Thermo Scientific Orbitrap Fusion Lumos User manual

Orbitrap Tribrid Series
Getting Started Guide
80000-97026 Revision A June 2018

© 2018 Thermo Fisher Scientific Inc. All rights reserved.
EASY-ETD, EASY-IC, FlexMix, Fusion, ID-X, LTQ Velos, Lumos, nanoViper, and Orbitrap ID-X are
trademarks; Unity is a registered service mark; and Hypersil GOLD, LTQ, Orbitrap, Orbitrap Fusion,
Orbitrap Fusion Lumos, Pierce, Thermo Scientific, Tribrid, and Xcalibur are registered trademarks of Thermo
Fisher Scientific Inc. in the United States. Fisher Scientific and Optima are registered trademarks of Fisher
Scientific Co. in the United States.
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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.
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
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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, June 2018
Software version: (Thermo) Foundation 3.1 SP5 and later, Xcalibur 4.2 and later, Tune 3.1 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 North American and European regulations. Your system meets the applicable requirements in the
electromagnetic compatibility (EMC) and product safety standards described in this section.
Unauthorized changes that you make to your system will void regulatory compliance and may defeat the built-in
protections for your instrument. Some examples of unauthorized changes include using replacement parts or adding
components, options, or peripherals that Thermo Fisher Scientific has not qualified and authorized. Unauthorized
changes can also result in bodily injury and/or damage to your system and laboratory.
Ensure continued compliance with regulatory standards:
• Follow all installation instructions provided in the documentation that comes with your system.
• Order replacement parts (as specified in the instrument manual) and additional components, options, and periph-
erals directly from Thermo Fisher Scientific or an authorized representative.
Regulatory compliance results for the following Thermo Scientific™ mass spectrometers:
•Orbitrap Fusion Lumos
•Orbitrap Fusion and Orbitrap ID-X
Orbitrap Fusion Lumos
Low Voltage Directive 2014/35/EU
This device complies with Low Voltage Directive 2014/35/EU and the harmonized safety standard IEC/EN/CSA/
UL 61010-1, 3rd Edition.
EMC Directive 2014/30/EU and other EMC test standards
This device was tested by TÜV Rheinland of North America and complies with the following EMC standards:
47 CFR 15, Subpart B, Class A: 2015 EN 61000-3-2: 2006 + A1 + A2 EN 61000-4-5: 2006
CISPR 11: 2009 + A1 EN 61000-3-3: 2008 EN 61000-4-6: 2009
ICES-003: 2014 EN 61000-4-2: 2009 EN 61000-4-8: 2010
EN 55011: 2009 + A1 EN 61000-4-3: 2006 + A1 + A2 EN 61000-4-11: 2004
EN 61326-1: 2013 EN 61000-4-4: 2004 + A1

Orbitrap Fusion and Orbitrap ID-X
FCC Compliance Statement
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.
Low Voltage Directive 2014/35/EU
This device complies with Low Voltage Directive 2014/35/EU and the harmonized safety standard IEC/EN/CSA/
UL 61010-1, 3rd Edition.
EMC Directive 2014/30/EU and other EMC test standards
This device was tested by TÜV Rheinland of North America and complies with the following EMC standards:
47 CFR 15, Subpart B, Class A: 2012 EN 61326-1: 2013 EN 61000-4-4: 2004 + A1
CISPR 11: 2009 + A1 EN 61000-3-2: 2006 + A1 + A2 EN 61000-4-5: 2006
AS/NZS CISPR 22: 2009 + A1 EN 61000-3-3: 2008 EN 61000-4-6: 2009
ICES-003: 2012 EN 61000-4-2: 2009 EN 61000-4-8: 2010
EN 55011: 2009 + A1 EN 61000-4-3: 2006 + A1 + A2 EN 61000-4-11: 2004
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.

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 [e-mail] 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.

Thermo Scientific Orbitrap Tribrid Series Getting Started Guide vii
C
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiii
Accessing Documentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiii
Viewing the Product Manuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv
Accessing the Help Menu Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv
Viewing Online User Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv
Providing Documentation Feedback. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
License for the 1M Option . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Special Notices, Symbols, and Cautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Model Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xviii
Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Ionization Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
H-ESI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
APCI Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
APPI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
NSI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
LC Flow Rate Ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Types of Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Templates in Thermo Xcalibur Instrument Setup (Method Editor) . . . . . . . . . . 7
Chapter 2 Setting Up the Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Preparing the Mass Spectrometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Installing the Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Preparing the Spray Insert for the Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Installing the Spray Insert . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Spray Insert Positions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Adjusting the Spray Direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Removing the Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Installing the NSI Source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chapter 3 Connecting the Inlet Plumbing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Guidelines for PEEK Fittings and Tubing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Setting Up the Syringe and Syringe Pump. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Plumbing Schematics for the Sample Introduction Techniques. . . . . . . . . . . . . 20
Contents

Contents
viii Orbitrap Tribrid Series Getting Started Guide Thermo Scientific
Setting Up the Inlet Plumbing for the Sample Introduction Techniques. . . . . . 21
Setting Up the Inlet for Direct Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Setting Up the Inlet for HPLC with an Autosampler. . . . . . . . . . . . . . . . . . . 22
Setting Up the Inlet for High-Flow Infusion Without an Autosampler . . . . . 23
Setting Up the Inlet for Loop Injections (Flow-Injection Analysis) . . . . . . . . 24
Connecting the Grounding Union to the H-ESI Spray Insert . . . . . . . . . . . . . . 26
Chapter 4 Preparing the System for Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Setting Up the Syringe Pump for Direct Infusion . . . . . . . . . . . . . . . . . . . . . . . 27
Setting Up the Mass Spectrometer for Calibration. . . . . . . . . . . . . . . . . . . . . . . 28
Chapter 5 Establishing a Stable Ionization Spray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Evaluating the Spray Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Optimizing the Ion Source Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Chapter 6 Calibrating the Mass Spectrometer in H-ESI Mode . . . . . . . . . . . . . . . . . . . . . . . .37
Calibration Solution Peak Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Peak Values for the Traditional Calibration Solutions . . . . . . . . . . . . . . . . . . 38
Peak Values for the Pierce FlexMix Calibration Solution. . . . . . . . . . . . . . . . 40
Running the Calibration Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Chapter 7 Using Tune to Acquire Sample Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
Setting Up the LC/MS System for Analyte Optimization . . . . . . . . . . . . . . . . . 47
Setting Up the Inlet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Configuring the Syringe Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Configuring the LC Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Configuring the Ion Source for Reserpine (Example). . . . . . . . . . . . . . . . . . . 49
Setting the Instrument’s Optimal Pressure. . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Defining the Scan Parameters for Precursor Optimization. . . . . . . . . . . . . . . . . 50
Defining the MS/MS Scan Parameters for Precursor Optimization . . . . . . . . 50
Optimizing the Isolation Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Optimizing the Ion Source Parameters on an Analyte . . . . . . . . . . . . . . . . . . 53
Optimizing the Fragmentation Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Defining the Scan Parameters for an MS3Scan . . . . . . . . . . . . . . . . . . . . . . . . . 54
Acquiring a Data File by Using the Tune Application . . . . . . . . . . . . . . . . . . 55
Chapter 8 Using the Xcalibur Data System to Acquire Data . . . . . . . . . . . . . . . . . . . . . . . . . .57
Selecting the External Start Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Acquiring a Data File by Using the Xcalibur Data System. . . . . . . . . . . . . . . . . 59
Appendix A Using Basic Tune Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
Opening the Tune Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Setting the Instrument System Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Setting the Instrument Power Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Checking the Instrument Readback Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Contents
Thermo Scientific Orbitrap Tribrid Series Getting Started Guide ix
Setting the Tune Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Using the MSnSetting Table in the Define Scan Pane . . . . . . . . . . . . . . . . . . . 67
Importing a Mass List from a File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Exporting a Mass List to a File. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Modifying Rows in the MSnSetting Table . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Adding or Removing Scan Parameters from the MSnSetting Table. . . . . . . . 69
Using the History Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Using the Favorites Pane to Save System Settings . . . . . . . . . . . . . . . . . . . . . . . 70
Saving a Favorite State. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Loading a Favorite State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Applying a Favorite State. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Deleting a Favorite State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Renaming a Favorite State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Appendix B Flushing the Inlet Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73
Supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Flushing the Inlet Components After Calibration . . . . . . . . . . . . . . . . . . . . . . . 75
Appendix C Preparing the Reserpine Sample Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
Appendix D Preparing the High Mass Range Calibration Solution. . . . . . . . . . . . . . . . . . . . . .79
Supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Preparing the Enfuvirtide Calibration Solution . . . . . . . . . . . . . . . . . . . . . . . . . 81
Preparing the 0.15% Ammonium Hydroxide Solution . . . . . . . . . . . . . . . . . 81
Preparing the 0.15% Ammonium Hydroxide/ACN-50:50 Solution . . . . . . . 82
Preparing the Enfuvirtide Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Preparing the 0.1% Acetic Acid/ACN-50:50 Solution. . . . . . . . . . . . . . . . . . 83
Preparing the Enfuvirtide Final Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Appendix E Installation Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
Orbitrap Tribrid Series Chemicals Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Calibration Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Performance Specification Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .89
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .95

Contents
xOrbitrap Tribrid Series Getting Started Guide Thermo Scientific

Thermo Scientific Orbitrap Tribrid Series Getting Started Guide xi
F
Figure 1. MS ion source mount assembly and ion sweep cone . . . . . . . . . . . . . . . . . . . . . 11
Figure 2. Proper connection for the PEEK tubing and fitting (syringe adapter
assembly) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Figure 3. Schematics of the sample introduction techniques (examples) . . . . . . . . . . . . . . 20
Figure 4. Plumbing connections for the grounding union (H-ESI mode) . . . . . . . . . . . . . 26
Figure 5. Syringe pump settings box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Figure 6. Plot Chromatogram dialog box with the TIC option selected . . . . . . . . . . . . . . 32
Figure 7. Optimization page of the Ion Source pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Figure 8. Report Generation Options dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Figure 9. Traditional n-Butylamine calibration solution’s normal mass-range spectrum
in positive ion polarity mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Figure 10. Traditional Ultramark 1621 calibration solution’s normal mass-range
spectrum in negative ion polarity mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Figure 11. FlexMix calibration solution’s low-mass range spectrum in positive ion
polarity mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Figure 12. FlexMix calibration solution’s low-mass range spectrum in negative ion
polarity mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Figure 13. FlexMix solution’s normal-mass range spectrum in positive ion polarity
mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Figure 14. FlexMix solution’s normal-mass range spectrum in negative ion polarity
mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Figure 15. FlexMix solution’s high-mass range spectrum in positive ion polarity
mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Figure 16. FlexMix solution’s high-mass range spectrum in negative ion polarity
mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Figure 17. Calibration pane (example for Orbitrap Fusion and Orbitrap Fusion Lumos
MSs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Figure 18. Syringe pump settings box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Figure 19. CID-MS/MS scan spectrum with fragmentation (reserpine example) . . . . . . . . 52
Figure 20. CID-MS3scan spectrum with fragmentation (reserpine example). . . . . . . . . . . 55
Figure 21. Data Acquisition pane in the Tune window . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Figure 22. Run Sequence dialog box (partial) showing the selected start instrument . . . . . 58
Figure 23. Change Instruments In Use dialog box showing the MS as the start
instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Figure 24. Orbitrap Fusion Lumos Tune window showing the Define Scan pane . . . . . . . 62
Figures

Figures
xii Orbitrap Tribrid Series Getting Started Guide Thermo Scientific
Figure 25. Tune Preferences dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Figure 26. Activation Type selected and added to the MSnSetting Table (CID
example) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Figure 27. Example labeling for the Enfuvirtide stock solution . . . . . . . . . . . . . . . . . . . . . 83

Thermo Scientific Orbitrap Tribrid Series Getting Started Guide xiii
P
Preface
The Orbitrap Tribrid Series Getting Started Guide is intended for the following Thermo
Scientific™ mass spectrometer (MS):
• Orbitrap Fusion™ (also known as Fusion™)
• Orbitrap Fusion Lumos™ (also known as Lumos™)
• Orbitrap ID-X™ (also know as ID-X™)
This guide describes how to set up and calibrate the MS system. Also, because this guide uses
drawings of various connections and component parts to help illustrate procedures, be sure to
start from , no matter where it appears.
Contents
• Accessing Documentation
• Providing Documentation Feedback
• License for the 1M Option
• Special Notices, Symbols, and Cautions
• Model Differences
•ContactingUs
Accessing Documentation
The Orbitrap Tribrid Series MS includes complete documentation.
•Viewing the Product Manuals
•Accessing the Help Menu Options
•Viewing Online User Documentation
For system requirements, refer to the release notes on the software DVD.

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xiv Orbitrap Tribrid Series Getting Started Guide Thermo Scientific
Viewing the Product Manuals
The Thermo Fisher Scientific service engineer installs the instrument control applications and
the instrument manuals on the data system computer.
To view the product manuals
From the Microsoft™ Windows™ taskbar, choose Start > All Apps (Windows 10) or
All Programs (Windows 7) > Thermo Instruments > model x.x, and then open the
applicable PDF file.
Accessing the Help Menu Options
Follow this procedure to view the Help systems for the instrument-control applications.
To view the Help
Do the following as applicable:
• Thermo Tune instrument-control application: Click the Options icon, , and
choose Tu n e H e l p .
• Thermo Xcalibur™ Method Editor application: Choose an option from the Help
menu (or press the F1 key).
Viewing Online User Documentation
Visit the Thermo Fisher Scientific website for product manuals and more.
To view user documentation from the Thermo Fisher Scientific website
1. Go to thermofisher.com.
2. Point to Services & Support and click Manuals on the left.
3. In the Refine Your Search box, search by the product name.
4. From the results list, click the title to open the document in your web browser, save it, or
print it.
To return to the document list, click the browser Back button.

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Thermo Scientific Orbitrap Tribrid Series Getting Started Guide xv
Providing Documentation Feedback
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.
License for the 1M Option
During the Orbitrap Fusion Lumos MS installation or upgrade process, the Thermo Fisher
Scientific field service engineer activates the optional 1M license (with purchase of the 1M
option).
Special Notices, Symbols, and Cautions
Make sure you understand the special notices, symbols, and caution labels in this guide. Most
of the special notices and cautions appear in boxes; those pertaining to safety also have
corresponding symbols. Some symbols are also marked on the instrument itself and can
appear in color or in black and white. For complete definitions, see Table 1.
Table 1. Notices, symbols, labels, and their meanings (Sheet 1 of 2)
Notice, symbol, or label Meaning
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
product.
Note Highlights information of general interest.
Tip Highlights helpful information that can make a task easier.

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xvi Orbitrap Tribrid Series Getting Started Guide Thermo Scientific
Caution: Read the cautionary information associated with this
task.
Chemical hazard: Observe safe laboratory practices and
procedures when handling chemicals. Only work with volatile
chemicals under a fume or exhaust hood. 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.
Heavy object: The Orbitrap Tribrid Series MS, excluding its
workbench, weighs over 227 kg (500 lb). Never try to detach
and move the instrument from its workbench; you can suffer
personal injury or damage the instrument.
Risk of electric shock: This instrument uses voltages that can
cause electric shock and personal injury. Before servicing the
instrument, shut it down and disconnect it from line power.
While operating the instrument, keep covers on.
Risk of eye injury: Eye injury can occur from splattered
chemicals, airborne particles, or sharp objects. Wear safety
glasses when handling chemicals or servicing the instrument.
Risk of laser radiation—Orbitrap Fusion Lumos MS with the
optional ultraviolet photodissociation (UVPD) module only:
Failure to understand and comply with laser cautions and
operating instructions in the UVPD Module User Guide can
result in property damage or serious injuries to the user.
Trip obstacle: Be aware of cords, hoses, or other objects located
on the floor.
Table 1. Notices, symbols, labels, and their meanings (Sheet 2 of 2)
Notice, symbol, or label Meaning

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Thermo Scientific Orbitrap Tribrid Series Getting Started Guide xvii
Model Differences
This table lists the required number of forepumps and the available options for the
Orbitrap Tribrid Series MSs.
Instrument Number of
forepumps
Available options
EASY-IC
ion source
EASY-ETD
ion source
Advanced
peak
determination
1M Orbitrap
resolution
UVPD
laser module
Orbitrap Fusion Lumos 2    
Orbitrap Fusion 1  
Orbitrap ID-X 1 

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xviii Orbitrap Tribrid Series Getting Started Guide Thermo Scientific
Contacting Us
Contact Email Telephone QR Codea
aYou can use your smartphone to scan a QR Code, which opens your email application or browser.
U.S. Technical Support [e-mail] (U.S.) 1 [phone]
U.S. Customer Service
and Sales
[e-mail] (U.S.) 1 [phone]
Global Support To find global contact information or customize your request
1. Go to thermofisher.com.
2. Click Contact Us, select the country, and then select the type of support
you need.
3. At the prompt, type the product name.
4. Use the phone number or complete the online form.
To find product support, knowledge bases, and resources
Go to thermofisher.com/us/en/home/technical-resources.
To find product information
Go to thermofisher.com/us/en/home/brands/thermo-scientific.
Note To provide feedback for this document, go to surveymonkey.com/s/PQM6P62 or send an email message to
Technical Publications ([e-mail]).

Thermo Scientific Orbitrap Tribrid Series Getting Started Guide 1
1
Introduction
This chapter provides general information about the Orbitrap Tribrid Series MS. 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 Hardware Manual.
Contents
• Ionization Techniques
• LC Flow Rate Ranges
• Types of Buffers
• Templates in Thermo Xcalibur Instrument Setup (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 ion source manuals.
•H-ESI Mode (Typically preferred for polar compounds)
•APCI Mode (Typically preferred for medium polar compounds)
•APPI Mode (Typically preferred for certain polar and nonpolar compounds)
•NSI Mode (Typically preferred for peptides and proteins)
Note To ensure the proper operation of the MS, Thermo Fisher Scientific recommends
that you perform the daily preventive maintenance described in the Hardware Manual.
Note For instruments with the optional EASY-ETD™ source, refer to the EASY-ETD and
EASY-IC Ion Sources User Guide. For an Orbitrap Fusion Lumos MS with the optional
UVPD device, refer to the UVPD Module User Guide.

1Introduction
Ionization Techniques
2Orbitrap Tribrid Series Getting Started Guide Thermo Scientific
H-ESI Mode
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 MS 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.
The upper limit for the system’s flow rate is 3000 μL/min. However, for optimum H-ESI
performance, vary the flow rate1into the MS over a range of 1–1000 μL/min, and configure
the H-ESI spray insert for the high- or low-flow2range as applicable. This flow rate range is
optimal for a wide range of separation techniques: capillary electrophoresis (CE), capillary
electrochromatography (CEC), analytical LC, capillary LC, and microbore LC. For the lower
range, make sure that the H-ESI spray insert contains the low-flow metal needle insert. See
Table 2 for H-ESI 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.
1The H-ESI spray insert can generate ions from liquid flows as low as 1 μL/min.
2For the range of 1–50 μL/min, install the low-flow needle insert (OPTON-30139) into the H-ESI spray insert.