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Creating a Method

Creating a MS Instrument Method

TSQ 8000 Evo User Guide

Thermo Scientific

Figure 59.

Acquisition-Timed Method Settings

a. In the

Scans

pane above list of scans, select

SIM

SRM

from the

Scan Type

list. If

you want to link to an external method, click

Link to External File

. You may link to

a .csv or .xml method file.

b. After clicking

Link to External File

, the

SIMBridge

dialog box opens. Choose the

language of your method file from the

Source Locale

drop-down menu. See

Figure 60

Note

In order to edit the scans within the TSQ 8000 Evo method editor, clear

the

Link to External File

Summary of Contents for TSQ 8000

Page 1: ...TSQ 8000 Evo Mass Spectrometer User Guide 1R120586 0002 Revision C December 2015 ...

Page 2: ...duct purchase to use in the product operation This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited except with the written authorization of Thermo Fisher Scientific Inc The contents of this document are subject to change without notice All technical information in this document is for reference purposes only System configuration...

Page 3: ...ous Substances Precautions xx Biological Hazard Warning Note xx Venting Toxic Gases xxi Contacting Us xxi Chapter 1 Introduction 1 Confirming Your Instrument is Working 1 Checking Power to the System 1 Verifying the Carrier Gas Flow Rate 2 Checking Your Carrier Gas Tank Pressure 4 Verify Collision Gas Tank Pressure 4 Checking the Vacuum and Temperature 4 Cleaning the Exterior of Your Instrument 6 ...

Page 4: ...y to Make an Injection 90 Improving the Way You Prepare Samples 90 Changing the Dwell Time or Scan Rate 90 Narrowing the Mass Range 91 Adjusting the Transfer Line and Ion Source Temperature 91 Optimizing an SRM Method 92 Optimizing an NCI Tune 92 Modifying an Automatic Tune 96 Chapter 6 Computer Settings 111 System Requirements 111 Computer Settings 111 Excluding the Xcalibur Directory from Virus ...

Page 5: ...MC and safety standards as described in the next section or sections by product name Changes that you make to your system may void compliance with one or more of these EMC and safety standards Changes to your system include replacing a part or adding components options or peripherals not specifically authorized and qualified by Thermo Fisher Scientific To ensure continued compliance with EMC and s...

Page 6: ... specified by Thermo Fisher Scientific could impair any protection provided by the instrument Notice on the Susceptibility to Electromagnetic Transmissions Your instrument is designed to work in a controlled electromagnetic environment Do not use radio frequency transmitters such as mobile phones in close proximity to the instrument THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES OPERATION IS S...

Page 7: ...ntific s compliance with these Directives and the recyclers in your country WEEE Konformität Dieses Produkt muss die EU Waste Electrical Electronic Equipment WEEE Richtlinie 2002 96 EC erfüllen Das Produkt ist durch folgendes Symbol gekennzeichnet Thermo Fisher Scientific hat Vereinbarungen mit Verwertungs Entsorgungsfirmen in allen EU Mitgliedsstaaten getroffen damit dieses Produkt durch diese Fi...

Page 8: ... compagnies de recyclage dans chaque état membre de l union européenne et ce produit devrait être collecté ou recyclé par celles ci Davantage d informations sur la conformité de Thermo Fisher Scientific à ces directives les recycleurs dans votre pays et les informations sur les produits Thermo Fisher Scientific qui peuvent aider la détection des substances sujettes à la directive RoHS sont disponi...

Page 9: ...atly reduced The extractable source incorporates a cartridge that contains the repeller source lenses and RF lens all of which can be removed from the instrument without breaking vacuum What once took hours or even an entire day is now accomplished in minutes In addition the TSQ 8000 Evo system includes innovative Thermo Scientific software that will help users new to triple quadrupole GC MS MS sy...

Page 10: ...1R120586 0002 TSQ 8000 Evo Hardware Manual PN 1R120586 0003 TSQ 8000 Evo Spare Parts Guide PN 1R120586 0004 TSQ 8000 Evo Auto SRM User Guide PN 1R120586 0005 Q Exactive GC ISQ Series and TSQ 8000 Evo Direct Probe System User Guide PN 1R120505 0006 To view product manuals Open the desktop folder Manuals To open Help From the TSQ Series window choose Help TSQ Series Help If available for a specific ...

Page 11: ... cause personal injury A CAUTION is intended to prevent improper actions that might cause personal injury or instrument damage You can find the following safety symbols on your instrument or in this guide System Requirements Hardware 4 6 GHz processor with 16 GB RAM CD R Rom or DVD drive 1000 GB or hard drive Video card and monitor capable of 1680 1050 resolution Quad core processor Software Micro...

Page 12: ...n contact with an ignition source This symbol indicates this risk could or might cause physical injury GLOVES REQUIRED Indicates that you must wear gloves when performing a task or physical injury could or might occur HAND AND CHEMICAL HAZARD Indicates that chemical damage or physical injury could or might occur INSTRUMENT DAMAGE Indicates that damage to the instrument or component might occur Thi...

Page 13: ...centration can accumulate You should also consider purging the hydrogen to further reduce hazards and ensure anyone who will be working with hydrogen has basic hydrogen safety training READ MANUAL Alerts you to carefully read your instrument s documentation to ensure your safety and the instrument s operational ability Failing to carefully read the documentation could or might put you at risk for ...

Page 14: ...be labeled as such at the supply or discharge point and at regular intervals along its length Where hydrogen gas piping passes through a wall the piping should be labeled on both sides of the wall There should also be contingency plans in place should an incident occur The site emergency response team as well as the local fire department should know the location of all hydrogen storage tanks Using...

Page 15: ...tion of temperature and pressure expected during service Piping and supports must be able to withstand static loading introduced by such things as ice and snow and dynamic loading from high wind and earthquake Caution should be used if burying hydrogen piping Proper controls should be used to protect against damage and corrosion and also to prevent Hydrogen from entering a building if there is any...

Page 16: ...piping system should have uninterruptible pressure relief The pressure relief system should be designed to provide a discharge rate sufficient to avoid further pressure increase and should vent to a safe location outside or to a ventilation system exhaust Purchasing Hydrogen Use the following guidelines when purchasing hydrogen Hydrogen Generator Because it minimizes the amount of hydrogen present...

Page 17: ...is precaution applies when the hydrogen is or is not in use Indoor storage of spare hydrogen cylinders has special requirements which is beyond the scope of this document Documentation for each vessel should include a description of the vessel a list of available drawings or other documents the most recent inspection results and the responsible person s name Prevent spare cylinders from toppling b...

Page 18: ...ectric power lines close to other flammable gases liquids or close to public areas It should be readily accessible to authorized personnel and delivery equipment but protected from physical damage or tampering As liquid hydrogen systems also have a cryogenic hazard additional safety considerations for the use of cryogenic liquids might be necessary ...

Page 19: ...A Code Of Practice CP33 The Bulk Storage of Gaseous Hydrogen at Users Premises CGA G 5 Hydrogen CGA G 5 4 Standard for Hydrogen Piping Systems at Consumer Locations CGA G 5 5 Hydrogen Vent Systems CGA G 5 6 Hydrogen Pipeline Systems CGA G 5 8 High Pressure Hydrogen Piping Systems at Consumer Locations FM Global Property Loss Prevention Data Sheets 7 50 Compressed Gases in Cylinders FM Global Prope...

Page 20: ...urface Burning Characteristics of Building Materials OSHA 29CFR1910 103 1910 103 Hydrogen Hazardous Substances Precautions Biological Hazard Warning Note In laboratories where samples with potential biological hazards are handled the user must label any equipment or parts which might become contaminated with biohazardous material The appropriate warning labels are included with the shipment of the...

Page 21: ...haust gases to a fume hood Consult local environmental and safety regulations for instructions in exhausting fumes from your system Contacting Us There are several ways to contact Thermo Fisher Scientific for the information you need To find out more about our products Go to www thermoscientific com en products mass spectrometry html for information about our products To get local contact informat...

Page 22: ......

Page 23: ...perature Checking Power to the System To confirm that the TSQ 8000 Evo system is powered on check that the power light on the front panel is lit and solid green See Figure 1 If it is not the instrument is not powered on IMPORTANT You will likely want to change the GC column before setting up a method See Chapter 2 Changing the Column for instructions on changing the column Contents Confirming Your...

Page 24: ...f the instrument and pull up on the large plastic ribbed power switch on the back To confirm that a TRACE 1300 is powered on Confirm that the power light on the status panel is solid green To confirm that a TRACE 1310 is powered on Confirm that the touchscreen main menu has appeared Verifying the Carrier Gas Flow Rate Once you confirm that the system is powered on verify that the carrier gas rate ...

Page 25: ...arrier gas flow rate on a TRACE 1300 1 Open the TraceFinder software by clicking the TraceFinder icon on the computer desktop 2 Click Real Time Status in the top right hand menu to open the Real Time Status pane 3 On the Real Time Status pane roadmap select the TRACE 1300 from the instrument list at the top of the pane 4 The TRACE 1300 status pane opens Figure 2 Status Pane in TraceFinder Software...

Page 26: ...hat the tank is connected to the mass spectrometer Checking the Vacuum and Temperature Use the lights on the front of your TSQ 8000 Evo system to check the vacuum and temperature of the instrument To check the vacuum look at the Vacuum light See Figure 3 When the light is a solid green the mass spectrometer is under sufficient vacuum If the Vacuum light is slowly blinking orange you have not yet a...

Page 27: ...nking orange the ion source ion optics and or transfer line are not at temperature If the Heaters light is not lit the heaters are not turned on You may also use the TSQ Series Dashboard to check the vacuum of your system If your system has achieved sufficient vacuum a green check mark appears next to Vacuum on the dashboard Note Until the Vacuum light is a solid green high vacuum is achieved the ...

Page 28: ...int free cloth Configuring Your Instrument Initially the field service engineer will configure your TSQ 8000 Evo instrument However if you have reinstalled Xcalibur or you have a new computer or device follow these instructions to reconfigure it To configure your TSQ 8000 Evo instrument 1 Close the Dashboard and any open Xcalibur windows 2 From the Start menu on your computer desktop choose Start ...

Page 29: ...figuration window opens you will see icons of all connected instruments 4 Select the TSQ Series icon in the Available Devices column and click Add to move it into the Configured Devices column Figure 5 Finding Available Devices 5 Select the icon for each instrument you want to configure See Figure 6 and click Add ...

Page 30: ...8 TSQ 8000 Evo User Guide Thermo Scientific Figure 6 Configuring a Device 6 Select the TSQ Series from the list of configured devices 7 Click Configure to open the TSQ Series Configuration dialog box 8 On the General page set the pressure units ...

Page 31: ...tarted a run When you configure the GC you can tell it what to send out to the instrument Make sure the value in this dialog box matches what you set on the GC The default is Active Low See Figure 8 Note You only need to set up the pressure units if you have an ion gauge or convectron gauge installed on your system The readbacks from these components will display in the units set in this dialog bo...

Page 32: ...ways Show Method Portability Between Instrument Methods if you need to create methods for older model TSQ 8000 or TSQ Duo systems Figure 9 Enabling Method Portability 11 Click Communications to reset the network IP address and assign a TCP port See Figure 10 This tells the instrument method how to find the TSQ 8000 Evo system if you ...

Page 33: ...ing the Maintenance Intervals 13 Select the Foreline Pump Oil check box to enable the maintenance intervals Then set the number of days after which you want to be reminded to check the oil The manufacturer recommends changing the oil every 125 days Select the Turbo Pump Oil Bearing check box to enable the maintenance reminder Then set the number of days after which you want to be reminded to check...

Page 34: ... want to be reminded to check the ion source The time between cleaning depends very strongly on your analysis You will have to determine the correct length of time between source cleanings 17 Select the Multiplier check box to enable the maintenance reminder Then set the number of days after which you want to be reminded to check the electron multiplier 18 Click OK to return to the main Instrument...

Page 35: ...tart In High to Low Inhibit Ready In When High End of Run Out High to Low Start of Run Out High to Low GC Ready Out When Low Prep Run Out When Low Note For the remainder of the GC configuration settings refer to the TRACE 1300 and TRACE 1310 Series GC User Guide Refer to your autosampler user documentation for the correct autosampler configuration settings Note If you are running samples with a Di...

Page 36: ... 14 TSQ 8000 Evo User Guide Thermo Scientific 22 Click Done to close the Instrument Configuration window 23 Check the status of the configured instruments in the Status Pane of the TSQ Series Dashboard Figure 14 TSQ Series Dashboard Status Pane ...

Page 37: ...of stationary phases and are available in many sizes that can be used with a mass spectrometer Large diameter columns made of steel are widely used in process gas analysis but they are not typically used on mass spectrometers There are also metal clad fused silica columns which have the advantages of fused silica but the metal makes them resistant to breakage These columns are less common and more...

Page 38: ...which can result in longer analysis time and peak tailing Large ID columns with thin films have very little interaction with the analytes which will result in very fast analysis times with little separation Typical film thicknesses are 0 25 μm for a column with an ID of 0 25 mm Other common film thicknesses are 0 1 0 5 and 1 0 μm Be careful when selecting columns for mass spectrometers Some column...

Page 39: ...ument 5 Look behind the right side of the vacuum interlock shield and twist the vent valve knob one and a half times in a counterclockwise direction to open the vent 6 Remove the current column a Make sure the heated zones of the GC are cooled down Refer to the GC documentation for instructions b Turn off the carrier gas and if used the detector gas See the GC documentation for information about u...

Page 40: ...e your fingers to tighten the retaining nut until it starts to grip the column h Tighten the column nut finger tight until it starts to grip the column plus a quarter turn i Remove the notched septum from the column 8 Set up the GC parameters a Set the oven and injector temperature to 50 C 122 F b Set the carrier gas flow to 1 0 mL min c Turn off vacuum compensation which is located on the Carrier...

Page 41: ...pass use the leak detector to find and fix any leaks c Repeat the leak check until no leaks are indicated 10 Calibrate the carrier gas flow column evaluation a Carefully push the capillary column end into the flowmeter section of the column flowmeter connector Figure 16 Column Flowmeter Connector b Connect the flowmeter to the dedicated fitting on the column flowmeter connector c If you have a TRA...

Page 42: ...mn evaluation until an appropriate K factor is achieved e a leak in the system while a K factor that is too high might indicate a blockage 11 Disconnect the column flowmeter a Disconnect the column from the column flowmeter connector b Remove the clear plastic component including its fittings from the oven and set them aside c Close the GC door 12 Condition the column before inserting it into the ...

Page 43: ...rdware manual for instructions 3 Unwind about one turn of the column from the column outlet end 4 Wipe approximately 300 mm 12 in of the column with a tissue soaked in methanol 5 Choose an appropriate ferrule for the outer diameter of your column 6 Insert the column through the spring loaded transfer line nut and ferrule entering through the tapered end of the ferrule 7 Wipe the column again with ...

Page 44: ... and break the column Use a magnifying glass to check for an even flat cut Repeat if necessary 10 Use a 5 16 in wrench to hold the column measuring tool steady Figure 18 Column Measuring Tool 11 While holding the column measuring tool steady tighten the spring loaded transfer line nut with a 1 4 wrench until the column just stops moving in the ferrule 12 Turn the spring loaded transfer line nut 1 ...

Page 45: ...line nut The septum marks the place on the column where it should exit the nut Figure 20 Positioning the Septum 15 Pull the column back from the spring loaded transfer line nut Do not move the septum from its position on the column Figure 21 Pulling the Column Back from the Spring Loaded Transfer Line Nut Column Outlet Spring Loaded Transfer Line Nut Septum Column Measuring Tool Column Column Spri...

Page 46: ...transfer line Figure 22 Inserting the Column into the Transfer Line 19 Tighten the spring loaded transfer line nut until it is just secure enough so that you cannot move it 20 Loosen the nut by turning it exactly 1 flat backward 21 Position the column in the transfer line using the cut septum to measure the correct length you should insert the column Note The ferrule should still be able to move o...

Page 47: ...o 40 C 104 F and allow it to cool before continuing 26 Close the front door of the GC 27 Restore working conditions a Raise the oven temperature to the initial temperature that you will use b Turn on vacuum compensation on the GC 28 Power on the TSQ 8000 Evo instrument See Chapter 1 Introduction for instructions 29 Once the instrument is pumped down and able to scan view air water spectra and look...

Page 48: ...gain with a tissue soaked in methanol Figure 24 Transfer Line Nut and SilTite Ferrule Orientation Note For best results we recommend you use the spring loaded transfer line nut See To connect the column using the spring loaded transfer line nut on page 21 CAUTION BURN HAZARD The injector detectors oven and transfer line may be hot Allow them to cool before touching them Note Wear clean lint and po...

Page 49: ...y 9 Use a 5 16 in wrench to hold the column measuring tool steady Figure 25 Column Measuring Tool 10 While holding the column measuring tool steady tighten the transfer line nut with a 1 4 wrench until the column just stops moving in the ferrule 11 Turn the transfer line nut 1 flat backward so the column is able to move in the ferrule with slight resistance 12 Line up the outlet of the column with...

Page 50: ...on on the column Figure 28 Pulling the Column Back from the Transfer Line Nut 15 Loosen the transfer line nut from the column measuring tool 16 Remove the column transfer line nut and ferrule from the column measuring tool making sure not to move the septum from its location on the column 17 Insert the column into the transfer line Transfer Line Nut Septum Column Measuring Tool Transfer Line Nut S...

Page 51: ...20 Position the column in the transfer line Use the septum as a guide to measure the correct length you should insert the column Be careful not to change the location of the septum on the column Figure 30 Positioning the Column in the Transfer Line 21 Tighten the nut 1 flat forward back to where it is secure enough in the transfer line that you cannot move it 22 Tighten the nut 1 additional quarte...

Page 52: ... on the TSQ 8000 Evo instrument See Chapter 1 Introduction for instructions d Once the TSQ 8000 Evo instrument is pumped own and able to scan view air water spectra and look for evidence of leaks with a large m z 28 signal If you observe a leak stop scanning and gently tighten the nut in small increments until no leaks appear when scanning Note If you are using a SilTite ferrule follow the instruc...

Page 53: ... the temperature setpoint If you exchanged the ion source without venting the source components take approximately 30 minutes to stabilize temperature You can tune the TSQ 8000 Evo system by using the TSQ Series Dashboard Accessing Auto Tune To access Auto Tune choose one of these two methods Double click the TSQ Series Dashboard shortcut on your desktop to open the TSQ Series Dashboard and then c...

Page 54: ...ser Guide Thermo Scientific Figure 31 TSQ Series Dashboard Tuning the Mass Spectrometer To tune your mass spectrometer 1 In the Tune Types field select EI CI or Both depending upon which ionization mode you are using 2 Select a tuning category See Figure 32 ...

Page 55: ...akes the longest amount of time to run but it has the advantage of reoptimizing nearly all the parameters affecting the signal This type of tune performs a mass calibration tunes the lenses and resolution and performs a leak check It adjusts the detector sensitivity to generate a m z 219 ion with an intensity of 20 000 000 counts Unless your SOP requires it this is not the best tune to use on a da...

Page 56: ... This allows the sample intensity to remain constant for longer periods without running a full retune of the instrument The daily tune check performs a leak check makes sure the mass calibration is correct and sets the detector sensitivity to generate a m z 219 ion with an intensity of 20 000 000 counts If your SOP allows it you can use this tune to rapidly verify that the previous lens tune is st...

Page 57: ...h a 1 0 mL min flow This tune does not set the detector gain The built in CI tune will start with the most recent AutoTune_NCI tune file so have an appropriate tune file saved on the instrument s PC k CI Tune Used to analyze samples with positive CI The standard PCI tune performs a mass calibration then tunes the lenses and sets the resolution This type of tune assumes you are using methane as the...

Page 58: ...the Start button to begin tuning Once the tune completes your tune report opens in the Tune Results Viewer If you did not select the Display Report When Complete check box you can click View Tune Report on the dashboard and view the report Note Make sure the power options on your computer are not set to go into Standby mode while you are acquiring data for your tune Otherwise it will interrupt you...

Page 59: ... want to display and change the name of your instrument 8 Select a mass for Calculate Relative Abundance Using The default is mass 69 which will reference all masses to mass 69 Some masses may have over 100 abundance If you do not wish to have a relative abundance above 100 select Base Peak This option will select the reference mass as the highest abundance mass in the tune 9 Click OK ...

Page 60: ...ning Tuning the Mass Spectrometer 38 TSQ 8000 Evo User Guide Thermo Scientific Figure 36 Tune Report Options 10 To print your tune report click Print Report to open a print dialog box and print your report ...

Page 61: ...report click the Save icon and save it as a Microsoft Excel file or an Adobe Acrobat PDF file You may find it useful to compare this tune with a previous tune report of the same tune type Some changes in peak height are normal but if the difference is significant see Troubleshooting 12 To browse to another tune report on your computer click Open Tune Results in the top of the window 13 Click Open ...

Page 62: ... samples you are ready to develop or run a method Updating Tunes for New RF Lens If you have purchased a new lens 3 RF lens PN 1R120574 0103 you must update the ion guide frequency in the Manual Tune utility and retune your instrument To update the ion guide frequency using the Manual Tune utility 1 Open the TSQ Series Dashboard 2 Click Manual Tune on the dashboard See Figure 39 ...

Page 63: ...Guide 41 Figure 39 Opening Manual Tune on the TSQ Series Dashboard 3 The Manual Tune utility opens 4 Select Frequency Tune on the top menu See Figure 40 Figure 40 Accessing Frequency Tune 5 From the Device drop down menu select Ion Guide See Figure 41 Manual Tune Frequency Tune ...

Page 64: ... has finished detecting the ion guide frequency go to Save To Instrument on the top menu A dialog box confirming that the tune settings have been saved to the instrument will open See Figure 42 Figure 42 Saving the New Ion Guide Frequency to the Instrument 8 To confirm that the intensities are still correct select Mass Resolution Tune on the top menu to go back to the Manual Tune home page Device ...

Page 65: ... See Figure 43 Figure 43 Selecting the Correct Number of Spectra to Scan 10 Select EI from the Cal Gas Level menu See Figure 44 Figure 44 Setting the Calibration Gas Level to EI 11 Click Start Scan Check the intensities for the masses 69 219 and 502 Set the masses using the Mass drop down menu below each spectrum See Figure 45 ...

Page 66: ...Scientific Figure 45 Checking the Mass Intensities 12 Once you have confirmed the intensities are sufficient for tuning retune the system Note The intensities might be lower than they were at the previous frequency set for the ion guide until the lenses are tuned in AutoTune ...

Page 67: ...or 1310 GC and your autosampler open the Method Editor Contents Accessing the Method Editor Creating an Autosampler Method Creating a MS Instrument Method Creating a GC Method Validating and Troubleshooting Methods Note For information about creating a method for other instruments and software refer to the appropriate documentation Note All of the configured instruments are shown in the left pane ...

Page 68: ...thod Editor window opens See Figure 46 Icons of all connected instruments appear in the left pane You may only configure one instrument at a time The bright green arrow on the lower right corner of the instrument icon indicates the active instrument The current instrument name also appears in the main menu Figure 46 Method Editor Window Instrument Name Icon Arrow ...

Page 69: ...ns GC Liquids GC Headspace or SPME 3 Enter the ID information for your samples You must fill in any field with an asterisk a Injectors Enter the injection ID into the corresponding injector port b Syringes Enter the syringe ID information c Wash Station Enter the wash station ID information d Optional Internal Standard Solvent Stations Enter ID information for your solvent station or interval stan...

Page 70: ...Enrichment Needle Solvent Option Internal Standard Double Internal Standard Post Needle Solvent Wash Solvent Flush Double or Solvent Flush Post c Rapid Mode Choose Disable After Sample Rinse After Bubble Elimination or After Sample Asp in Home d Syringe Type Enter your syringe volume in μL Enter the syringe needle length in mm e Sample Volume Enter the sample volume to be injected into the GC Typi...

Page 71: ... check box and enter the height from the bottom of the vial in mm at which the tip of the syringe needle will be placed in the sample vial when it is being filled j Injection Depth Select Standard or Minimum to indicate how the sample is introduced into the GC If you select Standard the autosampler inserts the needle all the way into the injection port If you select Minimum the autosampler barely ...

Page 72: ...elp ensure the sample being injected is not diluted by the residual rinse solvents By purging the syringe with your sample before injection the dilution is minimal The standard setting is between 1 and 3 rinses If you have a very limited amount of sample however you may want to set this field to 0 to conserve the sample e Post injection Use the Solvent and Cycles fields to set the number of solven...

Page 73: ... Method Setup pane to set the temperatures and acquisition threshold in General Acquisition mode See Figure 51 on page 52 Note The Maintenance method is used to bake out or cool down the instrument during a sequence This can be useful if you know you want to perform these tasks and you want to do them in an automated way as part of your sequence ...

Page 74: ...emperature depends on the analyte Higher temperatures will keep the ion source cleaner but will lead to increased fragmentation which may reduce sensitivity c Optional Select the Acquisition Threshold check box and enter a value for the minimum peak height for the data file if needed If your peak has an intensity that is below this threshold it will not be stored This setting may help reduce noise...

Page 75: ...ay only use one CI Gas Port per method Figure 52 Setting the CI Gas Ports Note Only use CI if you have installed a CI ion volume in the MS and connected CI reagent gas to your system If you have CI select a CI Gas Type from the pull down menu and set the CI Gas Flow Typical values for Methane CI for NCI are 1 0 1 5 mL min For Methane CI for PCI the gas flows are typically 1 5 2 0 mL min Click the ...

Page 76: ...the MS to run The end of the run can be between 0 and 1000 minutes With this option you can stop the acquisition when all the compounds of interest have eluted but the GC is still at an elevated temperature to keep the column clean Thermo Fisher Scientific recommends that you select this option to save burn time on the filament b In the Scans pane click a scan row to enter scan information under e...

Page 77: ...l scan finishes the SIM scans begin The SIM scans occur sequentially Two different SIM masses at 43 and 44 will be looked at for 0 05 and 0 08 seconds sequentially These simultaneous full scan and SIM scans will begin 2 5 minutes into the GC run You will get a complete set of scans every 0 342 seconds At 5 minutes into the GC run the scans change to a set of SRM scans The first SRM transition is 2...

Page 78: ...multiple SRM transitions in a single scan filter highlight the rows you want to combine and then right click and select Group SRM Scans Replace Selected Scans See Figure 55 You can also use this function to ungroup a group of SRM scans into individual rows Figure 55 Grouping SRM Scans iii Enter the product ion mass for SRM methods in the Product Mass column The product ion mass is the result of fr...

Page 79: ...f you have too many scans the MS signal becomes less precise This column must be set to a value between 0 0005 and 5 s for SRM and SIM methods The default is 0 2 s For full scan methods the minimum scan time depends on the mass range so that the scan rate does not exceed 13 000 amu s vii Use the Tune File Name to select a tune file to be used for this scan Select the autotune file you created in C...

Page 80: ...t setting is Normal which uses the tuned resolution values from the tune file specified in the Tune File Name column Selecting Wide or Widest increases the precursor peak width to 1 5 and 2 5 amu at 50 respectively Opening the precursor peak width will increase the ion signal into the collision cell and may improve the sensitivity of your method however it may also increase the background noise le...

Page 81: ...n The Time column in the group pane cannot be modified If necessary modify the scan time in the Scans pane ii The Total Scan Time sec column indicates the sum of all the scans in each segment See Figure 57 The total scan time also contains the stabilization time that occurs between each scan This method beginning 2 5 minutes into the GC run gives you a complete set of scans every 0 342 seconds At ...

Page 82: ...ected Use this column if you had analytes eluting before the solvent peak You can create a segment to turn off the filament during the solvent peak to preserve the filament vi Use the Emission Current column to set the emission current used during the acquisition For optimal analytical performance and stability use the emission current at which the system was tuned However if you want to use a dif...

Page 83: ... MS with the method Figure 58 Acquisition General CI Settings ix The MS MS Gain Multiplier column only applies to groups with MS MS events The MS MS gain multiplier only applies if the Use Last Tuned Detector Gain column is selected The default MS MS Gain Multiplier is 7 and is intended to give similar signal levels as a SIM scan on the precursor ion If you enter a specific gain the MS will use th...

Page 84: ... from the Scan Type list If you want to link to an external method click Link to External File You may link to a csv or xml method file b After clicking Link to External File the SIMBridge dialog box opens Choose the language of your method file from the Source Locale drop down menu See Figure 60 Note In order to edit the scans within the TSQ 8000 Evo method editor clear the Link to External File ...

Page 85: ...f Method Files using SIMBridge c Browse to your file See Figure 61 Figure 61 Linking to an External File using SIM Bridge d Click Open to open the method in SIMBridge e If necessary change the method headings in your original file to match those in the method editor A green check mark appears when your method is validated See Figure 62 ...

Page 86: ...od will be opened in the method editor See Figure 63 Figure 63 Viewing a Linked File in the Method Editor g If you open an older model TSQ 8000 or a TSQ Duo if you have a TSQ 8000 Evo system method the Instrument Model for Method box appears h Select your model from the drop down menu in Instrument Model for Method in order to make the imported method compatible with your model ...

Page 87: ...es the number of compounds analyzed in a segment If the windows do not overlap you will not notice an improvement by reducing the acquisition window l In the Mass column enter the precursor ion mass for SRM methods For SIM methods enter the mass of the ion you wish to monitor m In the Product Mass column enter the product ion mass for SRM methods The product ion mass is the result of fragmenting t...

Page 88: ... to generate positive or negative ions Only use this column if you are using CI mode In EI mode this column should always be set to Positive p Click the Show Analysis arrow see Figure 65 to validate your method A chart appears with all your analytes by name and in order of their retention times See Figure 66 Figure 65 Accessing the Show Analysis Feature Show Analysis Button ...

Page 89: ...ytes are shown in the analytes chart Increasing the size of the scrolling window allows you to view more analytes in your method If the number of analytes retention times being viewed exceeds 50 an evenly spaced sample of the analytes shows through the window You may also click the ladder icon walk your analytes have the software automatically run through your list of analytes Click the ladder ico...

Page 90: ...e 68 on page 69 Tip If your SRM or SIM windows are too congested to achieve the total scan time at the minimum dwell time the event will be highlighted in orange or red The orange warning indicates that you might get fewer scans across the peak than you want The red error indicates that the instrument cannot complete the scans in the time allotted in your method To correct this reduce the desired ...

Page 91: ...red Min Dwell Time cannot be met with the number of Requested Scans Per Peak If this occurs then the acquisition windows are reduced until either the Desired Min Dwell Time is met or the Minimum Window is reached If the Minimum Window is reached first the Minimum Dwell Time is reduced until the Requested Scans Per Peak is achievable If the absolute minimum dwell time on the instrument which is 0 5...

Page 92: ... compounds Include extra time in this window if there is any uncertainty in compound retention times in the method Note that the longer the dwell time for your compounds the narrower your acquisition windows will be Change the minimum acquisition window in the Minimum Window combo box The minimum window can be set from 0 24 5 min This is the smallest amount of time that should be scanned for a tra...

Page 93: ...ified in the Tune File Name column Opening the precursor peak width increases the ion signal into the collision cell which might improve the sensitivity of your method however it may also increase the background noise level Use the Product Q3 Resolution column to set the full width at 50 FWHM of the product mass for SRM scans The combo box options are Normal Wide 1 5 FWHM or Widest 2 5 FWHM for ea...

Page 94: ...ent negative CI automatic tune file you created in Chapter 3 Tuning If you are using positive CI select a tune file from the Tune File pull down menu Choose AutoTune_PCI to use the most recent positive CI automatic tune file you created in Chapter 3 Tuning The software defaults to the most recent AutoTune_NCI or AutoTune_PCI tune file you created ii In the Detector Gain area set the detector gain ...

Page 95: ... 7 To import a Thermo Scientific ISQ Series or TSQ Quantum Series MS method choose TSQ Series Import MS Method See Figure 70 Tip Click the arrow at the top right of the Instrument Settings panel to collapse or expand the panel Note This will only import the MS part of the method You must set the GC and autosampler parameters Instrument Settings ...

Page 96: ...ur open scan list As above you may import csv or xml files 10 Choose TSQ Series Export Timed Scans to export your method as a csv file If you prefer editing your methods in spreadsheet applications you may want to use this option 11 Choose TSQ Series Create Compound Data Store Export File to prepare your file for the compound database in the TraceFinder application a To create a new target ion fro...

Page 97: ...71 Creating a New Target Ion b Drag the confirming ion to the compound name at the top of the list Figure 72 Moving the Confirming Ion to the Compound Name c The confirming ion now appears in the list as a target ion See Figure 73 Confirming Ion Compound Name Compound Name ...

Page 98: ...w confirming ion to a target ion in the list select the confirming ion of interest See Figure 74 Figure 74 Adding a New Confirming Ion to a Target Ion e Drag the confirming ion under the target ion of interest See Figure 75 New Target Ion Confirming Ion ...

Page 99: ...eate Export File when you are through creating your ion list to export the list to the TraceFinder software compound database 12 Choose TSQ Series Create Segment List From Timed Scan List to import a general acquisition method 13 Choose TSQ Series View Tune Report to view the latest tune report the method will use Choose Report Options to open the Report Options dialog box see Figure 77 and add id...

Page 100: ...d time in any GC temperature program In the Initial row enter the initial temperature which must be 4 C 7 F above room temperature and less than the maximum operating temperature of your GC column If you set the initial temperature to a value below this limit the GC will not reach the initial temperature If you set the temperature above the limit the GC column will be damaged You can set the initi...

Page 101: ...re which should be set to the maximum temperature allowed by your method not the maximum allowed by your column The maximum temperature allowed by the GC is 450 C 5 Select the prep run timeout The prep run timeout is the maximum amount of time that the GC will wait before it gives up on an injection As an example with the default value of 10 minutes if the GC is ready to receive an injection but d...

Page 102: ... set the amount of pressure used to push the carrier gas through the column Refer to the TRACE 1300 1310 Series GC User Guide for more information 8 If you have a split splitless inlet SSL injector click the S SL Front or S SL Back tab to configure the injector port settings In the inlet area do the following a Select the Temperature check box and set the temperature to at least 10 C 18 F lower th...

Page 103: ...e how well the analytes are separated and how long the analysis will take If you use constant pressure as the column is heated in the oven the flow rate will fall as the viscosity of the carrier gas increases If you use constant flow the carrier gas pressure will increase as the column temperature increases to keep the flow constant Constant flow is more common Typical flow rates are 1 3 mL min Th...

Page 104: ...figure it See Figure 80 The PTV is a low thermal mass injector that allows the instrument to rapidly heat or cool the inlet You can use the PTV page to program the temperature of the injector Refer to the GC documentation for details about the PTV or other types of injectors Figure 80 PTV Settings ...

Page 105: ...or user interface a Attach the inlet or detector to the GC Refer to the GC documentation for instructions b Add the inlet or detector to the current instrument configuration Choose Start All Programs Thermo Foundation 3 1 Instrument Configuration c In the Configured Devices pane click the TRACE 1300 or TRACE 1310 icon Click Configure Note The user interface reflects the current configuration of yo...

Page 106: ...box select the Inlets or Detectors tab as appropriate e Click Get The software automatically detects the attached modules Select the gases as appropriate for your setup The default Channel names are Channel 1 and Channel 2 You may want to change them to something more descriptive Your hardware is now configured ...

Page 107: ...ted are incompatible with the number of points requested per peak Although using the window optimizer will avoid most of these problems extreme cases still exist where your method will fail to validate In these cases you can fix this by reducing the desired scans across the peak reducing the number of scan events or if the window optimizer is not selected by reducing the acquisition windows If you...

Page 108: ...sired scans across the peaks You can fix this by reducing the desired scans across the peak reducing the number of scan events or reducing the acquisition windows and the resolution will be lower than expected from your method To decrease the desired scans per peak In the Scan Settings area reduce the number of scans in the Desired Scans Per Peak box See Figure 85 Warning Icon Method Setup ...

Page 109: ...olute minimum limit and the only way to resolve the error is to remove transitions reduce the number of points across the peak and or ensure that the baseline peak width is set appropriately To reduce the acquisition windows 1 In the Scans pane go to the first problem analyte and click on the Window min column See Figure 86 2 Decrease the time 3 While holding down the shift key click the last prob...

Page 110: ... User Guide Thermo Scientific Figure 86 Reducing the Acquisition Windows When your method validates the warning icon and the red bars disappear from the method editor See Figure 87 Figure 87 Validated Method You may now save the method as described earlier Acquisition Windows ...

Page 111: ...temperature These temperatures can be adjusted for each ramp Oven changes are strongly dependent on the nature of the compounds you are analyzing At some point the GC oven has to be above the boiling point of the compounds you are looking for If the GC oven is not at the boiling point the compounds will not volatilize and they will become immobilized You can change ramp rates to separate coeluting...

Page 112: ...changing the type of cartridge you are using Changing the Dwell Time or Scan Rate The precision of your data depends on how well you define your chromatographic peak Typically you get good precision when sampling ten times across the chromatographic peak In a full scan analysis increasing the scan rate increases the number of times you have sampled across the peak However increasing the scan rate ...

Page 113: ...thod into groups you can create compound specific MS settings to optimize your data Adjusting the Transfer Line and Ion Source Temperature If your transfer line temperature is set too low the less volatile compounds may get stuck in the transfer line and never make it into the ion source On the other hand if your transfer line is too hot you could damage the column or cause a thermal breakdown in ...

Page 114: ...known the scan window can be reduced If this is in a high density compound area it can dramatically improve the ion signal and number of points across the peak Use AutoSRM to optimize collision energy and retention times This can reduce errors in collision energy and improve confidence in retention times to allow shorter windows Choose the optimum fragment ion for study if allowed by your method S...

Page 115: ...o User Guide 93 2 Go to Load Tune File to access the manual tune files Figure 90 Accessing Manual Tune Files 3 The Manual Tune Files dialog box opens Select a PCI Tune file Figure 91 Loading a PCI Tune File 4 Select the Low Mass tab on the right Note the ion guide voltage value ...

Page 116: ...oltage box under the Low Mass tab to activate it Figure 93 Activating the Ion Guide Voltage in a NCI Tune File 7 If the ion guide voltage value is positive change it to the PCI value you recorded earlier which should be negative You may also optimize the ion guide voltage value to any other negative value 8 Save the changes to the tune file or rename it and save it as a new file Ion Guide Voltage ...

Page 117: ...nd reinsert repeating until you get the highest m z 29 for the current flow and filament settings 3 Once the orientation of the ion volume is optimized if you are tuning in PCI adjust the methane flow so that the m z 41 greater than 20 of 29 at 50 μA and 70 eV If you are tuning in NCI use a flow between 1 and 1 5 mL min methane for most analytes 4 Perform full scan NCI with the calibration gas on ...

Page 118: ...ne file Follow these procedures To modify an automatic tune To modify advanced tune settings To modify an automatic tune 1 Open the TSQ Series Dashboard and click Tune Types 2 In the Tune Types dialog box select a tune type to edit and click Copy See Figure 95 Figure 95 Tune Types Dialog Box 3 The Tune Type Editor opens Configure the parameters under the General tab in the Tune Type Editor Figure ...

Page 119: ...tory made file on the instrument that can be used to begin tuning an instrument with a clean ion source Select Last Saved to use a tune file saved on the instrument by the most recent successful automatic tune Select a specific tune file if you have a reliable tune file you want to use as a starting point for a new tune file g Perform Mass Calibration Select this to enable the system to recalibrat...

Page 120: ...ission Current Defines the emission current that you use to run subsequent tunes but not the emission current that is used for data acquisition Tune File Select this option to use the value in the tune file that you selected on the General tab Default Select this option to use the default emission current which is 50 μA Custom Select this option if you want to use a value other than 50 μA when inc...

Page 121: ...ith the same value you are planning to use for your analysis This value affects the focusing of the electron beam into the source If you change your electron energy from 70 eV this value also changes This voltage must always be at least 45 V above the voltage applied to the filament The voltage applied to the filament is the same number but the opposite sign of the electron energy f Electron Lens ...

Page 122: ...to keep the ion source clean and maintain the right amount of sensitivity 5 Click the Targets tab to configure how you want to tune your targets Target tuning is used to adjust the way the mass spectrometer tunes to meet regulatory requirements Otherwise this is an optional setting Figure 98 Configuring Tuning Targets Tune Target Ion Ratios Select this check box to adjust the ratios based on the r...

Page 123: ...s the electron multiplier ages the voltage required for a given gain increases Depending on your sample load and if your system is leak tight oxygen is bad for the detector you should not have to perform this tune very often Limit to 1 2 times a year c Adjust Detector Sensitivity Select this check box to tune the detector to generate a consistent area count of a calibration gas ion for the tune re...

Page 124: ...Evo User Guide Thermo Scientific Figure 100 Setting Diagnostics 8 Click the Report tab configure how you want to view your data Figure 101 Selecting Data Report Options a Acquire Full Scan Spectrum Select this check box to display the full spectrum on your tune report ...

Page 125: ...indow 10 Click Close to return to the dashboard You can select the tune type in the automatic tune window 11 If you want to rename a tune you created open the Tune Types dialog box by clicking Tune Types on the TSQ Series Dashboard a Select Rename in the Tune Types dialog box Figure 102 Renaming a Tune Type b The Rename Tune Type dialog box opens Assign a valid new name to your tune You will not b...

Page 126: ... allowed values is how to move through the range how much the signal must change for a new value to be selected and what if anything can be done about the resolution of the peak and any errors that occur Select the Tune Lenses check box to access the data table See Figure 104 Figure 104 Tuning the Q1 Lenses a Device Click in this box to select the component you want to tune according to the settin...

Page 127: ...trols the ion guide s DC offset voltage It can potentially help focus the ions into the quadrupole while ensuring that neutral noise is eliminated The voltage on this component is mass dependent and should be set at several different masses This field is typically set between 1 and 15 V depending on the mass of the ion vi Ion Guide RF q Controls the ion guide s RF q amplitude This device is mass d...

Page 128: ...ity the tune will select the new intensity i On Error Click in this box to select how to handle an error in the tune Fail Stops the tune when an error occurs or if no tune points are found meeting the tune criteria Continue Allows the tune to continue on to the next device when an error occurs j Entrance lens offset and Exit lens offset Changing these voltages affects ion acceleration The default ...

Page 129: ...k in this box to set the occurrence of the resolution tune parameters before or after a lens tune Before and After Uses the same resolution parameters before and after a lens tune Before Uses the resolution parameters before a lens tune After Uses the resolution parameters after a lens tune 3 Select the Q3 Lenses tab to configure the Q3 lenses You must select the Show Advanced Settings check box t...

Page 130: ...s the ratio of the quadrupole DC and RF voltages to create the resolution required for your analysis You can set the desired peak width at a given mass and whether you measure the width at 10 or 50 of the peak height Because there is no static DC voltage involved the start stop and step values are not used c Mass Click in this box to select the ion to be used for tuning d Start Enter the starting ...

Page 131: ... an error in the tune Fail Stops the tune when an error occurs Continue Allows the tune to continue on to the next device when an error occurs k Entrance lens offset and Exit lens offset Changing these voltages affects ion acceleration The default levels will give the best performance changing the voltages will affect peak resolution l MS MS Voltages These overwrite the Q3 voltage tune values when...

Page 132: ...k width 10 Measures the width at 10 of the peak height 50 Measures the width at 50 of the peak height d Lens Tune Relation Click in this box to set the occurrence of the resolution tune parameters before or after a lens tune Before and After Uses the same resolution parameters before and after a lens tune Before Uses the resolution parameters before a lens tune After Uses the resolution parameters...

Page 133: ...e TSQ 8000 Evo system requires a Windows 7 32 bit or 64 bit operating system See System Requirements on page xi for a complete list of system requirements Computer Settings The following computer settings will help prevent communication interruptions in your TSQ 8000 Evo system To use the power save feature 1 From the Start menu choose Control Panel System and Security Power Options 2 On the left ...

Page 134: ...l System and Security System 2 On the left side of the window click Advanced System Settings 3 In the System Properties dialog box click Settings 4 In the Performance Options dialog box click the Advanced tab and then click Change to open the Virtual Memory window 5 Confirm that Automatically Manage Paging File Size for All Drives check box is selected See Figure 109 Figure 109 Virtual Memory Sett...

Page 135: ...d Settings 2 In the Actions Panel on the right see Figure select Properties 3 In the Windows Firewall with Advanced Security on Local Computer Properties window select Domain Profile State Customize Figure 110 Firewall Settings for the TSQ 8000 Evo System Computer 4 In the Protected Connections for the Domain Profile window make sure that any instrument checkboxes are not checked Figure 111 Settin...

Page 136: ...f you are using anti virus software on your computer exclude the following folders from scanning C Program Files Thermo C Thermo C Xcalibur Failure to exclude these folders might cause interruptions in data acquisition If you must for security reasons scan all system files set the virus scans so that they occur when you are not using the instrument to acquire data ...

Page 137: ...e good ion intensities good peak shapes and no air leak you might want to look first at the GC autosampler or carrier gas If you have air leaks locate and address them Pay particular attention to the transfer line ferrule vent valve knob front panel gas quality carrier gas vacuum compensation and vacuum interlock on the mass spectrometer as well as the inlet on the GC If your intensities are too l...

Page 138: ... full scan method to check if your system is functional Diagnostics Checks The following table provides you with possible corrective actions you can take after failing a diagnostics check on your system IMPORTANT When inserting a cold ion source cartridge such as after cleaning or when switching between EI and CI modes the ion source and lens stack will expand as the source cartridge heats often p...

Page 139: ...nts wear clean lint free gloves when performing any type of maintenance on a component in the vacuum manifold of the instrument Air Leaks Higher than normal vacuum pressure or poor sensitivity usually indicates an air leak Check the last o ring or ferrule that you installed Table 2 Diagnostics and Corrective Actions Diagnostic Cause of Failure Corrective Action Communication Check Unknown Contact ...

Page 140: ...leaks outgassing from Vespel ferrules 69 100 119 131 169 181 214 219 264 376 414 426 464 502 576 614 PFTBA and related ions PFTBA tuning compound 31 Methanol Cleaning solvent 43 58 Acetone Cleaning solvent 78 Benzene Cleaning solvent 91 92 Toluene or xylene Cleaning solvent 105 106 Xylene Cleaning solvent 151 153 Trichloroethane Cleaning solvent 149 Plasticizer phthalates Use of vinyl or plastic g...

Page 141: ...acuum compensation is on Carrier gas flow is drifting Check for leaks in injector or flow path Check the carrier gas tank Impurities have accumulated in column Run solvent blank to remove impieties If impurities persistent after multiple solvent blanks Inject solvent from a different source Change syringe liner and septum Clean injector Check impurity levels in your gas Use the correct gas purity ...

Page 142: ...rrier gas line Check pneumatic and inlet systems for leaks Use correct gas purity with low oxygen content Column is contaminated or damaged Condition or replace the column Oven temperature is higher than column s maximum allowable temperature Reduce oven temperature to the maximum allowable temperature of the column Leak at column fittings Find leak Tighten fittings if loose Replace ferrule if ove...

Page 143: ...e the flow to 40 50 ml min Performance of the column has degraded Test the column at the optimum flow rate Injector is dirty Clean or replace the liner Ion source is dirty Clean the ion source and tune the instrument Column is not far enough into the transferline The GC column does not extend into the MS source Use the column measuring tool to confirm column length If the end of the column is insi...

Page 144: ...t Stationary phase is not appropriate for your target compounds Replace the column and choose a more appropriate phase for your analysis Final hold oven temperature is too low crease the column oven temperature Do not exceed the recommended maximum temperature for the stationary phase Transferline temperature is too low If tailing occurs on late eluting compounds it is likely the source or transfe...

Page 145: ...ter Install an in line filter between the pneumatics and the inlet Missing Peaks Baseline or background present Column is broken Replace the column Column flow is incorrect Make sure the septa are sealing Make sure vacuum compensation is turned on Backflush settings are incorrect Set backflush to off until after injection Column position in S SL injector is incorrect too high Check the position of...

Page 146: ...tion Make sure the filament is not burned out Injection technique is not appropriate Use a different injection technique Injection parameters are not appropriate Verify the injection temperature and flow rates Sample injection technique is not reproducible Evaluate the sample preparation sequences Compare the results with a series of standard injections Syringe or septum is leaking Check and repla...

Page 147: ... Increase the temperature of the injector Voltage is not reaching the lens Replace the lens plate and springs if damaged Remove debris or broken pieces in the manifold Run a lens check diagnostic Check the connection by removing or inserting the ion source Retention Times Low reproducibility DCC is drifting or unstable Monitor the column pressure or flow Check and replace the controller if necessa...

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Page 149: ...esult issues investigating 124 diagnostics selecting tests to run 101 documentation survey xxi E electromagnetic compatibility vi EMC compliance v F FCC compliance vi filament voltage setting 99 filament turning off and on 60 fingerprint indications 117 full scan mode about 57 G GC creating a method 78 ghost peaks troubleshooting 123 I ion source temperature setting 52 100 L leak indications 117 l...

Page 150: ...ture of the ion source 52 temperature of the transfer line 91 temperature of transfer line 52 transfer line temperature setting 52 transfer line about 52 troubleshooting about 115 baseline issues 119 peak issues 121 results issues 124 tune report 39 TSQ 8000 creating a method 45 TSQ 8000 Evo changing the column 15 configuring 6 TSQ 8000 Evo AutoTune about 96 tune reports example report 39 printing...

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