TSQ Series TSQ Endura and TSQ Quantiva Getting Started Guide 80100-97025 Revision A July 2015 © 2015 Thermo Fisher Scientific Inc. All rights reserved. EASY-Max NG and Ion Max NG are trademarks; Unity is a registered service mark; and Hypersil GOLD AQ, TSQ Endura, TSQ Quantiva, Thermo Scientific, and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc. in the United States. The following are registered trademarks in the United States and other countries: Excel, Microsoft, and Windows are registered trademarks of Microsoft Corporation. Teflon is a registered trademark of E.I. du Pont de Nemours & Co. The following are registered trademarks in the United States and possibly other countries: APPI, PhotoMate, and Syagen are registered trademarks of Morpho Detection, Inc. Rheodyne is a registered trademark of IDEX Health & Science, LLC. Tygon is a registered trademark of the division of Saint-Gobain Performance Plastics Corporation. Chemyx is a trademark of Chemyx Inc. MX Series II is a trademark of IDEX Health & Science, LLC. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Thermo Fisher Scientific Inc. provides this document to its customers with a product 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 configurations and specifications in this document supersede all previous information received by the purchaser. This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of Sale shall govern all conflicting information between the two documents. Release history: Rev. A, July 2015 Software version: (Thermo) Foundation 3.0 and later, Xcalibur 3.0 and later, Tune 2.0 and later For Research Use Only. Not for use in diagnostic procedures. Regulatory Compliance Thermo Fisher Scientific performs complete testing and evaluation of its products to ensure full compliance with applicable domestic and international regulations. When the system is delivered to you, it meets all pertinent electromagnetic compatibility (EMC) 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 safety standards, replacement parts and additional components, options, and peripherals must be ordered from Thermo Fisher Scientific or one of its authorized representatives. EMC Directive 2004/108/EC EMC compliance has been evaluated by TÜV Rheinland of North America. EN 55011: 2009, A1: 2010 EN 61000-4-6: 2009 EN 61000-3-2: 2006, A2: 2009 EN 61000-4-11: 2004 EN 61000-3-3: 2008 EN 61326-1: 2013 EN 61000-4-2: 2009 CISPR 11: 2009, A1: 2010 EN 61000-4-3: 2006, A2: 2010 ICES-003 Issue 5: 2012 EN 61000-4-4: 2004, A1: 2010 CFR 47, FCC Part 15, Subpart B, Class A: 2012 EN 61000-4-5: 2006 Low Voltage Safety Compliance This device complies with Low Voltage Directive 2006/95/EC and harmonized standard EN/UL/CAN 61010-1. FCC Compliance Statement THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES. OPERATION IS SUBJECT TO THE FOLLOWING TWO CONDITIONS: (1) THIS DEVICE MAY NOT CAUSE HARMFUL INTERFERENCE, AND (2) THIS DEVICE MUST ACCEPT ANY INTERFERENCE RECEIVED, INCLUDING INTERFERENCE THAT MAY CAUSE UNDESIRED OPERATION. CAUTION Read and understand the various precautionary notes, signs, and symbols contained inside this manual pertaining to the safe use and operation of this product before using the device. FCC Compliance Statement THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES. OPERATION IS SUBJECT TO THE FOLLOWING TWO CONDITIONS: (1) THIS DEVICE MAY NOT CAUSE HARMFUL INTERFERENCE, AND (2) THIS DEVICE MUST ACCEPT ANY INTERFERENCE RECEIVED, INCLUDING INTERFERENCE THAT MAY CAUSE UNDESIRED OPERATION. CAUTION Read and understand the various precautionary notes, signs, and symbols contained inside this manual pertaining to the safe use and operation of this product before using the device. Notice on Lifting and Handling of Thermo Scientific Instruments For your safety, and in compliance with international regulations, the physical handling of this Thermo Fisher Scientific instrument requires a team effort to lift and/or move the instrument. This instrument is too heavy and/or bulky for one person alone to handle safely. Notice on the Proper Use of Thermo Scientific Instruments In compliance with international regulations: This instrument must be used in the manner specified by Thermo Fisher Scientific to ensure protections provided by the instrument are not impaired. Deviations from specified instructions on the proper use of the instrument include changes to the system and part replacement. Accordingly, order replacement parts from Thermo Fisher Scientific or one of its authorized representatives. WEEE Directive 2012/19/EU Thermo Fisher Scientific is registered with B2B Compliance (B2Bcompliance.org.uk) in the UK and with the European Recycling Platform (ERP-recycling.org) in all other countries of the European Union and in Norway. If this product is located in Europe and you want to participate in the Thermo Fisher Scientific Business-to-Business (B2B) Recycling Program, send an email request to [email protected] with the following information: • WEEE product class • Name of the manufacturer or distributor (where you purchased the product) • Number of product pieces, and the estimated total weight and volume • Pick-up address and contact person (include contact information) • Appropriate pick-up time • Declaration of decontamination, stating that all hazardous fluids or material have been removed from the product For additional information about the Restriction on Hazardous Substances (RoHS) Directive for the European Union, search for RoHS on the Thermo Fisher Scientific European language websites. IMPORTANT This recycling program is not for biological hazard products or for products that have been medically contaminated. You must treat these types of products as biohazard waste and dispose of them in accordance with your local regulations. Directive DEEE 2012/19/EU Thermo Fisher Scientific s'est associé avec une ou plusieurs sociétés de recyclage dans chaque état membre de l’Union Européenne et ce produit devrait être collecté ou recyclé par celle(s)-ci. Pour davantage d'informations, rendez-vous sur la page www.thermoscientific.fr/rohs. WEEE Direktive 2012/19/EU Thermo Fisher Scientific hat Vereinbarungen mit Verwertungs-/Entsorgungsfirmen in allen EU-Mitgliedsstaaten getroffen, damit dieses Produkt durch diese Firmen wiederverwertet oder entsorgt werden kann. Weitere Informationen finden Sie unter www.thermoscientific.de/rohs. C Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiii Mass Spectrometer Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiii Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv Installation Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv MS Calibration Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv Performance Specification Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xvi Chemical Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii Cautions and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xviii Thermo Scientific Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Ionization Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Using H-ESI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Using APCI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Using APPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Using NSI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 LC Flow Rates Ranges. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Types of Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Method Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Chapter 2 Setting Up the API Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 Preparing the Mass Spectrometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Installing or Removing the API Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Installing the API Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Removing the API Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Preparing the Spray Insert for the API Source . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Installing the Spray Insert . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Adjusting the Spray Direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Chapter 3 Connecting the Inlet Plumbing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 Sample Introduction Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Direct Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 High-Flow Infusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Loop Injection (Flow-Injection Analysis). . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 High-Performance Liquid Chromatography (HPLC) with an Autosampler. . 21 TSQ Endura and TSQ Quantiva Getting Started Guide vii Contents Plumbing Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Setting Up the Syringe Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Setting Up the Inlet Plumbing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Setting Up the Inlet for Direct Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Setting Up the Inlet for High-Flow Infusion . . . . . . . . . . . . . . . . . . . . . . . . . 26 Setting Up the Inlet for Manual or Auto-Loop Injections . . . . . . . . . . . . . . . 29 Setting Up the Inlet for an LC/MS System with an Autosampler . . . . . . . . . . . 31 Connecting the Grounding Union to the H-ESI Spray Insert . . . . . . . . . . . . . . 32 viii Chapter 4 Using the Syringe Pump and Divert/Inject Valve . . . . . . . . . . . . . . . . . . . . . . . . . .33 Syringe Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Divert/Inject Valve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Controlling the Divert/Inject Valve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Chapter 5 Preparing the System for Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37 Pumping Down the Mass Spectrometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Setting Up the Syringe Pump for Direct Infusion . . . . . . . . . . . . . . . . . . . . . . . 40 Setting Up the Mass Spectrometer for Calibration. . . . . . . . . . . . . . . . . . . . . . . 41 Chapter 6 Establishing a Stable Ionization Spray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43 Evaluating the Spray Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Optimizing the API Source Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Chapter 7 Performing the Tune, Calibration, or Calibration Check . . . . . . . . . . . . . . . . . . . .49 Performing System Tune and Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Spray Stability Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 System Tune and Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Check Mass Position and Resolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Calibrate Mass Position and Resolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Detector Gain Calibration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Chapter 8 Optimizing the API Source Parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55 Setting Up the Plumbing for Compound Optimization . . . . . . . . . . . . . . . . . . 55 Determining the Initial API Source Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Optimizing the RF Lens Voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Optimizing the Collision Energy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Chapter 9 Acquiring Sample Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63 Using the Tune Application to Acquire Sample Data . . . . . . . . . . . . . . . . . . . . 63 Using the Xcalibur Data System to Acquire Sample Data . . . . . . . . . . . . . . . . . 65 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific Contents Appendix A Using Basic Tune Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67 Opening the Tune Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Setting the Instrument Power Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Checking the Instrument Readback Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Controlling the Syringe Pump. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Setting the Data Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Setting the Ion Polarity Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Setting the Tune Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Using the Mass List Table in the Define Scan Pane . . . . . . . . . . . . . . . . . . . . . . 73 Using the History Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Using the Favorites Pane to Save System Settings . . . . . . . . . . . . . . . . . . . . . . . 76 Appendix B Flushing the Inlet Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79 Supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Flushing the Inlet Components after Calibration . . . . . . . . . . . . . . . . . . . . . . . 81 Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87 Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide ix Contents x TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific F Figures Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Figure 19. Figure 20. Figure 21. Figure 22. Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Figure 28. Figure 29. Figure 30. Figure 31. Figure 32. Figure 33. Figure 34. Thermo Scientific Thermo Xcalibur Instrument Setup window showing the scan types . . . . . . . . . . 7 MS API source mount assembly and ion sweep cone . . . . . . . . . . . . . . . . . . . . 11 EASY-Max NG API source with H-ESI spray insert (top, front view). . . . . . . . 13 API source connection to the MS mount assembly (installed ion sweep cone) . . 14 Front-to-back adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Rotational adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Schematics of the sample introduction techniques (examples) . . . . . . . . . . . . . . 22 Proper connection for the PEEK tubing and fitting (syringe adapter assembly). . 23 Plumbing connection for the syringe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Plumbing connections for direct infusion (H-ESI mode) . . . . . . . . . . . . . . . . . . 25 Plumbing connection between the LC union and the union Tee . . . . . . . . . . . 27 Plumbing connection between the union Tee and the divert/inject valve . . . . . 27 Plumbing connection between the union Tee and the grounding union . . . . . . 28 Divert/inject valve setup for manual loop injection . . . . . . . . . . . . . . . . . . . . . . 30 Plumbing connections for manual loop injection (APCI mode) . . . . . . . . . . . . . 31 Plumbing connections for the grounding union (H-ESI mode) . . . . . . . . . . . . . 32 Syringe pump setup (top view) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Divert/inject valve positions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Divert/inject valve plumbed as a loop injector and as a divert valve . . . . . . . . . . 36 Divert/inject valve (front view) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Power entry module (right side of the instrument) . . . . . . . . . . . . . . . . . . . . . . 38 By Board page in the Status pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Plot Chromatogram dialog box with the TIC option selected . . . . . . . . . . . . . . 44 Optimization page of the Ion Source pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Report Generation Options dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Calibration – Options page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Normalized intensity as a function of the M00 multipole offset voltage . . . . . . . 52 Isotopic peaks of the polytyrosine trimer at a peak width of m/z 0.4 . . . . . . . . . 53 Error range of the polytyrosine 1-3-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 LC flow rate of 200 μL/min on the Ion Source – Ion Source page . . . . . . . . . . 56 Define Scan – Optimization page showing the rf lens voltage optimization settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 RF optimization for the polytyrosine trimer ion . . . . . . . . . . . . . . . . . . . . . . . . 59 Define Scan – Optimization page showing the collision energy optimization settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Collision energy optimization curve and MS/MS spectrum of the polytyrosine trimer ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 TSQ Endura and TSQ Quantiva Getting Started Guide xi Figures Figure 35. Figure 36. Figure 37. Figure 38. Figure 39. Figure 40. Figure 41. Figure 42. Figure 43. Figure 44. Figure 45. Figure 46. Figure 47. Figure 48. xii Data Acquisition pane in the Tune window . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Run Sequence dialog box (partial) showing the selected start instrument . . . . . 65 Change Instruments In Use dialog box showing the MS as the start instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Tune window showing the Define Scan – Scan page . . . . . . . . . . . . . . . . . . . . . 68 Power mode icons showing the selected icon (mode) . . . . . . . . . . . . . . . . . . . . 69 Toggle button for the syringe modes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Syringe parameter box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Toggle button for the data types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Toggle button for the instrument polarity modes . . . . . . . . . . . . . . . . . . . . . . . 71 Tune Preferences dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Q3 Resolution selected and added to the SRM Table . . . . . . . . . . . . . . . . . . . . 74 History pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Favorites pane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 State name box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific P Preface The TSQ Endura and TSQ Quantiva Getting Started Guide describes how to set up and calibrate the Thermo Scientific™ TSQ Endura™ and TSQ Quantiva™ mass spectrometers (MSs), and acquire MS data. Contents • Mass Spectrometer Models • Related Documentation • Installation Kits • Cautions and Special Notices • Contacting Us To suggest changes to the documentation or to the Help Complete a brief survey about this document by clicking the button below. Thank you in advance for your help. Mass Spectrometer Models This guide is intended for the following mass spectrometer models: • TSQ Endura—Requires one forepump. • TSQ Quantiva—Requires two forepumps. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide xiii Preface Related Documentation The TSQ Endura and TSQ Quantiva mass spectrometers include complete documentation. In addition to this guide, you can also access the following documents as PDF files from the data system computer: • TSQ Endura and TSQ Quantiva Preinstallation Requirements Guide • TSQ Endura and TSQ Quantiva Getting Connected Guide • TSQ Endura and TSQ Quantiva Hardware Manual • Ion Max NG and EASY-Max NG Ion Sources User Guide • Safety and Regulatory Guide The TSQ Endura and TSQ Quantiva also ship with a printed copy of the Safety and Regulatory Guide. This guide contains important safety information about Thermo Scientific liquid chromatography (LC) and mass spectrometry (MS) systems. Make sure that all lab personnel have read and have access to this document. To view the product manuals From the Microsoft™ Windows™ taskbar, do the following: • For the Thermo Scientific mass spectrometer, choose Start > All Programs > Thermo Instruments > model x.x, and then open the applicable PDF file. • For an LC instrument controlled by a Thermo Scientific application, choose Start > All Programs > Thermo Instruments > Manuals > LC Devices and so on. The TSQ Endura and TSQ Quantiva application also provides Help. To view the Help Do the following as applicable: • To access the Tune application Help, click the Options icon, Tune Help. , and then choose • To access the Thermo Xcalibur™ Method Editor Help, choose the appropriate option from the Help menu. To download user documentation from the Thermo Scientific website 1. Go to www.thermoscientific.com. 2. In the Search box, type the product name and press Enter. 3. In the left pane, select Documents & Videos, and then under Refine By Category, click Operations and Maintenance. xiv TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific Preface 4. (Optional) Narrow the search results or modify the display as applicable: • For all related user manuals and quick references, click Operator Manuals. • For installation and preinstallation requirements guides, click Installation Instructions. • For documents translated into a specific language, use the Refine By Language feature. • Use the Sort By options or the Refine Your Search box (above the search results display). 5. Download the document as follows: a. Click the document title or click Download to open the file. b. Save the file. Installation Kits The TSQ Endura or TSQ Quantiva MS ships with several kits. However for the procedures in this guide, the following kits provide the necessary components: • MS Calibration Kit • Performance Specification Kit • Chemical Kit For a full list of the mass spectrometer kits and their contents, refer to the TSQ Endura and TSQ Quantiva Hardware Manual. MS Calibration Kit Table 1. MS Calibration Kit (P/N 80000-62013) (Sheet 1 of 2) Image Thermo Scientific Item Quantity Part number Ferrule, fingertight, natural PEEK 2 00101-18196 Fitting, fingertight, one-piece natural PEEK, 10-32 1 00109-99-00016 Fitting, fingertight, two-piece natural PEEK, two wings, 10-32 2 00101-18081 Fitting, fingertight, two-piece, one wing, 10-32 2 00101-18195 TSQ Endura and TSQ Quantiva Getting Started Guide xv Preface Table 1. MS Calibration Kit (P/N 80000-62013) (Sheet 2 of 2) Image Item Quantity Part number Grounding union, zero-dead-volume (ZDV), stainless steel, 1/16 in. orifice, 0.010 in. (0.25 mm) thru-hole, 10-32 1 00101-18182 HPLC union, black PEEK, 10-32, 0.01 in. thru-hole 1 00101-18202 — Syringe, GC, gas tight, 500 μL, 51 mm long 1 00301-01-00040 — Tubing, natural PEEK, 1/16 in. OD, 0.0025 in. ID, 28 cm (11 in.) long 2 80000-22032 Note Use this tubing with the calibration solutions and for flow rates less than 50 μL/min. — Tubing, red PEEK, 1/16 in. OD, 0.005 in. ID, 0.6 m (2 ft) long 1 00301-22912 — Tubing, red PEEK, 1/16 in. OD, 0.005 in. ID, 18 cm (7.1 in.) long 2 80000-22053 Note Use this tubing for flow rates equal to or greater than 50 μL/min. — Tubing, Teflon™ FEP, 1/16 in. OD, 0.03 in. ID, 3 cm (1.2 in.) long 1 00301-22915 Performance Specification Kit Table 2. Performance Specification Kit (P/N 80100-62008) (Sheet 1 of 2) Image Item — Column, HPLC, 20 × 2.1 mm ID, Hypersil GOLD AQ™ C18, 1.9 μm particles 1 00109-01-00013 Fitting, fingertight, one-piece natural PEEK, 10-32 10 00109-99-00016 Needle port, PEEK 1 00110-22030 Sample loop, 2 μL, PEEK 1 00110-16012 Syringe, gas tight, 500 μL 1 00301-19016 — xvi TSQ Endura and TSQ Quantiva Getting Started Guide Quantity Part number Thermo Scientific Preface Table 2. Performance Specification Kit (P/N 80100-62008) (Sheet 2 of 2) Image Item Quantity Part number — Tubing, red PEEK, 1/16 in. OD, 0.005 in. ID, 3 m (10 ft) long 1 00301-22912 Union Tee, HPLC, PEEK, 1/16 in. orifice, 0.020 in. (0.5 mm) thru-hole, 10-32 (provided with fingertight fittings) 1 00101-18204 Chemical Kit IMPORTANT Be aware of the following storage precautions for the calibration and reserpine solutions: Refrigerate the containers after opening. For long-term storage, keep refrigerated at 2–8 °C (36–46 °F). Table 3. Chemical Kit (P/N 80100-62006) Item Quantity Part number Calibration solution, polytyrosine 1-3-6, 10 mL 1 00301-22924 TSQ Reserpine Solution Kit (contains reserpine standard, 100 pg/μL, 5 × 1 mL, P/N HAZMAT-01-00081) 1 80100-62033 LCMS Functionality Test Kit (for field service use only) 1 HAZMAT-01-00044 Cautions and Special Notices Make sure that you follow the cautions and special notices presented in this guide. Cautions and special notices appear in boxes; those concerning safety or possible damage also have corresponding caution symbols. This guide uses the following types of cautions and special notices. CAUTION Highlights hazards to humans, property, or the environment. Each CAUTION notice is accompanied by an appropriate CAUTION symbol. IMPORTANT Highlights information necessary to prevent damage to software, loss of data, or invalid test results; or might contain information that is critical for optimal performance of the system. Note Highlights information of general interest. Tip Highlights helpful information that can make a task easier. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide xvii Preface The TSQ Endura and TSQ Quantiva Getting Started Guide contains the following cautionspecific symbols (Table 4). Table 4. Caution-specific symbols and their meanings Symbol Meaning Chemical hazard: Wear gloves and other protective equipment, as appropriate, when handling toxic, carcinogenic, mutagenic, corrosive, or irritant chemicals. Use approved containers and proper procedures to dispose of waste oil and when handling wetted parts of the instrument. Hot surface: Before touching the API source assembly, allow heated components to cool. Risk of electric shock: This instrument uses voltages that can cause electric shock and/or personal injury. Before servicing, shut down the instrument and disconnect it from line power. While operating the instrument, keep covers on. Risk of eye injury: Eye injury could occur from splattered chemicals or airborne particles. Wear safety glasses when handling chemicals or servicing the instrument. Sharp object: Avoid handling the tip of the syringe needle. Contacting Us There are several ways to contact Thermo Fisher Scientific for the information you need. You can use your smartphone to scan a QR code, which opens your email application or browser. Contact us xviii Customer Service and Sales Technical Support (U.S.) 1 (800) 532-4752 (U.S.) 1 (800) 532-4752 (U.S.) 1 (561) 688-8731 (U.S.) 1 (561) 688-8736 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific Preface Contact us Customer Service and Sales Technical Support us.customer-support.analyze @thermofisher.com us.techsupport.analyze @thermofisher.com To find global contact information or customize your request 1. Go to www.thermoscientific.com. 2. Click Contact Us, select the Using/Servicing a Product option, and then type the product name. 3. Use the phone number, email address, or online form. To find product support, knowledge bases, and resources Go to www.thermoscientific.com/support. To find product information Go to www.thermoscientific.com/lc-ms. Note To provide feedback for this document: • Send an email message to Technical Publications ([email protected]). • Complete a survey at www.surveymonkey.com/s/PQM6P62. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide xix Preface xx TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 1 Introduction This chapter provides general information about the TSQ Endura and TSQ Quantiva MSs. For information about using the Thermo Tune application, see Appendix A, “Using Basic Tune Functions.” For information about daily operation, maintenance, and system startup and shutdown, refer to the TSQ Endura and TSQ Quantiva Hardware Manual. Note • The Glossary defines some of the terms used in this guide. • To ensure the proper operation of the mass spectrometer, Thermo Fisher Scientific recommends that you perform the daily preventive maintenance described in the TSQ Endura and TSQ Quantiva Hardware Manual. Contents • Ionization Techniques • LC Flow Rates Ranges • Types of Buffers • Method Editor Ionization Techniques This section briefly describes the following ionization modes: heated-electrospray (H-ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and nanoelectrospray ionization (nanoESI or NSI). For additional information, refer to the API source’s manual. • Using H-ESI (Typically preferred for polar compounds) • Using APCI (Typically preferred for medium polar compounds) • Using APPI (Typically preferred for certain polar and nonpolar compounds) • Using NSI (Typically preferred for peptides and proteins) Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 1 1 Introduction Ionization Techniques Using H-ESI H-ESI is a soft gas phase ionization technique. The H-ESI source transfers ions in solution to the gas phase. H-ESI can analyze many samples that previously were not suitable for mass analysis (for example, heat-labile compounds or high molecular mass compounds). You can use H-ESI to analyze any polar compound that is an ion in solution, including adduct ions. Included in this class of compounds are biological polymers (such as proteins, peptides, glycoproteins, and nucleotides), pharmaceuticals and their metabolites, and industrial polymers. For example, you might analyze polyethylene glycols from a solution containing ammonium acetate because of adduct formation between NH4+ ions in the solution and oxygen atoms in the polymer. With H-ESI, the range of molecular masses that the mass spectrometer can analyze can exceed 50 000 Da if there is multiple charging. The H-ESI source can produce multiply-charged ions, depending on the structure of the analyte and the solvent. For example, the mass spectrum of a protein or peptide typically consists of a distribution of multiply-charged analyte ions. You can mathematically manipulate this mass spectrum to determine the molecular mass of the sample. Use H-ESI in either positive or negative ion polarity mode. The polarity of the ions in solution determines the ion polarity mode: acidic molecules form negative ions in high pH solution and basic molecules form positive ions in low pH solution. The installed H-ESI spray insert can be either positively or negatively charged. When it is positively charged, it generates positive ions. When it is negatively charged, it generates negative ions. Vary the flow rate into the mass spectrometer over a range of 1–1000 μL/min. See Table 5 for guidelines. In H-ESI, because both the buffer type and buffer concentration have a noticeable effect on sensitivity, you must choose these variables correctly. Large droplets with high surface tension, low volatility, low surface charge, strong ion solvation, and high conductivity negatively affect the H-ESI process. Conversely, H-ESI favors small droplets with low surface tension, high volatility, high surface charge, weak ion solvation, and low conductivity. Mixed organic-aqueous solvent systems that include organic solvents, such as methanol, acetonitrile, and isopropyl alcohol, are superior to water alone for H-ESI. Volatile acids and bases are good, but for best results do not use salts above 10 mM. Be aware that strong mineral acids and bases are extremely detrimental to the instrument. 2 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 1 Introduction Ionization Techniques IMPORTANT To obtain good H-ESI results, follow these guidelines: • Keep nonvolatile salts and buffers out of the solvent system. For example, avoid the use of phosphates and salts that contain potassium or sodium. Use acetate or ammonium salts instead. Do not use strong mineral acids and bases—they can damage the instrument. • Use organic/aqueous solvent systems and volatile acids and bases. Try to avoid the use of 100 percent aqueous solvents. • If possible, optimize the pH of the solvent system for the analyte. For example, if the analyte contains a primary or secondary amine, the mobile phase should be slightly acidic (pH 2–5). The acidic pH tends to keep positive ions in solution. Using APCI Like H-ESI, APCI is a soft gas phase ionization technique. Therefore, the gas phase acidities and basicities of the analyte and solvent vapor play an important role in the APCI process. APCI provides molecular mass information for compounds of medium polarity that have some volatility. APCI is typically used to analyze small molecules with molecular masses up to about 1000 Da. Use APCI in either positive or negative ion polarity mode. For most molecules, the positive ion mode produces a stronger ion current. This is especially true for molecules with one or more basic nitrogen (or other basic) atoms. Molecules that generally produce strong negative ions with acidic sites, such as carboxylic acids and acid alcohols, are an exception to this general rule. In general, APCI produces fewer negative ions than positive ions. However, the negative ion polarity mode can be more specific because it generates less chemical noise than does the positive mode. Consequently, the signal-to-noise ratio (S/N) might be better in the negative ion mode. The rate of solvent flowing from the LC into the mass spectrometer in APCI mode is typically high (200–2000 μL/min). See Table 6 for guidelines. APCI is a very robust ionization technique. It is not affected by minor changes in most variables, such as changes in buffer type or buffer strength. Using APPI APPI is also a soft ionization technique. In APPI an ion is generated from a molecule when it interacts with a photon from a light source, such as the Syagen Technology PhotoMate™ APPI™ light source. APPI generates molecular ions for molecules that have an ionization potential below the photon energy of the light being emitted by the light source. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 3 1 Introduction LC Flow Rates Ranges Molecules that include steroids, basic-drug entities, and pesticides have ionization potentials below the threshold. APPI reduces fragmentation because only a small amount of energy is deposited in the molecule. Molecules, such as the nitrogen sheath and auxiliary gas and the simple solvents used for LC/MS, are not ionized because their ionization potentials are greater than the photon energy. The result is selective ionization of an analyte versus the background. Using NSI Conventional electrospray (ESI) employs flow rates from 1 μL/min to 1 mL/min. Due to the high volume of liquid exiting the emitter, a drying gas, thermal heating, or both are often required to expedite desolvation and droplet shrinkage. NSI (or nanoESI) is a form of ESI that employs low flow rates of 10–1000 nL/min. NSI generally does not require a drying gas or thermal heating. Compared with ESI or H-ESI, NSI tolerates a wider range of liquid compositions including pure water. As you lower the flow rate, a lower volume of mobile phase passes through the emitter, producing smaller aerosol droplets. This makes NSI more effective than conventional ESI or H-ESI at concentrating the analyte at the emitter tip, producing significant increases in sensitivity demonstrated by the signal response of the mass spectrometer. See Table 7 for guidelines. LC Flow Rates Ranges The H-ESI spray insert can volatilize ions from liquid flows1 of 1–1000 μL/min. This flow rate range provides for a wide range of separation techniques: CE, CEC, analytical LC, capillary LC, and microbore LC. The APCI spray insert can volatilize ions from liquid flows2 of 200–2000 μL/min. This flow range provides for the use of separation techniques: analytical LC, microbore LC, and semi-preparative LC. While changing the flow rate of solvents entering the mass spectrometer, adjust the following parameters: • For H-ESI mode, adjust the spray voltage, the ion transfer tube and vaporizer temperatures, and the flow rates for the sheath, auxiliary, and sweep gases. • For APCI mode, adjust the corona discharge current, ion transfer tube and vaporizer temperatures, and the flow rates for the sheath, auxiliary, and sweep gases. • For NSI mode, adjust the spray voltage and the ion transfer tube temperature. 1 The H-ESI spray insert can generate ions from liquid flows as low as 1 μL/min. However, flows below 5 μL/min require more care. 2 For the APCI spray insert, flows below 200 μL/min require more care to maintain a stable spray. 4 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 1 Introduction LC Flow Rates Ranges The following tables list the guidelines (default parameter values for the API source) for system operation using H-ESI (Table 5), APCI (Table 6), and NSI (Table 7) for a range of LC solvent flow rates. Table 5. Guidelines for setting operating parameters for LC/H-ESI/MS LC flow rate (μL/min) Spray voltage (V)a Sheath gas (arbb units) Auxiliary gas (arb units) Sweep gas (arb units) Ion transfer tube temperature (°C) Vaporizer temperature (°C) Typical nitrogen gas consumption (L/min) 5 Pos: 3500 Neg: –2500 5 5 0 275 20 Less than 1 200 Pos: 3500 Neg: –2500 35 10 0 325 275 8 500 Pos: 3500 Neg: –2500 50 15 2 350 400 13 1000 Pos: 3500 Neg: –2500 60 20 2 380 500 17 a Positive and negative polarity modes b Arbitrary Table 6. Guidelines for setting operating parameters for LC/APCI/MS LC flow rate (μL/min) Sheath gas (arba units) Auxiliary gas (arb units) Sweep gas (arb units) Ion transfer tube temperature (°C) Vaporizer temperature (°C) Corona discharge current (μA)b 200 25 5 0 250 325 Pos: 4 Neg: –10 1000 45 5 2 275 500 Pos: 4 Neg: –10 a Arbitrary b Positive and negative polarity modes Table 7. Guidelines for setting operating parameters for LC/NSI/MS Spray voltage (V) Sweep gas (arbitrary units) Ion transfer tube temperature (°C) Positive mode: 1200 Negative mode: –600 0 325 Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 5 1 Introduction Types of Buffers Note Use the Ion Source page of the Ion Source pane to determine the initial spray voltage, sheath gas pressure, auxiliary gas pressure, sweep gas pressure, ion transfer tube temperature, and vaporizer temperature for the LC flow rate of your experiment. See “Determining the Initial API Source Settings” on page 55. Use the Ion Source – Optimization page to optimize the spray voltage, sheath gas pressure, auxiliary gas pressure, and sweep gas pressure for your experiment. See “Determining the Initial API Source Settings” on page 55. Types of Buffers Many LC applications use nonvolatile buffers such as phosphate and borate. Avoid using nonvolatile buffers because they can cause salt buildup in parts of the API source, such as the ion transfer tube and nozzle of the spray insert. Using nonvolatile buffers without also cleaning the API source to remove salt deposits might compromise the integrity of the spray. For LC/MS experiments, replace nonvolatile buffers with the following volatile buffers: • Acetic acid • Ammonium acetate • Ammonium formate • Ammonium hydroxide • Formic acid • Triethylamine (TEA) For a list of recommended solvents, refer to the TSQ Endura and TSQ Quantiva Preinstallation Requirements Guide. CAUTION Avoid exposure to potentially harmful materials. By law, producers and suppliers of chemical compounds are required to provide their customers with the most current health and safety information in the form of Material Safety Data Sheets (MSDSs) or Safety Data Sheets (SDSs). The MSDSs and SDSs must be freely available to lab personnel to examine at any time. These data sheets describe the chemicals and summarize information on the hazard and toxicity of specific chemical compounds. They also provide information on the proper handling of compounds, first aid for accidental exposure, and procedures to remedy spills or leaks. Read the MSDS or SDS for each chemical you use. Store and handle all chemicals in accordance with standard safety procedures. Always wear protective gloves and safety glasses when you use solvents or corrosives. Also, contain waste streams, use proper ventilation, and dispose of all laboratory reagents according to the directions in the MSDS or SDS. 6 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 1 Introduction Method Editor For LC applications that require nonvolatile buffers, follow these guidelines for best performance: • Optimize the spray insert position. • Install the mass spectrometer’s optional ion sweep cone. • Reduce the concentration of buffers to an absolute minimum. Note You might need to increase the frequency of API source maintenance when you use nonvolatile buffers. Method Editor Use the Method Editor (Figure 1) that opens in the Xcalibur Instrument Setup window to create an instrument method for your experiment. To save time entering the parameters for an instrument method, open the system template designed for the experiment type that you want to perform, enter the parameters specific to the experiment, and then save the entries as part of an Xcalibur instrument method (.meth file name extension). For additional information, refer to the Xcalibur Instrument Setup Help. Figure 1. Thermo Xcalibur Instrument Setup window showing the scan types Scan Types icon bar Thermo Scientific Mass table Properties pane TSQ Endura and TSQ Quantiva Getting Started Guide 7 1 Introduction Method Editor 8 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 2 Setting Up the API Source This chapter provides information about setting up the API source for H-ESI, APCI, and APPI experiments. The API source ships with the mass spectrometer and consists of the source housing, a heater assembly, and the H-ESI spray insert. For APCI experiments, order the APCI Installation Kit (P/N OPTON-30159), which includes the APCI spray insert. For APPI experiments, order the APPI Interface Kit (P/N OPTON-30185). IMPORTANT For H-ESI, APCI, and APPI experiments, install the correct API source as follows: • TSQ Endura MS—Use the Thermo Scientific EASY-Max NG™ API source. • TSQ Quantiva MS—Use the Thermo Scientific Ion Max NG™ API source. For NSI experiments, use one of the compatible Thermo Scientific nanospray sources. Contents • Preparing the Mass Spectrometer • Installing or Removing the API Source • Preparing the Spray Insert for the API Source Preparing the Mass Spectrometer Before you install the API source, install or remove the ion sweep cone as specified in the following procedure. IMPORTANT For best results, wear clean gloves before you handle the API source’s spray insert or the mass spectrometer’s ion sweep cone. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 9 2 Setting Up the API Source Preparing the Mass Spectrometer To prepare the mass spectrometer 1. Complete all data acquisition, if any. 2. Open the Tune window (see page 68). 3. Place the mass spectrometer in Off mode (see page 69). The LC/MS system is now in off mode. After the API source housing, spray insert, or both have cooled to room temperature, you can safely remove these components. Note Always place the system in off mode before removing the spray insert or the API source housing. When the system is in off mode, the API gases, high voltage, and syringe pump are off. 4. If you want to change the installed API source, wait until it has cooled to room temperature. For instructions on how to remove the API source, refer to the Ion Max NG and EASY-Max NG Ion Sources User Guide. CAUTION Hot surface. Avoid touching the API source housing when the mass spectrometer is in operation. The external surface of the EASY-Max NG API source housing can become hot enough to burn your skin. 5. Depending on the ionization mode, do the following (Figure 2): • For H-ESI, APCI, or APPI mode, install the ion sweep cone over the mass spectrometer’s spray cone (Figure 2). • For NSI mode, remove the ion sweep cone from the mass spectrometer by grasping its outer ridges and pulling it off. 10 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 2 Setting Up the API Source Installing or Removing the API Source Figure 2. MS API source mount assembly and ion sweep cone Sheath gas line to the API source Auxiliary gas line to the API source Spray cone (do not remove) Ion sweep cone (remove for NSI mode only) Source drain routes to the back of the mass spectrometer. Installing or Removing the API Source The EASY-Max NG source holds the H-ESI or APCI spray insert. All the wiring and gas plumbing for this API source are internal. This means you can install or remove the API source or change the spray insert—all without the use of tools. Note For instructions on how to configure the EASY-Max NG source for H-ESI, APCI, or APPI mode, refer to Chapter 3 in the Ion Max NG and EASY-Max NG Ion Sources User Guide. The TSQ Endura and TSQ Quantiva MS internally routes the solvent waste from the bottom of the API source to the back Drain/Waste port. This section provides the following procedures: • Installing the API Source • Removing the API Source Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 11 2 Setting Up the API Source Installing or Removing the API Source Installing the API Source Complete the appropriate procedure: • To install the EASY-Max NG API source (instrument calibration and experiments) • To install the NSI source (experiments) CAUTION Use these guidelines for the API source drain: • Use the Tygon™ tubing provided with the solvent waste container to connect the solvent waste container to a fume exhaust system. • To prevent solvent waste from backing up into the mass spectrometer, make sure that all Tygon tubing is above the level of liquid in the waste container as follows: – From the mass spectrometer to the solvent waste container – From the waste container to the exhaust system Equip your lab with at least two fume exhaust systems: • The analyzer optics become contaminated if the drain/waste tubing and the exhaust tubing from the forepump connect to the same fume exhaust system. Route the exhaust tubing from the forepump to a dedicated fume exhaust system. – 12 Do not vent the Tygon drain tube (or any vent tubing connected to the waste container) to the same fume exhaust system that connects to the forepump. Vent the waste container to a dedicated fume exhaust system. The exhaust system for the API source must accommodate a flow rate of up to 30 L/min (64 ft3/h). TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 2 Setting Up the API Source Installing or Removing the API Source To install the EASY-Max NG API source 1. Follow the procedure To prepare the mass spectrometer. 2. For APCI mode, check that the corona discharge needle assembly is installed in the API source housing. For instructions, refer to the Ion Max NG and EASY-Max NG Ion Sources User Guide. 3. Unlock the source’s locking levers (down position, Figure 3). Figure 3. EASY-Max NG API source with H-ESI spray insert (top, front view) H-ESI spray insert Locking lever (down unlocked position) Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 13 2 Setting Up the API Source Installing or Removing the API Source 4. Align the two guide pin holes on the back of the source with the guide pins on the front of the mass spectrometer (Figure 4), and then carefully press the source onto the mass spectrometer. Figure 4. API source connection to the MS mount assembly (installed ion sweep cone) High voltage (HV) connection Guide pins on the MS API source mount assembly (left) and guide pin holes on the back of the source (right) 5. Lock the source’s locking levers (up position). 6. To switch between ionization modes, refer to the Ion Max NG and EASY-Max NG Ion Sources User Guide. 7. Verify that the solvent waste system connects to the back Drain/Waste port. During the initial installation of the mass spectrometer, a Thermo Fisher Scientific service engineer installs the solvent waste system. For instructions, refer to the TSQ Endura and TSQ Quantiva Getting Connected Guide. To install the NSI source 1. Follow the procedure To prepare the mass spectrometer. 2. For additional instructions, refer to the NSI source manual. 14 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 2 Setting Up the API Source Preparing the Spray Insert for the API Source Removing the API Source To access the ion sweep cone, API source interface, ion transfer tube, internal APCI corona needle (APCI-configured source), or internal APPI lamp (APPI-configured source), you must remove the API source from the mass spectrometer. To remove the API source 1. Complete all data acquisition, if any. 2. Turn off the liquid flow from the LC (or other sample introduction device) to the API source. 3. In the Tune window, place the mass spectrometer in Off mode (see page 69). CAUTION Hot surface. The maximum safety limit for heated surfaces is 70 °C (158 °F). Although the source falls below this maximum, it can still severely burn you. Allow the source to cool to room temperature (approximately 20 minutes) before you touch it. 4. Disconnect the sample line from the grounding union or spray insert, as applicable. 5. Unlock the source’s locking levers. For the EASY-Max NG API source, see Figure 3. 6. Pull the source straight off of the mass spectrometer. 7. Place the source in a safe location for temporary storage. Preparing the Spray Insert for the API Source For detailed instructions, refer to the Ion Max NG and EASY-Max NG Ion Sources User Guide. • Installing the Spray Insert • Adjusting the Spray Direction Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 15 2 Setting Up the API Source Preparing the Spray Insert for the API Source Installing the Spray Insert For H-ESI mode, install the H-ESI spray insert and turn on the source heater. For APCI mode, install the APCI spray insert. For APPI mode, you can use either of the spray inserts; refer to “Spray Insert Selection” in Chapter 1 of the Ion Max NG and EASY-Max NG Ion Sources User Guide. IMPORTANT For the calibration procedures and experiments with flow rates that are less than 50 μL/min, make sure that the H-ESI spray insert contains the low-flow metal needle insert (P/N 80000-60152). To install the spray insert 1. Follow the procedure “Installing the API Source.” 2. To switch between ionization modes or change the needle insert, refer to the Ion Max NG and EASY-Max NG Ion Sources User Guide. Adjusting the Spray Direction To maximize sensitivity or robustness, you can adjust the spray direction by a few millimeters. Typically, you adjust the spray direction while optimizing the API source parameters for the analytes. Make the adjustment according to the guidelines in Table 8. Note The depth and angle of the spray insert and heater assembly are not adjustable. Table 8. Guidelines for adjusting the heater and spray insert position Adjustment control Description Front-to-back position 1 For H-ESI mode, use position 1 for calibrating the mass spectrometer and for low liquid flow rates (less than 50 μL/min). In position 1, the spray is closest to the entrance of the mass spectrometer. 2 Use position 2 (default) for liquid flow rates greater than 50 μL/min. 3 Use position 3 for enhanced robustness, for example, when you use a biological matrix. In position 3, the spray is farthest from the entrance of the mass spectrometer. Rotational position Left, center, right (marks) 16 TSQ Endura and TSQ Quantiva Getting Started Guide Use the center mark to position the spray closest to the entrance of the mass spectrometer. Thermo Scientific 2 Setting Up the API Source Preparing the Spray Insert for the API Source To adjust the spray direction CAUTION Hot surface. Avoid touching the API source housing when the mass spectrometer is in operation. The external surface of the housing can become hot enough to burn your skin. Allow the housing to cool before you touch it. 1. Loosen the top two retainer knobs that secure the heater assembly. 2. Do any of the following (see Table 8): • Move the heater forward or backward to the desired position (Figure 5). Figure 5. Front-to-back adjustment Retainer knob for the spray insert and the heater Front-to-back position indicator Front-to-back • Turn the side rotational adjustment knob to rotate the heater (Figure 6). Figure 6. Rotational adjustment Rotational position indicator Rotational adjustment knob 3. Using your fingers, tighten the top two retainer knobs to secure the heater assembly in its new position. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 17 2 Setting Up the API Source Preparing the Spray Insert for the API Source 18 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 3 Connecting the Inlet Plumbing This chapter provides information about the sample introduction techniques and how to set up the inlet plumbing for these techniques. For operational information about the syringe pump and divert/inject valve, see Chapter 4, “Using the Syringe Pump and Divert/Inject Valve.” The MS Calibration Kit (see Table 1) and Performance Specification Kit (see Table 2) contain the required components for the inlet plumbing connections. Contents • Sample Introduction Techniques • Plumbing Connections • Setting Up the Syringe Pump • Setting Up the Inlet Plumbing • Setting Up the Inlet for an LC/MS System with an Autosampler • Connecting the Grounding Union to the H-ESI Spray Insert Sample Introduction Techniques The TSQ Endura and TSQ Quantiva MSs have an external syringe pump and divert/inject valve. The following techniques are available to introduce samples into the API source: • Direct Infusion • High-Flow Infusion • Loop Injection (Flow-Injection Analysis) • High-Performance Liquid Chromatography (HPLC) with an Autosampler Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 19 3 Connecting the Inlet Plumbing Sample Introduction Techniques Figure 7 shows schematic drawings of these sample introduction techniques. IMPORTANT Compound optimization solutions, such as the reserpine sample solution, can contaminate your system at high concentrations. For best results, use the LC flow technique of automatic-loop injection to introduce optimization solutions into the mass spectrometer. Direct Infusion The direct infusion technique uses the syringe pump to infuse sample directly into the API source. Use this technique to introduce the calibration solution for calibrating in H-ESI mode. You can also use this technique to introduce a solution of pure analyte at a steady rate for qualitative analyses and perform experiments at a low flow rate with the syringe pump. For plumbing instructions, see “Setting Up the Inlet for Direct Infusion.” High-Flow Infusion The high-flow infusion technique uses an LC union Tee to direct the solvent flow from the syringe pump into the solvent flow produced by an LC pump. The combined solvent flow goes through the divert/inject valve into the API source. Use this infusion method to perform experiments at a higher flow rate with an LC system. The high-flow infusion method puts a comparatively large amount of solvent into the mass spectrometer, which means you might need to clean the ion spray cone more frequently. When the divert/inject valve is in the Load position, solvent flow from the LC pump enters the valve through port 6 and exits the valve through port 5, which connects to the API source. When the divert/inject valve is in the Inject position, solvent flow from the LC pump enters the valve through port 6 and exits the valve through port 1 to waste. For plumbing instructions, see “Setting Up the Inlet for High-Flow Infusion.” For information about the valve configurations, see “Configurations.” 20 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 3 Connecting the Inlet Plumbing Sample Introduction Techniques Loop Injection (Flow-Injection Analysis) Use the loop injection technique when there is a limited amount of sample. To use this technique, attach a sample loop, an injection port fitting, and an LC pump to the divert/inject valve, and then connect the divert/inject valve to the API source. With the valve in the Load position, use a syringe to load sample through the injection port fitting into the sample loop, and then switch the position of the inject valve to the Inject position. Switching the valve to the Inject position allows the solvent flow from the LC pump to backflush the sample out of the loop and into the API source. Additionally, follow these guidelines: • Use a manual loop injection without chromatographic separation for qualitative or quantitative analysis when there is a limited amount of a pure sample. • Use a manual loop injection with chromatographic separation for qualitative or quantitative analysis when there is a limited amount of a sample mixture. Requires an LC column between the injection valve and the API source. • Use an automatic loop injection to optimize the mass spectrometer’s sensitivity to a compound for an MS/MS experiment. For plumbing instructions, see “Setting Up the Inlet for Manual or Auto-Loop Injections.” High-Performance Liquid Chromatography (HPLC) with an Autosampler To perform loop injection by using the liquid chromatography (LC) technique, install an LC column between the sample inlet of the API source and port 6 of the divert/inject valve, or connect an LC system with an autosampler to the mass spectrometer. To automatically inject a set of samples, connect an LC system with an autosampler to the divert/inject valve and connect the divert/inject valve to the API source. Use the autosampler to inject sample solution into the flow from an LC pump. In a typical LC/MS experiment, direct the solvent flow through an LC column to separate the compounds of a mixture before they are directed into the API source. For plumbing instructions, see “Setting Up the Inlet for an LC/MS System with an Autosampler.” Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 21 3 Connecting the Inlet Plumbing Sample Introduction Techniques Figure 7. Schematics of the sample introduction techniques (examples) Legend Red PEEK tubing Teflon FEP tubing Direct infusion Syringe pump High-flow infusion (divert valve) API source LC pump Waste 2 1 6 3 4 Syringe pump 5 API source Manual loop injection (loop injector) LC pump 2 1 6 3 Waste 4 5 API source Automatic loop injection (loop injector) Syringe pump LC pump 2 1 6 3 Waste 4 5 API source HPLC with autosampler (divert valve) 2 1 6 3 4 Waste 5 Column Autosampler LC pump API source 22 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 3 Connecting the Inlet Plumbing Plumbing Connections Plumbing Connections The modular divert/inject valve shipped with your order is a six-port, two-position, Rheodyne™ injection valve. The ports use standard 10-32 fittings for high-pressure and 1/16 in. OD tubing. To connect the high-pressure tubing to the valve, use the one-piece fingertight fittings provided in the MS Calibration Kit (see Table 1 in the Preface). IMPORTANT To help ensure spray stability, make sure that all PEEK tubing is not crimped, kinked, or otherwise damaged. Ensure the following when you make the plumbing connections: • The ends of the PEEK tubing are squarely cut (Figure 8). For best results, use a polymeric tubing cutter to ensure square cuts. Poorly cut tubing can cause flow restrictions. • The PEEK tubing makes contact with the bottom of the receiving port. Tubing that is not properly seated can add dead volume to a chromatographic system. • The fittings are not overtightened. Tighten the PEEK fittings by using your fingers only, not a wrench. Overtightening the PEEK fittings can cause leaks. Figure 8. Proper connection for the PEEK tubing and fitting (syringe adapter assembly) Fingertight PEEK fitting Red PEEK tubing Union with 10-32, coned-bottom, receiving port Properly seated, square-cut end Setting Up the Syringe Pump Use the syringe pump to directly infuse sample into the API source, to infuse sample into the solvent stream that is produced by an LC pump, or to automatically load sample into the divert/inject valve. IMPORTANT To minimize the possibility of cross-contamination, do the following: • Use a different syringe and length of PEEK tubing for each type of solution. • Wipe off the needle tip with a clean, lint-free tissue before reinserting the syringe into the syringe adapter assembly. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 23 3 Connecting the Inlet Plumbing Setting Up the Inlet Plumbing To set up the syringe for infusion or high-flow infusion experiments 1. Load a clean, 500 μL syringe with the sample solution. CAUTION Sharp object. The syringe needle can puncture your skin. Handle it with care. 2. Using one of the two-piece, fingertight fittings, connect a 4 cm (1.5 in.) length of Teflon tubing to the (black) LC union (Figure 9). The LC union has a 10-32, coned-bottom receiving port. Figure 9. Plumbing connection for the syringe LC union Fingertight fitting Fingertight ferrule Syringe Teflon tube 3. Hold the plunger of the syringe in place and carefully insert the tip of the syringe needle into the free end of the tubing. Note If necessary, use the syringe needle tip to enlarge the opening slightly in the end of the tubing. 4. Place the syringe into the syringe holder of the syringe pump. 5. Squeeze the release button on the syringe pump’s pusher block and slowly move the pusher block until it contacts the syringe plunger. Setting Up the Inlet Plumbing This section describes how to set up the inlet plumbing for the following techniques: • Setting Up the Inlet for Direct Infusion • Setting Up the Inlet for High-Flow Infusion • Setting Up the Inlet for Manual or Auto-Loop Injections IMPORTANT To help ensure spray stability, make sure that all PEEK tubing is not crimped, kinked, or otherwise damaged. 24 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 3 Connecting the Inlet Plumbing Setting Up the Inlet Plumbing Setting Up the Inlet for Direct Infusion Figure 10 shows the inlet plumbing connections to introduce sample into the API source by using direct infusion. For instrument calibration, remember to use the natural PEEK tubing. To connect an infusion line between the LC union and the grounding union 1. Set up the syringe pump; see “Setting Up the Syringe Pump.” 2. Use red PEEK tubing (infusion line) to connect the LC union to the grounding union as follows (Figure 10): • Using a two-piece fingertight fitting, connect one end of the tubing to the free end of the LC union that connects to the syringe. • Using a two-piece fingertight fitting, connect the other end to the grounding union. 3. Follow the procedure in “Connecting the Grounding Union to the H-ESI Spray Insert.” This completes the inlet setup for the direct infusion technique. Figure 10. Plumbing connections for direct infusion (H-ESI mode) Two-piece, two-wing fingertight fitting Syringe LC union and fingertight fitting Red PEEK tubings Two-piece fingertight fitting Sample inlet (part of the source) Grounding union holder (part of the source) For APCI mode, this path through the grounding union is optional. (The parts are in the Source LC Connection Kit.) Grounding union Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 25 3 Connecting the Inlet Plumbing Setting Up the Inlet Plumbing Setting Up the Inlet for High-Flow Infusion Table 9 lists the plumbing connections required to set up the system for a high-flow infusion experiment. (You can make the connections in any order.) Table 9. Connections for high-flow infusion Connection Location Reference 1 Connect the syringe to the union Tee. Connecting the Syringe to the Union Tee 2 Connect the LC pump to the union Tee. Connecting the LC pump to the Union Tee 3 Connect the union Tee to the divert/inject Connecting the Union Tee to valve. the Divert/Inject Valve 4 Connect port 1 of the divert/inject valve to Connecting the Divert/Inject a waste container. Valve to a Waste Container 5 For H-ESI mode, connect the union Tee to Connecting the Union Tee to the grounding union. For APCI mode, the API Source connect the union Tee directly to the sample inlet. 6 For H-ESI mode, connect the grounding union to the sample inlet of the H-ESI spray insert. Connecting the Grounding Union to the H-ESI Spray Insert Connecting the Syringe to the Union Tee To connect the syringe to the union Tee 1. Set up the syringe pump; see “Setting Up the Syringe Pump.” 2. Use red PEEK tubing (infusion line) to connect the LC union to the union Tee as follows (Figure 11): • Using a two-piece fingertight fitting, connect one end of the tubing to the free end of the LC union that connects to the syringe. • Using a two-piece fingertight fitting, connect the other end to the union Tee. 26 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 3 Connecting the Inlet Plumbing Setting Up the Inlet Plumbing Figure 11. Plumbing connection between the LC union and the union Tee Two-piece fingertight fitting LC union Union Tee Two-piece fingertight fitting Red PEEK tubing Connecting the LC pump to the Union Tee To connect the LC pump to the divert/inject valve • Using an appropriate fitting, connect a length of red PEEK tubing to the outlet of the LC pump. • Using a two-piece fingertight fitting, connect the other end of the tubing to the union Tee (Figure 12). Figure 12. Plumbing connection between the union Tee and the divert/inject valve Connect this end to the LC pump. Union Tee Sample input to port 6 on the divert/inject valve Two-piece fingertight fitting to union Tee One-piece fingertight fitting to port 6 Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 27 3 Connecting the Inlet Plumbing Setting Up the Inlet Plumbing Connecting the Union Tee to the Divert/Inject Valve To connect the union Tee to the divert/inject valve • Using a one-piece fingertight fitting, connect a length of red PEEK tubing to the union Tee (Figure 12). • Using a one-piece fingertight fitting, connect the other end of the tubing to port 6 of the divert/inject valve. Connecting the Divert/Inject Valve to a Waste Container To connect the divert/inject valve to a waste container • Using a one-piece fingertight fitting, connect a length of the Teflon tubing to port 1 of the divert/inject valve. • Insert the other end of the tubing into a suitable waste container. Connecting the Union Tee to the API Source To connect the union Tee to the API source 1. Using a fingertight fitting and a ferrule, connect a length of red PEEK tubing to the union Tee (Figure 13). Figure 13. Plumbing connection between the union Tee and the grounding union Union Tee Red PEEK tubing Two-piece fingertight fitting Grounding union 28 TSQ Endura and TSQ Quantiva Getting Started Guide Two-piece fingertight fitting Thermo Scientific 3 Connecting the Inlet Plumbing Setting Up the Inlet Plumbing 2. Do one of the following to connect the other end of the tubing: • For H-ESI mode, use a two-piece fingertight fitting to connect the other end of the tubing to the grounding union (Figure 10). For instructions on how to connect the other end of the grounding union, see “Connecting the Grounding Union to the H-ESI Spray Insert.” –or– • For APCI mode, use a two-piece fingertight fitting to connect the other end of the tubing directly to the sample inlet of the APCI spray insert. Note The plumbing path through the grounding union of the API source is optional for APCI mode. A knurled nut secures the grounding bar to the API source housing. You do not need to remove the grounding bar if you choose not to use that plumbing path in the APCI mode. This completes the inlet setup for the high-flow infusion technique. Setting Up the Inlet for Manual or Auto-Loop Injections Figure 14 shows the inlet plumbing connection to introduce sample into the API source by using manual or auto-loop injection. To set up the inlet for loop injections 1. Do one of the following: • To load sample automatically with the syringe pump, set up the syringe pump; see “Setting Up the Syringe Pump.” Using a red PEEK infusion line, make the following connections: – Using a two-piece fingertight fitting, connect one end of the infusion line to the free end of the LC union that connects to the syringe. – Using a one-piece fingertight fitting, connect the other end to port 2 of the divert/inject valve. –or– • To load sample manually with a hand-held syringe, connect the needle port to port 2 of the divert/inject valve (Figure 14). Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 29 3 Connecting the Inlet Plumbing Setting Up the Inlet Plumbing Figure 14. Divert/inject valve setup for manual loop injection Port 2 to a loop filler (needle port) Sample loop from port 1 to port 4 Port 6 to the LC pump 1 3 Port 3 to a waste container 6 4 5 Port 5 to the grounding union (H-ESI mode) or the spray insert’s sample inlet (APCI mode) 2. Connect a sample loop from port 1 to port 4 of the divert/inject valve. 3. Use red PEEK tubing to connect port 6 of the divert/inject valve to the LC pump as follows: • Using an appropriate fitting and ferrule, connect one end of the tubing to the outlet of the LC pump. • Using a one-piece fingertight fitting, connect the other end to port 6 of the divert/inject valve. 4. Connect port 5 of the divert/inject valve to the API source: a. Using a one-piece fingertight fitting, connect a length of red PEEK tubing to port 5 of the divert/inject valve. b. Depending on whether you installed the H-ESI or APCI spray insert, do one of the following: • For H-ESI mode, use a two-piece fingertight fitting to connect the other end of the red PEEK tubing that connects to port 5 of the divert/inject valve to the grounding union. For instructions on how to connect the other end of the grounding union, see “Connecting the Grounding Union to the H-ESI Spray Insert.” –or– • For APCI mode, connect the other end of the red PEEK tubing to the sample inlet of the APCI spray insert (Figure 15). Or, you can connect the tubing to the installed grounding union and associated flow path (Figure 10 or Figure 16). 30 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 3 Connecting the Inlet Plumbing Setting Up the Inlet for an LC/MS System with an Autosampler Figure 15. Plumbing connections for manual loop injection (APCI mode) Loop filler (needle port) APCI spray insert Port 6 of the divert/inject valve to the LC pump Port 3 to waste Port 5 of the divert/inject valve to the sample inlet of the APCI spray insert 5. Use Teflon tubing to connect port 3 of the divert/inject valve to a waste container as follows: • Using a Rheodyne fitting, connect one end of the tubing to port 3 of the divert/inject valve. • Place the other end into an appropriate waste container. This completes the inlet setup for the manual and auto-loop injection techniques. Setting Up the Inlet for an LC/MS System with an Autosampler This section describes how to connect the inlet plumbing to introduce sample into the API source from the autosampler in an LC system. To connect the inlet plumbing for an LC/MS system with an autosampler 1. Use red PEEK tubing to connect port 6 of the divert/inject valve to the outlet of the autosampler as follows: • Using an appropriate fitting and ferrule, connect one end of the tubing to the outlet of the autosampler. • Using a one-piece fingertight fitting, connect the other end to port 6 of the divert/inject valve. 2. Use Teflon tubing to connect port 1 of the divert/inject valve to a waste container as follows: • Using a Rheodyne fitting, connect one end of the tubing to port 1 of the divert/inject valve. • Place the other end into an appropriate waste container. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 31 3 Connecting the Inlet Plumbing Connecting the Grounding Union to the H-ESI Spray Insert 3. Do one of the following to connect port 5 of the divert/inject valve to the API source: • For H-ESI mode, use a one-piece fingertight fitting to connect a length of red PEEK tubing between port 5 of the divert/inject valve and the grounding union. For instructions on how to connect the other end of the grounding union, see “Connecting the Grounding Union to the H-ESI Spray Insert.” –or– • For APCI mode, use a one-piece fingertight fitting to connect a length of red PEEK tubing between port 5 of the divert/inject valve and the sample inlet of the APCI spray insert. This completes the inlet setup for using an autosampler in an LC/MS system. Connecting the Grounding Union to the H-ESI Spray Insert Figure 16 shows the connection between the grounding union and the sample inlet of the H-ESI spray insert. The grounding union is not required for the APCI mode plumbing. CAUTION To prevent electric shock, verify that the grounding union is made of stainless steel. A grounding union made of a nonconductive material, such as PEEK, creates an electric shock hazard. Figure 16. Plumbing connections for the grounding union (H-ESI mode) For APCI mode, the grounding union and this plumbing path are not required. H-ESI spray insert Sample input Grounding union installed in the grounding union holder (bar) API source housing 32 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 4 Using the Syringe Pump and Divert/Inject Valve This chapter describes the external syringe pump and divert/inject valve that ship with the TSQ Endura or TSQ Quantiva mass spectrometer. For information about installing these components, refer to the TSQ Endura and TSQ Quantiva Getting Connected Guide. Contents • Syringe Pump • Divert/Inject Valve Syringe Pump The external Chemyx™ Fusion 100T syringe pump delivers sample solution from an installed syringe, through the sample transfer line (red PEEK), and into the API source. The motorized pusher block (Figure 17) depresses the syringe plunger at the flow rate specified in the data system. (The default flow rate for calibration is 5 μL/min.) You can start and stop the syringe pump from the data system; refer to the data system Help for instructions. You can also start and stop the syringe pump by pressing the syringe pump buttons. Note If you choose to provide a syringe pump other than the Fusion 100T, ensure that it can provide a steady, continuous flow of 1–5 μL/min. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 33 4 Using the Syringe Pump and Divert/Inject Valve Divert/Inject Valve Figure 17. Syringe pump setup (top view) Teflon tubing Fingertight fittings LC union, internal view Syringe pump Red PEEK tubing Pusher block Release knob Syringe holder Syringe Divert/Inject Valve The external Rheodyne MX Series II™ divert/inject valve is a 6-port motorized valve that switches between two positions. In the first position, port one connects internally to port two, port three connects to port four, and port five connects to port six. In the second position, the valve rotates one position so that port one connects internally to port six, port two connects to port three, and port four connects to port five. Figure 18 shows the valve’s internal flow paths for both positions. The Method Editor in the Xcalibur application identifies the valve’s two positions as “1–2” (port 1 to 2) and “1–6” (port 1 to 6). 34 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 4 Using the Syringe Pump and Divert/Inject Valve Divert/Inject Valve Figure 18. Divert/inject valve positions Internal connection path (light gray) 2 1 2 Port 1 internally switches between port 2 (position 1–2) and port 6 (position 1–6, shown). 1 Valve screw 6 3 4 5 Position 1–2 3 6 4 5 Position 1–6 Configurations You can configure (plumb) the divert/inject valve as a loop injector (for flow injection analysis) or as a divert valve. The divert valve can switch the solvent front, gradient endpoint, or any portion of the LC run to waste. Figure 19 shows both of these configurations. In the loop injector valve configuration, the valve switches between these two positions: • Load (position 1–2)—The sample loop is isolated from the solvent stream. Solvent flow from the LC pump enters and exits the valve through ports five and six, respectively. When you load the sample into port two, the sample enters and exits the sample loop through ports one and four, respectively. As you overfill the sample loop, the excess sample exits the valve through port three to waste. • Inject (position 1–6)—The sample loop is open to the solvent stream. The solvent flow from the LC pump flushes sample out of the sample loop, and then exits through port six into the API source. In the divert valve configuration, the valve switches between these two positions: • Detector (position 1–2)—Solvent flow from the LC pump enters the valve through port five and exits through port six into the API source. • Waste (position 1–6)—Solvent flow from the LC pump enters the valve through port five and exits through port four to waste. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 35 4 Using the Syringe Pump and Divert/Inject Valve Divert/Inject Valve Figure 19. Divert/inject valve plumbed as a loop injector and as a divert valve Sample loop Sample input 1 2 6 3 Waste 4 1 2 6 3 4 5 5 API source API source Waste LC pump LC pump Loop injector (Position 1–2 with load configuration) Divert valve (Position 1–2 with detector configuration) Controlling the Divert/Inject Valve You can control the divert/inject valve as follows: • Use the mass spectrometer’s data system to specify the parameters in the Divert Valve Properties pane of the Method Editor. For instructions, refer to the Xcalibur Method Editor Help. • Use the valve’s control buttons (Figure 20) to divert the LC flow between the mass spectrometer and waste when the valve is in the divert valve configuration, or switch between load and inject modes when the valve is in the loop injector configuration. For instructions, refer to the manufacturer’s manual. Figure 20. Divert/inject valve (front view) Valve position indicator Six-port, two-position valve Valve control buttons 36 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 5 Preparing the System for Calibration This chapter describes how to prepare the TSQ Endura or TSQ Quantiva system before you calibrate the mass spectrometer. IMPORTANT You must pump down the instrument for the full 15 hours before you start the instrument calibration process. Contents • Pumping Down the Mass Spectrometer • Setting Up the Syringe Pump for Direct Infusion • Setting Up the Mass Spectrometer for Calibration Pumping Down the Mass Spectrometer To help optimize the performance of the mass spectrometer, pump down the vacuum system for at least 15 hours. To pump down the mass spectrometer 1. Check that the forepumps’ exhaust tubing connect to the exhaust system and that any valves in the exhaust path are open. 2. Turn on the forepumps’ power switches. 3. Place the electronics service switch in the Service Mode (down) position (Figure 21). Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 37 5 Preparing the System for Calibration Pumping Down the Mass Spectrometer Figure 21. Power entry module (right side of the instrument) Electronics service switch Main Power switch 4. Turn on the Main Power switch. The LEDs on the front panel remain off. 5. Verify that the forepumps are running and that there are no leaks in the connection between the forepumps and the mass spectrometer. 6. Wait 1 hour. 7. Place the electronics service switch in the Operating Mode (up) position. The mass spectrometer’s restart sequence begins. The Power LED on the front panel turns green, and the Vacuum, Communication, and System LEDs remain off. When the instrument startup process is complete, the Vacuum and Communication LEDs are green and the System LED is yellow. 8. Open the Status pane in the Tune window (see page 68), click the downward arrow, and then choose By Board (Figure 22). 38 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 5 Preparing the System for Calibration Pumping Down the Mass Spectrometer Figure 22. By Board page in the Status pane Opens and closes the selected pane. Includes the source vacuum gauges. Click to select By Function or By Board. 9. Check the readback values for the source pressure gauges as follows: • Double-click Source Board, and then verify that the Source Pressure and Analyzer Pressure readback values are below the operating threshold limits (see Table 10). Table 10. Threshold limits for the vacuum pressure gauges Instrument Source pressure (Torr) TSQ Endura 2.5 TSQ Quantiva 4.5 Analyzer pressure (Torr) 9.0 × 10–6 Normal readback measurements show a green square ( ). If the vacuum pressure values are normal, follow the next procedure “Setting Up the Syringe Pump for Direct Infusion.” Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 39 5 Preparing the System for Calibration Setting Up the Syringe Pump for Direct Infusion Setting Up the Syringe Pump for Direct Infusion Use the syringe pump to infuse the calibration solution directly into the H-ESI source. For information about the syringe pump, see “Syringe Pump” on page 33. IMPORTANT To minimize the possibility of cross-contamination, use a different syringe and length of PEEK tubing for each type of solution. To set up the syringe pump for direct infusion of the calibration solution 1. Turn on the syringe pump’s power switch. The power switch is located on the back of the device. 2. In the Tune window, place the mass spectrometer in Standby mode. 3. Load a clean, 500 μL syringe with 500 μL of the ESI positive calibration solution. For a list of provided solutions, see “Chemical Kit.” Note To minimize the possibility of cross-contamination of the assembly, be sure to wipe off the tip of the needle with a clean, lint-free tissue before reinserting it into the syringe adapter assembly (Figure 8). 4. Plumb the inlet for direct infusion as follows: a. Follow steps 2–5 in “To set up the syringe for infusion or high-flow infusion experiments” on page 24. b. Follow steps 2 and 3 in “To connect an infusion line between the LC union and the grounding union” on page 25. CAUTION To prevent electric shock, verify that the grounding union is made of stainless steel and is completely inserted into the grounding union holder. Go to the next section, “Setting Up the Mass Spectrometer for Calibration.” 40 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 5 Preparing the System for Calibration Setting Up the Mass Spectrometer for Calibration Setting Up the Mass Spectrometer for Calibration Before you calibrate the mass spectrometer, set up the operational parameters. CAUTION Before beginning normal operation of the mass spectrometer each day, verify that there is sufficient nitrogen for the API source. If you run out of nitrogen, the mass spectrometer automatically turns off to prevent atmospheric oxygen from damaging the source. The presence of oxygen in the source when the mass spectrometer is on can be unsafe. In addition, if the mass spectrometer turns off during an analytical run, you might lose data. To set up the mass spectrometer for calibration 1. In the Tune window, place the mass spectrometer in On mode. 2. Open the Ion Source page in the Ion Source pane, and then do the following: a. In the Current LC Flow (μL/min) box, enter 3. b. Click Get Defaults, and then click Apply. The Tune application sets the default parameters for the H-ESI source. 3. Set the syringe pump parameters as follows: a. Click the dropdown arrow, syringe parameters box. , next to the Syringe On/Off button to open the b. In the Flow Rate (μL/min) box, type 3. c. In the Volume (μL) list, select 500. The Tune application automatically sets the internal diameter (ID) for the syringe volume. d. Click Syringe On (Off ) to start the syringe pump. 4. Click Positive (Negative) to select the positive ion polarity mode. 5. Click Standard Pressure Mode (Intact Protein Mode) to select the standard pressure mode. 6. Verify that the system readback is normal (see page 69). This completes the setup for calibrating the mass spectrometer. Go to Chapter 6, “Establishing a Stable Ionization Spray.” Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 41 5 Preparing the System for Calibration Setting Up the Mass Spectrometer for Calibration 42 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 6 Establishing a Stable Ionization Spray Before you calibrate the mass spectrometer, make sure that you establish stable ionization spray conditions. The intensity and stability of the ionization spray largely depend on the performance of the API source. IMPORTANT • Failure to maintain a stable spray might compromise the data quality or result in a poor calibration or diagnostic result. • If the spray becomes unstable with your analyte solution, return to this chapter to evaluate the spray stability. Contents • Evaluating the Spray Stability • Optimizing the API Source Parameters Evaluating the Spray Stability Use the Plot Chromatogram tool ( ) to evaluate the API source’s ionization spray. To evaluate the spray stability 1. Open the Tune window. 2. Set up the system to use the calibration solution as follows: a. Verify that the syringe contains the appropriate calibration solution. b. In the Tune window, verify the following syringe and instrument settings: – 2 μL/min in the Current LC Flow box – 500 μL/min flow rate and 500 μL syringe volume – Positive ion polarity mode – Profile data type c. Go to step 4. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 43 6 Establishing a Stable Ionization Spray Evaluating the Spray Stability 3. (Optional) Set up the system to use an analyte solution as follows: • Verify that the LC device or the syringe contains a sufficient amount of the analyte. • Open the Ion Source page in the Ion Source pane, and verify the value in the Current LC Flow (μL/min) box. 4. Place the mass spectrometer in On mode (see page 69). The mass spectrometer begins scanning and applies high voltage to the spray insert. A real-time mass spectrum appears in the Tune window. 5. Turn on the flow for the solution as follows: • For the calibration solution, click Syringe On (Off ) to start the syringe pump. –or– • For an analyte solution, turn on the flow from the LC device or the syringe pump. A real-time plot of the solution’s mass spectrum appears. 6. Plot the full total ion current (TIC) and relative standard deviation (RSD) graphs as follows: a. Click the Plot Chromatogram icon, box (Figure 23). , to open the Plot Chromatogram dialog Figure 23. Plot Chromatogram dialog box with the TIC option selected b. Select the Spray Stability check box to monitor the relative standard deviation (RSD) of the desired ion current. c. Select the TIC option. 44 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 6 Establishing a Stable Ionization Spray Optimizing the API Source Parameters d. Click OK to plot the TIC chromatogram. The Plot Chromatogram tool generates a real-time graph (plot) of the full TIC where you can observe the signal stability and the effects of changes to various parameters. The tool also generates a real-time graph of the RSD of the TIC for a 10 Da-selected ion monitoring (SIM) scan that is centered around the most abundant mass-to-charge ratio in the current spectrum. 7. Observe the RSD graph, and review the signal stability rating and maximum %RSD value. Table 11 lists the criteria for a stable spray in either positive or negative ion polarity mode. Table 11. Recommended %RSD values and ratings for the calibration solutions Ion polarity mode Acceptable signal stability rating Maximum %RSD (threshold) Positive Excellent or Good 15 Negative Excellent or Good 15 8. If the signal stability rating is poor or the %RSD value is above the threshold, follow the procedure in the next section, “Optimizing the API Source Parameters.” IMPORTANT Thermo Fisher Scientific recommends that you optimize the API source parameters only if the preceding spray evaluation determines that the ionization spray is unstable. This completes the spray stability evaluation. Optimizing the API Source Parameters If the ionization spray is unstable, follow the procedure in this section to optimize the API source parameters. IMPORTANT Thermo Fisher Scientific recommends that you optimize the API source parameters only if the preceding spray evaluation determines that the ionization spray is unstable. To optimize the API source parameters 1. Verify that the syringe has a sufficient amount of the calibration solution. 2. In the Tune window, click Syringe On (Off ) to start the syringe pump. For an analyte solution, turn on the flow from the syringe pump or the LC device. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 45 6 Establishing a Stable Ionization Spray Optimizing the API Source Parameters 3. Open the Optimization page of the Ion Source pane, and then do the following: a. Select the Polarity Ion Spray Voltage (V) option (Figure 24). Figure 24. Optimization page of the Ion Source pane Optimization tab (selected) b. In the Signal Type list, select TIC. c. Click Optimize. The status area displays the message “Optimization In Progress.” After Tune completes the optimization, the optimized value and the Accept and Reject buttons appear. d. Click Accept. The Report Generation Options dialog box opens (Figure 25). Figure 25. Report Generation Options dialog box 46 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 6 Establishing a Stable Ionization Spray Optimizing the API Source Parameters e. Select an option, and then click OK. Tip To turn off the Report Generation Options dialog box, see “Setting the Tune Preferences” on page 72. 4. Optimize the remaining source parameters. 5. (Optional) Save the parameters’ state in the Favorites pane (see page 76). For additional information about the Favorites pane, refer to the Tune Help. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 47 6 Establishing a Stable Ionization Spray Optimizing the API Source Parameters 48 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 7 Performing the Tune, Calibration, or Calibration Check This chapter describes how to tune, calibrate, or check the calibration of the TSQ Endura and TSQ Quantiva MSs in H-ESI mode. The tune, calibrate, and calibration evaluation procedures require that you infuse the calibration polytyrosine 1-3-6 calibration solution into the instrument at a steady flow rate. Contents • Performing System Tune and Calibration • Spray Stability Evaluation • System Tune and Check • Check Mass Position and Resolution • Calibrate Mass Position and Resolution • Detector Gain Calibration Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 49 7 Performing the Tune, Calibration, or Calibration Check Performing System Tune and Calibration Performing System Tune and Calibration Calibration parameters are instrument parameters that affect the mass accuracy and resolution. Calibrate the TSQ Endura and TSQ Quantiva MSs in H-ESI mode before acquiring data in H-ESI or APCI mode. Generally, you must calibrate the mass spectrometer every one to three months of operation for optimum performance over the entire mass range of the mass analyzer. Tune parameters are instrument parameters that affect the magnitude of the ion signal. There are two types of tune parameters: mass dependent and compound dependent. • Mass-dependent tune parameters include the rf voltage of the rf lens, the dc offset voltages of multipoles M00 and M0, and the dc offset voltages of lenses L11, L12, L21, L23, L31, L33, and L4. • Compound-dependent tune parameters include the spray voltage (H-ESI or NSI mode) or spray current (APCI mode), sheath gas pressure, auxiliary gas pressure, sweep gas pressure, vaporizer temperature, and ion transfer tube temperature. Use the Optimization page of the Ion Source pane to optimize the spray voltage or spray current, sheath gas pressure, auxiliary gas pressure, and sweep gas pressure for your compound, whenever you change experiments. See Chapter 8, “Optimizing the API Source Parameters.” Note The Tune application writes the calibration parameters and the mass-dependent tune parameters to the calibration file. It writes the compound-dependent tune parameters to a change record in the History pane. You can rename and save the change record to the Favorites pane for future use in the Tune application or the Method Editor. See “Using the History Pane” on page 75. To perform system tune and calibration 1. Set up to infuse the polytyrosine 1-3-6 calibration solution at 2 μL/min into the API source. See “Setting Up the Syringe Pump for Direct Infusion.” 2. Click Calibration to display the Calibration Status page, and then click Calibrate to display the Calibration Options pane (Figure 26). 50 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 7 Performing the Tune, Calibration, or Calibration Check Spray Stability Evaluation Figure 26. Calibration – Options page 3. Clear the Skip Spray Stability Evaluation check box. 4. Under Quad Selection, select the Q1MS, Q3MS, or Both option. 5. Under System Tune and Calibration Options, select one of the following options: • System Tune and Check • Check Mass Position and Resolution • Calibrate Mass Position and Resolution • Detector Gain Calibration 6. Click Start. 7. When the procedure is complete, select a report generation option and click OK. Note When it completes a calibration, the mass spectrometer writes the calibration parameters to a calibration file, which overwrites the previous calibration file. You cannot replace or modify the calibration file. Spray Stability Evaluation The spray stability evaluation generates real-time graphs of both the TIC and the RSD of the TIC. See “Evaluating the Spray Stability” on page 43. The %RSD must be less than 15 percent for the spray stability evaluation to pass. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 51 7 Performing the Tune, Calibration, or Calibration Check System Tune and Check System Tune and Check When you select the System Tune and Check option (Figure 26), the mass spectrometer maximizes the ion signal by optimizing the rf voltage of the rf lens, the dc offset voltages of multipoles M00 and M0, and the dc offset voltages of lenses L11, L12, L21, L23, L31, L33, and L4. The system optimizes for polytyrosine 1, 3, and 6, and for quadrupoles Q1 and Q3. Figure 27 shows the M00 multipole offset voltage optimization for polytyrosine 1, 3, and 6. After the optimizations are complete, the system performs a mass position and resolution test. For a description of the multipoles and lenses, refer to the TSQ Quantiva and TSQ Endura Hardware Manual. Figure 27. Normalized intensity as a function of the M00 multipole offset voltage Check Mass Position and Resolution On a regular basis, run the check mass position and resolution evaluation by selecting its check box (Figure 26). If the evaluation fails, run the mass position and resolution calibration. During the mass position and resolution evaluation, the Tune application compares the measured isotopic peaks of polytyrosine 1-3-6 (red curve) with the theoretical isotopic peaks (blue curve). Figure 28 shows the experimental (red curve) and theoretical (blue curve) isotopic peaks of the polytyrosine trimer at a peak width of m/z 0.4. This procedure repeats for the polytyrosine 1, 3, and 6, for m/z 0.4 and 0.7 peak widths, and for quadrupoles Q1 and Q3. 52 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 7 Performing the Tune, Calibration, or Calibration Check Check Mass Position and Resolution Figure 28. Isotopic peaks of the polytyrosine trimer at a peak width of m/z 0.4 Figure 29 shows the error range boxes for polytyrosine 1, 3, and 6. The y axis is the error range of the peak widths from the expected peak width of m/z 0.4 (in mDa), and the x axis is the error range of the mass positions from the expected mass position (in mDa). The mass spectrometer measures the peak width and mass position over a large number of scans. The error ranges boxes must be within ±20 mDa from the expected peak width and mass positions for the evaluation to pass. Figure 29. Error range of the polytyrosine 1-3-6 Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 53 7 Performing the Tune, Calibration, or Calibration Check Calibrate Mass Position and Resolution Calibrate Mass Position and Resolution Use the Calibrate Mass Position and Resolution option (Figure 26) if the mass position and resolution evaluation fails. During the mass position and resolution calibration, the calibration procedure varies the rf and dc voltages that it applies to quadrupoles Q1 and Q3 to best fit the measured peak widths and positions of the polytyrosine isotopic peaks (red curve in Figure 28) to the theoretical peak widths and positions of those peaks (blue curve in Figure 28). This procedure repeats for polytyrosine 1, 3, and 6, for m/z 0.4 and 0.7 peak widths, and for quadrupoles Q1 and Q3. Detector Gain Calibration The detector gain decreases as the electron multiplier ages. The detector gain calibration increases the voltage on the electron multiplier to maintain a gain of 5 × 105 for MS mode and 2 × 106 for MS/MS mode. Select the Detector Gain option (Figure 26) if you notice a falloff of the ion signal intensity. You can now start using your analyte solution for data acquisition. IMPORTANT Before you start using your analyte, follow the procedure “To flush the inlet components” on page 81. 54 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 8 Optimizing the API Source Parameters This chapter describes how to optimize the API source parameters, including those that are compound-dependent, such as the spray voltage or spray current and the pressures for the API gases that maximize the ion signal of your analyte. Contents • Setting Up the Plumbing for Compound Optimization • Determining the Initial API Source Settings • Optimizing the RF Lens Voltage • Optimizing the Collision Energy Setting Up the Plumbing for Compound Optimization Use the high-flow infusion technique to introduce the analyte into the mass spectrometer’s API source. The high-flow infusion technique uses a Tee union to direct the analyte from the syringe pump into an LC flow that is appropriate (flow rate and composition) for your experiment. For plumbing instructions, see “Setting Up the Inlet for High-Flow Infusion.” Proceed to the next section, Determining the Initial API Source Settings. Determining the Initial API Source Settings Use the Ion Source page of the Ion Source pane to determine the initial spray voltage, pressures for the API gases, ion transfer tube temperature, and vaporizer temperature for the LC flow rate of your experiment. These initial API source settings provide a starting point for optimizing system performance. The optimal settings for your application depend on the compounds of interest, the solvent matrix, and the chromatographic conditions. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 55 8 Optimizing the API Source Parameters Determining the Initial API Source Settings To determine the initial API source settings 1. Open the Tune window. 2. Click Ion Source to display the Ion Source page of the Ion Source pane. 3. In the Current LC Flow (μL/min) box, type the flow rate (for example, 200) and click Get Defaults. Figure 30 shows the Ion Source page with the initial API source settings that are appropriate for the 200 μL/min flow rate. Figure 30. LC flow rate of 200 μL/min on the Ion Source – Ion Source page 4. Click Apply. The Tune application makes a change record in the History pane. Proceed to the next section, Optimizing the RF Lens Voltage. 56 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 8 Optimizing the API Source Parameters Optimizing the RF Lens Voltage Optimizing the RF Lens Voltage The rf lens transfers ions from the API source to the ion optics. Use the Define Scan – Optimization page to find the optimum rf lens voltage for transmitting a precursor ion through the rf lens. Note The rf lens voltage is a mass-dependent tune parameter until you run the rf lens voltage optimization. After that it is a compound-dependent tune parameter. The magnitude of the rf lens voltage affects the mass spectrum as follows: • Decreases the rf lens voltage, which does the following: – Decreases the amount of fragmentation of fragile ions in the rf lens. – Decreases the transmission of high m/z ions through the rf lens and increases the transmission of low m/z ions. • Increases the rf level, which does the following: – Increases the amount of fragmentation of fragile ions in the rf lens. – Increases the transmission of high m/z ions through the rf lens and decreases the transmission of low m/z ions. To optimize the rf lens voltage 1. Set up the system for direct infusion or high-flow infusion (see “Setting Up the Inlet Plumbing” on page 24). 2. Introduce the compound into the instrument at a steady flow rate. 3. Click Define Scan, and then click the Optimization tab to display the Optimization page (Figure 31). Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 57 8 Optimizing the API Source Parameters Optimizing the RF Lens Voltage Figure 31. Define Scan – Optimization page showing the rf lens voltage optimization settings 4. Specify the mass input option, either Formula or m/z. 5. Enter a name for the compound. 6. Specify the charge state. 7. Select the Precursor - Optimize RF Lens check box. 8. Click Optimize. Figure 32 shows the intensity of the polytyrosine trimer ion signal (m/z 508.2) as a function of the rf lens voltage. The optimization algorithm determined that the optimum rf lens voltage is 118.0 V. 58 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 8 Optimizing the API Source Parameters Optimizing the Collision Energy Figure 32. RF optimization for the polytyrosine trimer ion After the optimization is complete, the optimized value, and the Accept and Reject buttons appear. 9. Click Accept to accept the optimized value or Reject to reject it. Optimizing the Collision Energy The collision energy determines the product ion intensities from collision-induced dissociation (CID) of the precursor ions in an MS/MS experiment. Use the Define Scan – Optimization page to find the optimum collision energy for the MS/MS transitions of a single precursor ion. The following example uses the polytyrosine trimer ion as the precursor ion. To optimize the collision energy 1. Set up the system for direct infusion or high-flow infusion (see “Setting Up the Inlet Plumbing” on page 24). 2. Introduce the compound into the instrument at a steady flow rate. 3. Click Define Scan, and then click the Optimization tab to display the Optimization page. Figure 33 shows the settings for the collision energy optimization of the m/z 508.2 to 299 MS/MS transition of the polytyrosine trimer ion. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 59 8 Optimizing the API Source Parameters Optimizing the Collision Energy Figure 33. Define Scan – Optimization page showing the collision energy optimization settings 4. Specify the mass input option for the precursor ion. 5. Enter a name for the compound. 6. Specify the charge state of the precursor ion. 7. Select the Product check box. 8. Select the CID gas pressure (in milliTorr). 60 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 8 Optimizing the API Source Parameters Optimizing the Collision Energy 9. Select the input option: • Select Unknown Product Ions to optimize the collision energy for the top n most intense product ions. • Select Known Product Ions to optimize the collision energy for the product ions that you list in the Product Mass table. 10. Click Optimize. The Tune application plots the product ion intensity as a function of collision energy and determines the optimum collision energy. Figure 34 shows the intensity of the m/z 508.2 to 299.15 MS/MS transition of the polytyrosine trimer ion as a function of the collision energy (top) and the MS/MS spectrum of the polytyrosine trimer ion (bottom). The optimization algorithm determined that the optimum collision energy is 24.0 V. Figure 34. Collision energy optimization curve and MS/MS spectrum of the polytyrosine trimer ion After the optimization is complete, the optimized value, and the Accept and Reject buttons appear. 11. Click Accept to accept the optimized value or Reject to reject it. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 61 8 Optimizing the API Source Parameters Optimizing the Collision Energy 62 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 9 Acquiring Sample Data This chapter describes how to use the Tune application to manually acquire sample data and how to use the Xcalibur data system to set the start (trigger) instrument before you run an instrument method to acquire sample data. Note • Before you begin the analysis of the sample solution, make sure that you have calibrated the mass spectrometer in H-ESI mode within the last three months. • The data system computer automatically saves the acquired data to its hard drive. Contents • Using the Tune Application to Acquire Sample Data • Using the Xcalibur Data System to Acquire Sample Data Using the Tune Application to Acquire Sample Data To acquire a sample data file 1. Open the Data Acquisition pane (Figure 35), and then do the following: a. (Optional) To change the destination folder for the raw data file, click the Browse icon. The default folder location is in C:\Thermo\Data. b. In the File Name box, type reserpine (or the name of the analyte). If the base file name already exists in the save location, the Tune application adds a time-stamp suffix that consists of the year (YYYY), month (MM), day (DD), and time (HHMMSS). c. In the Sample Name box, type the name of the analyte (or other suitable label). Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 63 9 Acquiring Sample Data Using the Tune Application to Acquire Sample Data d. In the Comment box, type a comment about the experiment. For example, describe the ionization mode, scan type, scan rate, sample amount, or method of sample introduction. The data system includes the comment in the header information for the raw data file. You can also add this information to reports created with the Xcalibur XReport reporting application. To open this application, choose Start > All Programs > Thermo Xcalibur > XReport. e. Under Timed Acquisition, select the Continuously option (acquires data until you stop the acquisition). Figure 35. Data Acquisition pane in the Tune window Start/stop the data acquisition recording. Click to open/close the Data Acquisition pane. File name box 2. Click Record to start data acquisition. After the Tune parameters reach their specified settings, the data acquisition process begins and the small circle on the Record button turns red ( ). 3. When you are ready, click Record again to stop the acquisition. The small circle on the Record button turns gray (not recording). For more information about reviewing the acquired data, refer to the Thermo Xcalibur Qual Browser User Guide or the Qual Browser Help. 64 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific 9 Acquiring Sample Data Using the Xcalibur Data System to Acquire Sample Data Using the Xcalibur Data System to Acquire Sample Data Thermo Scientific mass spectrometry applications, such as the Xcalibur data system, can control the connected external device. If the Xcalibur application can control the external device, it selects the autosampler as the default start (trigger) instrument for a sequence run. If the Xcalibur application cannot control the external device, it selects the mass spectrometer as the start instrument, which means that you must change the start instrument to the appropriate instrument as part of the Xcalibur sequence setup. Follow these procedures: 1. To select the external start instrument 2. To acquire a data file by using the Xcalibur data system To select the external start instrument 1. Open the Xcalibur data system, and then choose View > Sequence Setup View to open the Sequence Setup window. 2. Open the sequence that you want to run as follows: a. Click the Open button and browse to the appropriate folder. b. Select the sequence (.sld) file and click Open. 3. Choose Actions > Run Sequence or Actions > Run This Sample to open the Run Sequence dialog box (Figure 36). The Yes in the Start Instrument column indicates the default start instrument for the sequence run. Figure 36. Run Sequence dialog box (partial) showing the selected start instrument The LC device is the start instrument. Change Instruments Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 65 9 Acquiring Sample Data Using the Xcalibur Data System to Acquire Sample Data 4. If Yes appears in the Start Instrument column for the mass spectrometer or if you need to change the start instrument to another device, click Change Instruments to open the Change Instruments In Use dialog box (Figure 37). Figure 37. Change Instruments In Use dialog box showing the MS as the start instrument a. In the Start Instrument column, click the blank field to the right of the appropriate triggering device (typically an autosampler) to move “Yes” to that field. b. Click OK. 5. In the Run Sequence dialog box, complete the remaining selections. 6. Click OK. This completes the start instrument setup. To acquire a data file by using the Xcalibur data system For instructions, refer to the Instrument Setup and Sequence Setup topics in the Xcalibur Help. 66 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific A Using Basic Tune Functions This appendix describes some of the basic Tune functions that are used throughout this guide. You enable several of the functions by pressing a toggle button. For additional information about the Tune window, refer to the Tune Help. Contents • Opening the Tune Window • Setting the Instrument Power Mode • Checking the Instrument Readback Status • Controlling the Syringe Pump • Setting the Data Type • Setting the Ion Polarity Mode • Setting the Tune Preferences • Using the Mass List Table in the Define Scan Pane • Using the History Pane • Using the Favorites Pane to Save System Settings Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 67 A Using Basic Tune Functions Opening the Tune Window Opening the Tune Window To open the Tune window From the Windows taskbar, choose Start > All Programs > Thermo Instruments > model x.x Tune (Figure 38). For information about the buttons and icons in the Tune application and what they control, refer to the Tune Help. Figure 38. Tune window showing the Define Scan – Scan page Three power mode icons (On/Standby/Off) Manual data acquisition Instrument readback status Chromatogram view Spectrum view Define Scan – Scan page Plot Chromatogram tool Controls for the graphs Panes: Status, History, and Favorites 68 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific A Using Basic Tune Functions Setting the Instrument Power Mode Setting the Instrument Power Mode Use the three power mode icons in the Tune window (Figure 38) to set the mass spectrometer’s power mode (on, standby, or off ). Before you remove the API source or the spray insert, always place the system in standby mode. The mass spectrometer then automatically switches to off mode. In standby mode, the System LED on the front panel turns yellow and the mass spectrometer turns off the electron multipliers, conversion dynodes, 8 kV power to the API source, main rf voltage, and ion optic rf voltages. The auxiliary, sheath, and sweep gas flows remain on and return to their standby default settings (2 arbitrary). For a list of the on/off status of the components under varying power conditions, refer to Chapter 3 in the TSQ Endura and TSQ Quantiva Hardware Manual. To set the instrument power mode Click the icon for the power mode that you want (Figure 39). The center of the selected icon changes from white to green. Figure 39. Power mode icons showing the selected icon (mode) On mode Standby mode Off mode Checking the Instrument Readback Status The system readback icon is located in the top, right corner of the Tune window. Table 12 lists the various readback states. Table 12. Instrument readback icons and their meanings (Sheet 1 of 2) Icon Thermo Scientific Background color Meaning Green The system parameters are within tolerance. Green The system is initializing. Amber One or more settings are changing. Red An error has occurred. TSQ Endura and TSQ Quantiva Getting Started Guide 69 A Using Basic Tune Functions Controlling the Syringe Pump Table 12. Instrument readback icons and their meanings (Sheet 2 of 2) Icon Background color Meaning Gray The API source is off. Dark gray There is no communication between the mass spectrometer and the data system. Controlling the Syringe Pump Follow these procedures, as applicable: • To turn the syringe pump on or off • To set the syringe pump parameters To turn the syringe pump on or off Click Syringe On (Off ) to switch between on and off (Figure 40). Figure 40. Toggle button for the syringe modes To set the syringe pump parameters 1. Click the dropdown arrow, parameter box (Figure 41). , next to the Syringe On/Off button, to open the syringe Figure 41. Syringe parameter box 2. Type the parameter values that you want. The Tune application automatically saves the values. 3. Click the dropdown arrow again or click elsewhere in the Tune window to close the syringe parameter box. 70 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific A Using Basic Tune Functions Setting the Data Type Setting the Data Type To set the data type Click Centroid (Profile) to select the data type that you want (Figure 42). Figure 42. Toggle button for the data types Centroid data type Profile data type Setting the Ion Polarity Mode To set the ion polarity mode Click Positive (Negative) to select the polarity mode that you want (Figure 43). Figure 43. Toggle button for the instrument polarity modes Positive polarity Thermo Scientific Negative polarity TSQ Endura and TSQ Quantiva Getting Started Guide 71 A Using Basic Tune Functions Setting the Tune Preferences Setting the Tune Preferences You can set a few preferences for how the Tune application works. To set the Tune preferences 1. Click the Options icon, and then choose Tune Preferences to open the Tune Preferences dialog box (Figure 44). Figure 44. Tune Preferences dialog box 2. Under General and Report Content Options, select all check boxes that apply. 3. In the center under Report Options, select one of the options, and then click OK. 72 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific A Using Basic Tune Functions Using the Mass List Table in the Define Scan Pane Using the Mass List Table in the Define Scan Pane The mass list table appears when you select the SIM Scan (Q1), SIM Scan (Q3), or SRM scan type in the Define Scan pane. Use this table to specify scan parameters. To set different scan parameters for the precursor ions, add the parameters to the table. • To add a row to the table • To delete a row from the table • To delete multiple rows from the table • To add or remove scan parameters from the table To add a row to the table Do one of the following: • Click the Add Row icon, . • Right-click the table, and then choose Add Row from the shortcut menu. To delete a row from the table 1. Select the row number to highlight the entire row. 2. Do one of the following: • Click the Delete Selected Rows icon, . • Right-click the selected row, and then choose Delete Selected Rows from the shortcut menu. • Press the DELETE key on your keyboard. To delete multiple rows from the table 1. Select the first row’s number to highlight the entire row. 2. Do one of the following: • For an adjacent row or group of sequential rows, hold down the SHIFT key and select another row number. • For an adjacent row or nonsequential rows, hold down the CTRL key and select each additional row number. 3. Do one of the following: • Click the Delete Selected Rows icon, . • Right-click the selected row, and then choose Delete Selected Rows from the shortcut menu. • Press the DELETE key on your keyboard. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 73 A Using Basic Tune Functions Using the Mass List Table in the Define Scan Pane To add or remove scan parameters from the table Click the Table icon once to add the adjacent scan parameter to the table (Figure 45). Click it again to remove the parameter from the table. Figure 45. Q3 Resolution selected and added to the SRM Table Q3 Resolution is selected. Q3 Resolution appears in the table. To import a mass list from a file 1. Click Import to open the Open dialog box. 2. Browse to an XML or a CVS (Microsoft Excel™) file, and then click Open. The list of mass-to-charge ratio values appears in the table. To export a mass list to a file 1. Complete the list of mass-to-charge ratio values. 2. Click Export to open the Save As dialog box. 3. Browse to a location, enter a file name, and then select a file type (.csv, .txt, or .xml). 4. Click Save. 74 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific A Using Basic Tune Functions Using the History Pane Using the History Pane When you click Apply in the Ion Source pane or the Define Scan pane, the Tune application adds a change record to the History pane (Figure 46). The change record records all changes to the instrument state that originated from the Tune application. Change records in the History pane work as follows: • The Tune application creates a change record when you change parameters in one of the Ion Source or Define Scan panes and then click Apply. • The History pane displays the change records as subitems under the date that they were created. The maximum number of change records is 100. • Click a change record to display its parameters, or right-click it and choose Load from the shortcut menu. Parameters that are colored red differ from their default values. • Double-click a change record to submit its parameters to the mass spectrometer, or right-click it and choose Apply from the shortcut menu. • A change record is inactive if the API source type of the change record differs from the current API source type. Figure 46. History pane Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 75 A Using Basic Tune Functions Using the Favorites Pane to Save System Settings Using the Favorites Pane to Save System Settings You can manually save the current settings for the API source and scan parameters in the Favorites pane. • To create a favorite state • To load only or apply a favorite state • To rename a favorite state • To delete a favorite state To create a favorite state 1. In the Tune window, modify the parameters in one of the Ion Source or Define Scan panes. 2. Click Apply or Export. 3. Click the Favorites tab to display the Favorites pane (Figure 47). Figure 47. Favorites pane 4. Click Save Current State, and then type a unique name in the box (Figure 48). Figure 48. State name box State name box 76 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific A Using Basic Tune Functions Using the Favorites Pane to Save System Settings 5. Click Save Current State again to save the state. The most recent state appears at the top of the User Settings list. To load only or apply a favorite state Under User Settings, right-click the state name, and then choose one of the following from the shortcut menu: • Load to only display the key parameters in the applicable parameter boxes. • Apply to submit the key parameters to the mass spectrometer. You can click Apply without first loading the parameters. To rename a favorite state 1. Under User Settings, right-click the state name, and then choose Rename from the shortcut menu. 2. Type a different name and press ENTER. To delete a favorite state Under User Settings, right-click the state name, and then choose Delete from the shortcut menu. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 77 A Using Basic Tune Functions Using the Favorites Pane to Save System Settings 78 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific B Flushing the Inlet Components This appendix describes how to flush the inlet components (sample transfer line, sample tube, and spray insert) after both the positive and negative calibration processes, and also before you change from one analyte solution to another. In addition, Thermo Fisher Scientific recommends that you clean the ion sweep cone, spray cone, and ion transfer tube, on a regular basis to prevent corrosion and to maintain optimum performance of the API source. A good practice is to wash or flush the ion sweep cone and ion transfer tube at the end of each operating day after you pump a solution of 50:50 methanol/water from the LC system through the inlet components. If you use a mobile phase that contains a nonvolatile buffer or inject high concentrations of sample, you might need to clean these parts more often. Be aware that it is not necessary to vent the system to flush the ion sweep cone and ion transfer tube. For instructions on how to clean the ion sweep cone, spray cone, and ion transfer tube, refer to Chapter 6 in the TSQ Endura and TSQ Quantiva Hardware Manual. CAUTION When the ion transfer tube is installed, do not flush it with cleaning solution, which flushes the residue into the mass spectrometer. Contents • Supplies • Flushing the Inlet Components after Calibration Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 79 B Flushing the Inlet Components Supplies Supplies Table 13 lists the necessary supplies for flushing and cleaning specific components. CAUTION Avoid exposure to potentially harmful materials. By law, producers and suppliers of chemical compounds are required to provide their customers with the most current health and safety information in the form of Material Safety Data Sheets (MSDSs) or Safety Data Sheets (SDSs). The MSDSs and SDSs must be freely available to lab personnel to examine at any time. These data sheets describe the chemicals and summarize information on the hazard and toxicity of specific chemical compounds. They also provide information on the proper handling of compounds, first aid for accidental exposure, and procedures to remedy spills or leaks. Read the MSDS or SDS for each chemical you use. Store and handle all chemicals in accordance with standard safety procedures. Always wear protective gloves and safety glasses when you use solvents or corrosives. Also, contain waste streams, use proper ventilation, and dispose of all laboratory reagents according to the directions in the MSDS or SDS. Table 13. Flushing and cleaning supplies Description Part number Gloves, nitrile Fisher Scientific™ 19-120-2947a Unity Lab Services: • 23827-0008 (size medium) • 23827-0009 (size large) a 80 Acetone, LC/MS-grade Fisher Scientific: AX0120-2 Methanol, LC/MS-grade Fisher Scientific: A456-1 Water, LC/MS-grade Fisher Scientific: W6-1 Multiple sizes are available. TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific B Flushing the Inlet Components Flushing the Inlet Components after Calibration Flushing the Inlet Components after Calibration This section describes how to flush the inlet components (sample transfer line, sample tube, and spray insert) with the syringe after calibration. For best results, follow this procedure before you acquire data on an analyte. Tip You can also use an LC pump to flush the 50:50 methanol/water solution through the inlet components to the API source at a flow rate of 200–400 μL/min for approximately 15 minutes. To flush the inlet components 1. Turn off the flow from the syringe pump (see page 70). 2. Place the mass spectrometer in Standby mode (see page 69). 3. Remove the syringe from the syringe pump as follows: a. Lift the syringe holder off of the syringe. b. Press the pusher block’s release knob and slide the block to the left. c. Remove the syringe from the holder. d. Carefully remove the syringe needle from the Teflon tube on the syringe adapter assembly (Figure 8). 4. Clean the syringe as follows: a. Rinse the syringe with a solution of 50:50 methanol/water. b. Rinse the syringe with acetone several times. 5. Flush the sample transfer line, sample tube, and spray insert as follows: a. Load the cleaned syringe with a solution of 50:50 methanol/water (or another appropriate solvent). b. Carefully reinsert the syringe needle into the Teflon tube on the syringe adapter assembly. c. Slowly depress the syringe plunger to flush the sample transfer line, sample tube, and spray insert with the solution. d. Remove the syringe needle from the syringe adapter assembly. This completes the procedure to flush the inlet components. Repeat this procedure after you complete the negative polarity calibrations. Thermo Scientific TSQ Endura and TSQ Quantiva Getting Started Guide 81 B Flushing the Inlet Components Flushing the Inlet Components after Calibration 82 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific G Glossary A B C D E F G H I J K L M N O P Q R S T U V W X Y Z A C APCI spray current The ion current carried by the charged particles in the APCI source. The APCI corona discharge voltage varies, as required, to maintain the set spray current. centroid data Data used to represent mass spectral peaks in terms of two parameters: the centroid (the weighted center of mass) and the intensity. The data is displayed as a bar graph. The normalized area of the peak provides the mass intensity data. API source The sample interface between the liquid chromatograph (LC) and the mass spectrometer (MS). atmospheric pressure chemical ionization (APCI) A soft ionization technique done in an ion source operating at atmospheric pressure. Electrons from a corona discharge initiate the process by ionizing the mobile phase vapor molecules, forming a reagent gas. atmospheric pressure ionization (API) Ionization performed at atmospheric pressure by using atmospheric pressure chemical ionization (APCI), heated-electrospray (H-ESI), or nanospray ionization (NSI). atmospheric pressure photoionization (APPI) A soft ionization technique that shows an ion generated from a molecule when it interacts with a photon from a light source. auxiliary gas The outer-coaxial gas (nitrogen) that assists the sheath (inner-coaxial) gas in dispersing and/or evaporating sample solution as the sample solution exits the ESI or APCI (optional) spray insert. Thermo Scientific charge state The imbalance between the number of protons (in the nuclei of the atoms) and the number of electrons that a molecular species (or adduct ion) possesses. If the species possesses more protons than electrons, its charge state is positive. If it possesses more electrons than protons, its charge state is negative. collision energy The energy used when ions collide with the collision gas. collision gas A neutral gas used to undergo collisions with ions. collision-induced dissociation (CID) A method of fragmentation where ions are accelerated to highkinetic energy and then allowed to collide with neutral gas molecules such as argon. The collisions break the bonds and fragment the ions into smaller pieces. conversion dynode A highly polished metal surface that converts ions from the mass analyzer into secondary particles, which enter the electron multiplier. TSQ Endura and TSQ Quantiva Getting Started Guide 83 Glossary: divert/inject valve D H divert/inject valve A valve on the mass spectrometer that can be plumbed as a loop injector or as a divert valve. heated-electrospray (H-ESI) A type of atmospheric pressure ionization that converts ions in solution into ions in the gas phase by using electrospray (ESI) in combination with heated auxiliary gas. E electron multiplier A device used for current amplification through the secondary emission of electrons. Electron multipliers can have a discrete dynode or a continuous dynode. electrospray (ESI) A type of atmospheric pressure ionization that is currently the softest ionization technique available to transform ions in solution into ions in the gas phase. electrospray ionization (ESI) See electrospray (ESI). F flow rate, syringe pump status The syringe pump injection flow rate in milliliters per minute (mL/min) or microliters per minute (μL/min) for the current sample, as defined in the current experiment method. heated-electrospray ionization (H-ESI) See heatedelectrospray (H-ESI). high performance liquid chromatography (HPLC) Liquid chromatography where the liquid is driven through the column at high pressure. Also known as high pressure liquid chromatography. I ion detection system A high sensitivity, off-axis system for detecting ions. It produces a high signalto-noise ratio (S/N) and allows for switching of the voltage polarity between positive ion and negative ion modes of operation. The ion detection system includes two ±12 kVdc conversion dynodes and a discrete dynode electron multiplier. ion optics Focuses and transmits ions from the API source to the mass analyzer. forepump The pump that evacuates the foreline. A rotary-vane pump is a type of forepump. It might also be referred to as a backing, mechanical, rotaryvane, roughing, or vacuum pump. ion polarity mode The mass spectrometer can operate in either of two ion polarity modes: positive or negative. fragment ion A charged dissociation product of an ionic fragmentation. Such an ion can dissociate further to form other charged molecular or atomic species of successively lower formula weights. ion sweep cone A removable cone-shaped metal cover that fits on top of the API ion transfer tube and acts as a physical barrier to protect the entrance of the tube. full-scan type Provides a full mass spectrum as opposed to the selected ion monitoring (SIM) scan type, which produces only one mass. With the fullscan type, the mass analyzer is scanned from the first mass to the last mass without interruption. Also known as single-stage full-scan type. isolation window The baseline width of a window for a mass peak (or peak cluster) of interest for an MS/ MS or MSn scan. L LC pump A high pressure solvent pump in the LC that provides the pressure on the input side of a column to drive the eluent and sample through the column. 84 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific Glossary: lens lens An element that provides focusing of the ion beam. M mass analysis A process that produces a mixture of ionic species that is then separated according to the mass-to-charge ratios (m/z) of the ions to produce a mass spectrum. mass analyzer A device that determines the mass-tocharge ratios (m/z) of ions by one of a variety of techniques. mass spectrometer An instrument that ionizes sample molecules and then separates the ions according to their mass-to-charge ratio (m/z). The resulting mass spectrum is a characteristic pattern for the identification of a molecule. mass spectrum A graphical representation (plot) of measured ion abundance versus mass-to-charge ratio. The mass spectrum is a characteristic pattern for the identification of a molecule and is helpful in determining the chemical composition of a sample. mass-to-charge ratio (m/z) An abbreviation used to denote the quantity formed by dividing the mass of an ion (in Da) by the number of charges carried by the ion. For example, for the ion C7H72+, m/z = 45.5. molecular ion An ion formed by the removal (positive ion) or addition (negative ion) of one or more electrons to/from a molecule without fragmentation of the molecular structure. MS scan modes Scan modes where only one stage of mass analysis is performed. The scan types used with the MS scan modes are full-scan type and selected ion monitoring (SIM) scan type. N nano liquid chromatography (nanoLC) Liquid chromatography with typical flow rates of 10–1000 nL/min and 10–150 μm diameter columns. Thermo Scientific nanoelectrospray ionization (nanoESI or NSI) A type of electrospray (ESI) that accommodates very low flow rates of sample and solvent at 1–20 nL/min (for static nanoelectrospray) or 100–1000 nL/min (for dynamic nanoelectrospray, which is also called nanoESI nanoLC gradient separation). nanoESI nanoLC gradient separation Employs microscale capillary columns to separate the analytes in complex mixtures. The sample is loaded onto a column using an injection valve or a gas pressure vessel. The mixture components are then eluted by a solvent gradient and pumped through the emitter. nanoESI (NSI) spray current The flow of charged particles in the nanoESI (NSI) source. The voltage on the NSI spray needle supplies the potential required to ionize the particles. nanoESI (NSI) spray voltage The high voltage that is applied to the spray needle in the nanoESI (NSI) source to produce the NSI spray current as liquid emerges from the nozzle. The NSI spray voltage is selected and set; the NSI spray current varies. nanospray ionization (NSI) See nanoelectrospray ionization (nanoESI or NSI). P precursor ion An electrically charged molecular species that can dissociate to form fragments. The fragments can be electrically charged or neutral species. A precursor ion can be a molecular ion or an electrically charged fragment of a molecular ion. precursor mass The mass-to-charge ratio of a precursor ion. The location of the center of a target precursor-ion peak in mass-to-charge ratio (m/z) units. product ion An electrically charged fragment of an isolated precursor ion. product mass The mass-to-charge ratio of a product ion. The location of the center of a target production peak in mass-to-charge ratio (m/z) units. TSQ Endura and TSQ Quantiva Getting Started Guide 85 Glossary: profile data profile data Data representing mass spectral peaks as point-to-point plots, with each point having an associated intensity value. Q qualitative analysis Chemical analysis designed to determine the identity of the components of a substance. quantitative analysis Chemical analysis designed to determine the quantity or concentration of a specific substance in a sample. R relative standard deviation (RSD) A measure of the dispersion of a group of measurements relative to the mean of the group. Relative standard deviation is expressed as a percentage of the average value. The percent relative standard deviation is calculated as: %RSD = 100 S X where S is the standard deviation and X is the sample mean. retention time (RT) The time after injection at which a compound elutes. The total time that the compound is retained on the chromatograph. selected ion monitoring (SIM) scan type A scan type where the mass spectrometer acquires and records ion current at only one or a few selected mass-to-charge ratio values. sheath gas The inner coaxial gas (nitrogen), which is used in the API source to help nebulize the sample solution into a fine mist as the sample solution exits the H-ESI or APCI nozzle. signal-to-noise ratio (S/N) The ratio of the signal height (S) to the noise height (N). The signal height is the baseline corrected peak height. The noise height is the peak-to-peak height of the baseline noise. source See API source. static nanoelectrospray A device that performs continuous analysis of small analyte solution volumes over an extended period of time. sweep gas Nitrogen gas that flows out from behind the sweep cone in the API source. Sweep gas aids in solvent declustering and adduct reduction. syringe pump A device that delivers a solution from a syringe at a specified rate. T total ion current (TIC) The sum of the ion current intensities across the scan range in a mass spectrum. S sample loop A loop of calibrated volume that is used to perform flow injection analysis. scan Comprised of one or more microscans. Each microscan is one mass analysis (ion injection and storage/scan-out of ions) followed by ion detection. After the microscans are summed, the scan data is sent to the data system for display and/or storage. The process of ramping the amplitude of the rf and dc voltages on the multipole rods in the mass analyzer to transmit ions from the lowest mass to the highest mass of a specified scan range. 86 TSQ Endura and TSQ Quantiva Getting Started Guide Thermo Scientific I Index A APCI mode description 3 plumbing connection, direct infusion 25 spray insert, installing 16 API source cautions 12 high voltage connector 14 installing or removing 11 solvent waste container, connecting 11 spray insert 16 APPI mode, selecting the spray insert 16 auto-loop injection schematic 22 setup 29 uses 19 autosampler injection 31 B buffers, description 6 buttons, on/standby/off 68 C Calibration pane See panes, Calibration Options calibration parameters 50 caution symbols, description xviii Change Instruments In Use dialog box 66 cleaning inlet components 81 ion sweep cone 79 ion transfer tube 79 spray cone 79 syringe 81 compliance FCC iii–iv regulatory iii Thermo Scientific contacting us xviii contamination, preventing 23, 40 D data acquisition button 64 Tune 63 Xcalibur 66 Data Acquisition pane 64 data type, setting 71 Define Scan pane See panes, Define Scan direct infusion connecting the plumbing for 25 description 20 schematic 22 directive, WEEE v divert/inject valve configurations as divert valve 35 as loop injector 35 schematic of 36 controlling 36 description 34 positions 35 schematic 22 valve position indicator 36 documentation accessing xiv downloading xiv related xiv E electromagnetic compatibility iii EMC compliance iii TSQ Endura and TSQ Quantiva Getting Started Guide 87 Index: F F L favorite states saving as 76 using to set parameters 76 Favorites pane 76 FCC compliance iii–iv figures, list of xi flow rates, setting 4 flow-injection analysis, description 21 flushing inlet components 81 forepump, fume exhaust system cautions 12 LC pump, connecting to the divert/inject valve 28 LC union xvi LC with autosampler injection, schematic 22 LC/MS experiments, connecting the plumbing for 31 LC/MS operational guidelines APCI mode 5 H-ESI mode 5 NSI mode 5 loop injection connecting the plumbing for 29 liquid chromatography description, and 21 G gas flow rates, adjusting for LC flow rate 4 grounding (ZDV) union xvi, 29 H H-ESI mode description 2 plumbing connection, direct infusion 25 spray insert, installing 16 high-flow infusion connecting the plumbing for 26 description 20 schematic 22 History pane 75 HPLC with autosampler injection schematic 22 uses 21 I infusion line, connecting to grounding union 25 ion polarity mode, setting 71 Ion Source pane See panes, Ion Source ion sweep cone, cleaning 79 ion transfer tube cleaning 79 temperature, adjusting for LC flow rate 4 K kits Chemical xvii MS Calibration xv Performance Specification xvi 88 TSQ Endura and TSQ Quantiva Getting Started Guide M manual loop injection schematic 22 setup 29 uses 19 mass list exporting from Tune 74 importing into Tune 74 mass spectrometers API source, installing or removing 11 calibration parameters 50 flow rates, setting 4 plumbing connections 19 power modes, setting 69 pumping down the vacuum 37 sample introduction techniques 19 spray insert, installing or removing 16 tune parameters 50 MSDS 6 MSn setting table, using 73 N NSI mode, description 4 O optimization API source parameters, general procedure 45 note 45 signal type, list 46 Optimization page Define Scan pane 58 Ion Source pane 46 Thermo Scientific Index: P P panes Calibration Options page 51 Status page 50 Data Acquisition 64 Define Scan Optimization page 58 Scan page 68 Favorites 76 History 75 Ion Source Ion Source page 56 Optimization page 46 Status 39 PEEK tubing, special notice 23 plumbing connections, inlet 19 polarity mode See ion polarity mode, setting power mode button 68 pump down, MS 37 R readback status, description 69 record button 64 regulatory compliance iii Report Generation Options dialog box 46 Run Sequence dialog box (acquisition options) 65 S safety standards iii sample introduction techniques schematic diagrams 22 summary of connections 19 Scan page, Define Scan pane 68 SDS 6 sequence run, start instrument in Xcalibur 65 solvents description 6 waste 11 source See API source spray cone, cleaning 79 spray insert, API source adjustments front-to-back 17 rotational 17 installing or removing 16 Thermo Scientific start instrument configuring with Xcalibur 65 description 65 Run Sequence dialog box 65 Status pane 39 syringe adapter assembly 23 avoid contamination 40 cleaning 81 syringe adapter assembly, drawing 34 syringe pump controlling 70 default flow rate 33 description 33 setting up 24 T Tune application opening 68 preferences, setting 72 tune parameters compound dependent 50 mass dependent 50 U union types, plumbing grounding (ZDV) xvi LC xvi Tee xvii V vaporizer temperature, adjusting for LC flow rate 4 W waste container, solvent 14 WEEE directive v X Xcalibur file type, raw data (.raw) 63 Xcalibur file type, sequence (.sld) 65 Z ZDV union xvi TSQ Endura and TSQ Quantiva Getting Started Guide 89
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