps Speed Setting 1 Aspirate From Reagent4 10ul 3 normal 5 mm 3 normal 5 mm Action Position Amount Height Comment 8 Compressor w ES 9 SetOutput w INJECT Destination Vial none Reagent A Vial C30 Reagent C Vial none Reagent B Vial none Reagent D Vial none Start Run on INJECT Marker C Start Run at End of User Program Cancel JUserProg_1 v 109 You can select when to Start the Run either on INJECT Marker step 9 in the above example or at the End of User Program After entering all the steps required for handling the sample enter a name for this
210. verses the Directory outline and the Window toolbar positions Method windows Hold the mouse cursor over each button to see a Tooltip describing its function A status bar is at the bottom of the Method Builder window The toolbars and the status bar may be hidden or shown using the View menu Use the Method Builder to display and edit multiple Method files If the Method Builder is already displaying one method you can open another using the File Open command The Methods can be selected cascaded or tiled to compare the same sections in different Method files from the window menu on the window toolbar Method Parameters The Parameters pane displays the content of the method section selected in the Directory Four types of windows can be displayed in the Parameters pane method file information method notes hardware module descriptions and editable parameters for the instrument modules data handling and reports File Information 3 Method Notes Location C VananWS 500 MS Methods 500 M5 test mth BY ProStar 430 AutoSampler Control Address 1 Createct Wednesday October 21 2009 16 17 38 Y Configuration x a E 212 LC Solvent Delivery System Control Addre Modited Wednesday October 21 2009 16 18 35 B Pump Program Size 90086 bytes Y S00 MS Mass Spec Address 3 Method Fla Athibutes ET 500 M5 Mass Spec Control dd EJ MS Acquisition Method C Readony LJ Hidden i sta 0O Channel 1 M5 Data z i E Standard
211. w a Chromatographic Time Segments 1 MSMS reserpine SS 10 00 ES Auto MS MS Acquisition Data Type Centroid Profile Special Applications 99 To set ESI Parameters 1 Select a value between 10 and 15 psi for the Drying Gas Pressure a Check that Use Ramp Rate is not selected b Enter the End Temperature for the drying gas c Click OK to save the changes Electrospray lonization Setpoints Spray Chamber Temperature si C Needle Voltage Positive 4500 wolts Nebulizer Gas Pressure 35 0 psi Needle Voltage Negative 45004 walts Drying Gas Pressure 10 0 psi Spray Shield oltage Positive 400 0 volts Spray Shield Voltage Negative Eli Drying Gas Temperature FP Use Ramp Rate Initial Hold Time 0 203 min Start Temperature Jall Ramp End Time 0 20 min End Temperature 350 Ramp Rate 0 00 C min E 10 00 min e Spray Shield Voltage Overiden Segments Click Special Applications in the Method tab and click Diverter Special Applications 6 Prort Valve Syringe Pump f Manual TIR Flow Rate 200 00 microliters min W Tum Off at Method End Diverter r Relays m er m 3 Injector M Use Relays Diwert to Waste Load To API Inject Defaults Restore Cancel Save the method and click Activate Changes Hide Keypad W Trap Select Views 6 Port Valve Infusion Pugip Method Active Method Segment lon Gauge Ta f Det o Postion g Stork Ac
212. xecution of automation after it was suspended 116 Reset Modules Stops the current run and resets all modules simultaneously Automation proceeds to the next injection after all the modules go to the Ready state Select from the following in the Automation menu in System Control Automation Recalculate Instrument Y Begin SarmpleList Begi Ber Begi n Sequence in l gin t Selected SampleList Line In At Selecte d Sequence Line Suspend Automation Resume Automation Stop Automation Reset Modules Enable Automated Printing Setting a Collect Divert Delay Use the diverter valve to divert the injection front away from the API source Frequently the solvent front contains un retained non volatile salts from the sample These salts deposited in the source build up over time and the source must be cleaned regularly Diverting the solvent front reduces maintenance on the Source and API Interface The delay parameter is used to switch the diverter valve to waste In the Method Segment section enable Syringe Pump On Method Segment IY Detector On Scan Type Ful Scan Mode Standard Load Click Edit Method and add a segment to the 500 MS Method Segment section Give the segment an appropriate name in this case divert and set the time for the end of the segment 1 min in this example Note you can also have a divert segment at the
213. xt Select Configuration Select an existing instrument configuration for the method or select a custom configuration for the method Then click Next to continue Select a Configuration r Configuration Description Custom Address Instrument 1 25 Y 500 MS lt Back Cancel Select the channels for post run processing Create sections for post run processing For the following module 500 MS at address 3 Select the channels ta Select the Post Alun pIocezs processes to peshornn W Standard MS Reports WMS Data Handling A reene rreme errena eem a eee e AE 59 After you select the data channels and type s of post run processing for each detector the wizard creates a method The method sections contain default values for all parameters which you can edit Data handling and reports are discussed in the MS Workstation Software Reference Manual Confirm configuration Added configuration To be added to method ProStar Dynamax Address 24 ProStar Dynamax Control 410 Control Address 25 Click Finish to add the module control 500 MS Address 56 and or post run processing to the method 500 MS Control Channel 1 MS Data You may click Back to go back and Standard MS Reports change any configuration information MS Data Handling lt Back Cancel Method Builder Window Overview E Method Builder 500 MS test mth T File Edit View Window H

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