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