Orbitrap Fusion Series Getting Started Guide Version A

Orbitrap Fusion Series Getting Started Guide Version A
Orbitrap Fusion Series
Getting Started Guide
80011-97003 Revision A
July 2015
© 2015 Thermo Fisher Scientific Inc. All rights reserved.
EASY-Max NG, LTQ Velos, Optima, and Orbitrap Fusion Lumos are trademarks; Unity is a registered service
mark; and Accela, Hypersil GOLD AQ, LTQ, Orbitrap, Orbitrap Fusion, Pierce, Thermo Scientific, Tribrid,
and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc. in the United States. Fisher Scientific is
a registered trademark of Fisher Scientific Co. in the United States.
The following are registered trademarks in the United States and other countries: Excel, Microsoft, and
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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. 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.
Regulatory compliance results for the following Thermo Scientific™ products:
•
•
Orbitrap Fusion Lumos
Orbitrap Fusion
Orbitrap Fusion Lumos
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 61000-4-8: 2010
EN 61000-3-3: 2008
EN 61000-4-11: 2004
EN 61000-4-2: 2009
EN 61326-1: 2013
EN 61000-4-3: 2006, A2: 2010
CISPR 11: 2009, A1: 2010
EN 61000-4-4: 2004, A1: 2010
ICES-003 Issue 5: 2014
EN 61000-4-5: 2006
CFR 47, FCC Part 15, Subpart B, Class A: 2015
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 2006/95/EC and harmonized standard EN 61010-1:2010
(3rd Edition).
Orbitrap Fusion
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 61010-1:2010
(3rd Edition).
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 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
Orbitrap Fusion Series Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiii
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv
Installation Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Calibration Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Performance Specification Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xvi
Orbitrap Fusion Series Chemicals Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Cautions and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xix
Thermo Scientific
Chapter 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Ionization Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Using H-ESI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Using APCI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Using APPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Using NSI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
LC Flow Rate Ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Types of Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Templates in Thermo Xcalibur Instrument Setup (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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Removing the API Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Preparing the Spray Insert for the EASY-Max NG API Source . . . . . . . . . . . . . 14
Installing the Spray Insert . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Adjusting the Spray Direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chapter 3
Connecting the Inlet Plumbing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Sample Introduction Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Direct Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
High-Flow Infusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Loop Injection (Flow-Injection Analysis). . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
High-Performance Liquid Chromatography (HPLC) with an Autosampler. . 19
Orbitrap Fusion Series Getting Started Guide
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Plumbing Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Setting Up the Syringe Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Setting Up the Inlet Plumbing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Setting Up the Inlet for Direct Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Setting Up the Inlet for High-Flow Infusion . . . . . . . . . . . . . . . . . . . . . . . . . 24
Setting Up the Inlet for Manual or Auto-Loop Injections . . . . . . . . . . . . . . . 27
Setting Up the Inlet for an LC/MS System with an Autosampler . . . . . . . . . . . 29
Connecting the Grounding Union to the H-ESI Spray Insert . . . . . . . . . . . . . . 30
Chapter 4
Using the Syringe Pump and Divert/Inject Valve . . . . . . . . . . . . . . . . . . . . . . . . . .31
Syringe Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Divert/Inject Valve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Controlling the Divert/Inject Valve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Chapter 5
Preparing the System for Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
Pumping Down the Mass Spectrometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Setting Up the Syringe Pump for Direct Infusion . . . . . . . . . . . . . . . . . . . . . . . 39
Setting Up the Mass Spectrometer for Calibration. . . . . . . . . . . . . . . . . . . . . . . 40
Chapter 6
Establishing a Stable Ionization Spray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
Evaluating the Spray Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Optimizing the API Source Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Chapter 7
Calibrating the Mass Spectrometer in H-ESI Mode . . . . . . . . . . . . . . . . . . . . . . . .47
Running the Positive Ion Polarity Calibrations . . . . . . . . . . . . . . . . . . . . . . . . . 48
Running the Negative Ion Polarity Calibrations . . . . . . . . . . . . . . . . . . . . . . . . 49
Chapter 8
Acquiring Sample Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Using the Tune Application to Acquire Sample Data . . . . . . . . . . . . . . . . . . . . 51
Setting Up the LC/MS System for Analyte Optimization . . . . . . . . . . . . . . . 52
Defining the Scan Parameters for Precursor Optimization. . . . . . . . . . . . . . . 54
Optimizing the Fragmentation Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Defining the Scan Parameters for an MS3 Scan . . . . . . . . . . . . . . . . . . . . . . . 57
Acquiring a Data File by Using the Tune Application . . . . . . . . . . . . . . . . . . 58
Using the Xcalibur Data System to Acquire Sample Data . . . . . . . . . . . . . . . . . 60
Appendix A Using Basic Tune Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
Opening the Tune Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Setting the Instrument Power Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Checking the Instrument Readback Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Controlling the Syringe Pump. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Setting the Data Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Setting the Ion Polarity Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Setting the Instrument Pressure Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
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Orbitrap Fusion Series Getting Started Guide
Thermo Scientific
Contents
Setting the Tune Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Using the MSn Setting Table in the Define Scan Pane . . . . . . . . . . . . . . . . . . . 69
Using the History Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Using the Favorites Pane to Save System Settings . . . . . . . . . . . . . . . . . . . . . . . 71
Appendix B Flushing the Inlet Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75
Supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Flushing the Inlet Components After Calibration . . . . . . . . . . . . . . . . . . . . . . . 77
Appendix C Preparing the Reserpine Sample Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79
Appendix D Preparing the High Mass Range Calibration Solution . . . . . . . . . . . . . . . . . . . . . .81
Supplies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Preparing the Enfuvirtide Calibration Solution . . . . . . . . . . . . . . . . . . . . . . . . . 83
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .93
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Orbitrap Fusion Series Getting Started Guide
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Orbitrap Fusion Series Getting Started Guide
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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.
Thermo Scientific
Thermo Xcalibur Instrument Setup window showing the system templates
(Orbitrap Fusion Lumos MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
MS API source mount assembly and ion sweep cone . . . . . . . . . . . . . . . . . . . . 10
EASY-Max NG API source with H-ESI spray insert (top, front view). . . . . . . . 12
API source connection to the MS mount assembly (installed ion sweep
cone) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Front-to-back adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Rotational adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Schematics of the sample introduction techniques (examples) . . . . . . . . . . . . . . 20
Proper connection for the PEEK tubing and fitting (syringe adapter
assembly) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Plumbing connection for the syringe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Plumbing connections for direct infusion (H-ESI mode) . . . . . . . . . . . . . . . . . . 23
Plumbing connection between the LC union and the union Tee . . . . . . . . . . . 25
Plumbing connection between the union Tee and the divert/inject valve . . . . . 25
Plumbing connection between the union Tee and the grounding union . . . . . . 26
Divert/inject valve setup for manual loop injection . . . . . . . . . . . . . . . . . . . . . . 28
Plumbing connections for manual loop injection (APCI mode) . . . . . . . . . . . . . 29
Plumbing connections for the grounding union (H-ESI mode) . . . . . . . . . . . . . 30
Syringe pump setup (top view) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Divert/inject valve positions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Divert/inject valve plumbed as a loop injector and as a divert valve . . . . . . . . . . 34
Divert/inject valve (front view) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Power entry module (right side of the instrument) . . . . . . . . . . . . . . . . . . . . . . 36
By Board page in the Status pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Diagnostics pane showing the Bake Orbitrap Chamber parameter table . . . . . . 38
Plot Chromatogram dialog box with the TIC option selected . . . . . . . . . . . . . . 42
Optimization page of the Ion Source pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Report Generation Options dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Calibration pane (example). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
CID-MS/MS scan spectrum with fragmentation (reserpine example) . . . . . . . . 56
CID-MS3 scan spectrum with fragmentation (reserpine example). . . . . . . . . . . 58
Data Acquisition pane in Tune . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Run Sequence dialog box (partial) showing the selected start instrument . . . . . 60
Change Instruments In Use dialog box showing the MS as the start
instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Tune window showing the Define Scan pane . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Orbitrap Fusion Series Getting Started Guide
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Figures
Figure 34.
Figure 35.
Figure 36.
Figure 37.
Figure 38.
Figure 39.
Figure 40.
Figure 41.
Figure 42.
Figure 43.
Figure 44.
xii
Power mode icons showing the selected icon (mode) . . . . . . . . . . . . . . . . . . . .
Toggle button for the syringe modes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Syringe parameter box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Toggle button for the data types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Toggle button for the instrument polarity modes . . . . . . . . . . . . . . . . . . . . . . .
Toggle button for the instrument pressure modes . . . . . . . . . . . . . . . . . . . . . . .
Tune Preferences dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Activation Type selected and added to the MSn Setting Table (CID
example) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Favorites pane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
State name box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Example labeling for the Enfuvirtide stock solution . . . . . . . . . . . . . . . . . . . . .
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Preface
The Orbitrap Fusion Series Getting Started Guide describes how to set up and calibrate the
Thermo Scientific™ Orbitrap Fusion™ Series Tribrid™ mass spectrometer (MS).
Contents
• Orbitrap Fusion Series 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.
Orbitrap Fusion Series Models
The Orbitrap Fusion Series documentation is intended for the following instrument models:
• Orbitrap Fusion—Requires one forepump.
• Orbitrap Fusion Lumos™—Requires two forepumps.
Thermo Scientific
Orbitrap Fusion Series Getting Started Guide
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Preface
Related Documentation
The Orbitrap Fusion Series mass spectrometer includes complete documentation. In addition
to this guide, you can also access the following documents as PDF files from the data system
computer:
• Orbitrap Fusion Series Preinstallation Requirements Guide
• Orbitrap Fusion Series Getting Connected Guide
• Orbitrap Fusion Series Hardware Manual
• Ion Max NG and EASY-Max NG Ion Sources User Guide
• EASY-ETD and EASY-IC Ion Sources User Guide (for instruments with the ETD or
Internal Calibration configuration)
• Safety and Regulatory Guide
The Orbitrap Fusion Series also ships 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 a 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 software application, choose Start >
All Programs > Thermo Instruments > Manuals > LC Devices and so on.
The Orbitrap Fusion Series 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 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
Orbitrap Fusion Series Getting Started Guide
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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 Orbitrap Fusion Series MS ships with several kits. However, for the procedures in this
guide, the following kits provide the necessary components:
• Calibration Kit
• Performance Specification Kit
• Orbitrap Fusion Series Chemicals Kit
For a full list of the Orbitrap Fusion Series kits and their contents, refer to the Orbitrap Fusion
Series Hardware Manual.
Calibration Kit
Table 1. Calibration Kit (P/N 80000-62078) (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
Orbitrap Fusion Series Getting Started Guide
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Preface
Table 1. Calibration Kit (P/N 80000-62078) (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, gas tight, 500 μL
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
—
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Quantity
Part number
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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
Orbitrap Fusion Series Chemicals Kit
IMPORTANT Be aware of the following storage precautions.
• Calibration and reserpine solutions—Refrigerate the containers after opening. For
long-term storage, keep refrigerated at 2–8 °C (36–46 °F).
• Enfuvirtide—Refrigerate the container after opening. For long-term storage, keep
refrigerated at –25 to –15 °C (–13 to 5 °F).
Table 3. Orbitrap Fusion Series Chemicals Kit (P/N 80000-62049)
Item
Quantity
Part number
Positive calibration solution, n-Butylamine, 10 mL
(Pierce™ LTQ™ Velos™ ESI Positive Ion Calibration Solution, P/N 88323)
2
HAZMAT-01-00061
Negative calibration solution, Ultramark 1621, 10 mL
(Pierce LTQ ESI Negative Ion Calibration Solution, P/N 88324)
2
HAZMAT-01-00062
Enfuvirtide, 90 mg
1
HAZMAT-01-00083
Reserpine standard solution, 100 pg/μL, 1 mL
5
HAZMAT-01-00081
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.
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Preface
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.
The Orbitrap Fusion Series Getting Started Guide contains the following caution-specific
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.
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Preface
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
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
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
Orbitrap Fusion Series Getting Started Guide
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Preface
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1
Introduction
This chapter provides general information about the Orbitrap Fusion Series Tribrid 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 Orbitrap Fusion Series 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
Orbitrap Fusion Series 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 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)
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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.
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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.
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1
Introduction
LC Flow Rate 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 Rate 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 ion transfer tube temperature and the flow rates for the
sheath, auxiliary, and sweep gases.
• For APCI mode, adjust the ion transfer tube and vaporizer temperatures, and the flow
rates for the sheath, auxiliary, and sweep gases.
• For NSI mode, adjust 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.
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1 Introduction
LC Flow Rate 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
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
LC flow
rate
(μL/min)
a
Positive and negative polarity modes
b
Arbitrary
Table 6. Guidelines for setting operating parameters for LC/APCI/MS
Corona
discharge
current (μA)b
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)
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
2
275
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Introduction
Types of Buffers
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 Orbitrap Fusion Series 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.
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.
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1 Introduction
Templates in Thermo Xcalibur Instrument Setup (Method Editor)
Templates in Thermo Xcalibur Instrument Setup (Method Editor)
Use the Method Editor (Figure 1) that opens in the Thermo Xcalibur™ Instrument Setup
window to create the instrument methods for your experiments. 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.
Method Editor provides default system templates for several types of experiments:
metabolomics, proteomics, and small molecules.
Figure 1.
Thermo Xcalibur Instrument Setup window showing the system templates (Orbitrap Fusion Lumos MS)
Click the tree
icon to display
the templates.
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Introduction
Templates in Thermo Xcalibur Instrument Setup (Method Editor)
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2
Setting Up the API Source
This chapter provides information about setting up the API source. Use the Thermo Scientific
EASY-Max NG™ API source for H-ESI, APCI, and APPI experiments. For NSI experiments,
use one of the compatible Thermo Scientific nanospray sources.
The EASY-Max NG source ships with the Orbitrap Fusion Series MS 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 80000-62060), which includes the APCI spray insert. For
APPI experiments, order the APPI Interface Kit (P/N OPTON-30185).
Contents
• Preparing the Mass Spectrometer
• Installing or Removing the API Source
• Preparing the Spray Insert for the EASY-Max NG 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.
 To prepare the mass spectrometer
1. Complete all data acquisition, if any.
2. Open the Tune window (see page 64).
3. Place the mass spectrometer in Off mode (see page 65).
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.
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2
Setting Up the API Source
Preparing the Mass Spectrometer
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.
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.
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2 Setting Up the API Source
Installing or Removing the API Source
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 Orbitrap Fusion Series 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
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.
–
Thermo Scientific
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).
Orbitrap Fusion Series Getting Started Guide
11
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)
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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 Orbitrap Fusion
Series 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.
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Setting Up the API Source
Preparing the Spray Insert for the EASY-Max NG 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 65).
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 EASY-Max NG 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
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.
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2 Setting Up the API Source
Preparing the Spray Insert for the EASY-Max NG API Source
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)
Thermo Scientific
Use the center mark to position the spray closest to the
entrance of the mass spectrometer.
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Setting Up the API Source
Preparing the Spray Insert for the EASY-Max NG 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.
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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 Calibration Kit and Performance Specification Kit contain the required components for
the inlet plumbing connections (see Table 1 and Table 2 in the Preface).
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 Orbitrap Fusion Series MS has 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
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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.”
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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.”
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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
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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 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.
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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.
LC union
Plumbing connection for the syringe
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.
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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 tubing
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
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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.
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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
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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 tubings
Two-piece
fingertight fitting
Grounding
union
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Two-piece
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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.”
• 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).
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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).
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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.
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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
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4
Using the Syringe Pump and Divert/Inject Valve
This chapter describes the external syringe pump and divert/inject valve that ship with the
Orbitrap Fusion Series MS. For information about installing these components, refer to the
Orbitrap Fusion Series 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 3 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.
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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).
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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.
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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 in 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
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5
Preparing the System for Calibration
This chapter describes how to prepare the Orbitrap Fusion Series system before you calibrate
the mass spectrometer.
Note
• You must pump down the instrument for the full 15 hours and complete the bakeout
period before you start the instrument calibration process.
• The figures shown in this chapter exclude the features for the ETD and Internal
Calibration (IC) options. If the optional EASY-ETD or EASY-IC ion source is
installed in your mass spectrometer, refer to the EASY-ETD and EASY-IC Ion Sources
User Guide for the applicable figures.
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 spectrometers, pump down the vacuum system
for at least 15 hours and bakeout the Orbitrap FT chamber for 12 hours during that period.
 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).
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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 forepump 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.
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Preparing the System for Calibration
Pumping Down the Mass Spectrometer
8. Open the Status pane in the Tune window (see page 64), click the downward arrow, and
then choose By Board (Figure 22).
Figure 22. By Board page in the Status pane
Opens and closes the
selected pane.
Click to select By Function
or By Board.
Includes the source
vacuum gauges.
Includes the Orbitrap
ultra-high vacuum (UHV)
gauge.
9. Check the readback values for the source and Orbitrap (UHV) pressure gauges as follows:
• Double-click Source, and then verify that the Source Pressure and Ion Gauge
Pressure readback values are below the operating threshold limits (see Table 10).
• Double-click FT Vacuum, and then verify that the UHV Pressure readback value is
below the operating threshold limit (see Table 10).
Table 10. Threshold limits for the vacuum pressure gauges
Instrument
Source pressure
(Torr)
Orbitrap Fusion Lumos
Less than 4.5
Orbitrap Fusion
Less than 3.0
Ion gauge
pressure (Torr)
UHV pressure
(Torr)
1.5 × 10–4
5 × 10–8
Normal readback measurements show a green square ( ). If the vacuum pressure
values are normal, follow the next procedure “To bakeout the Orbitrap FT chamber.”
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Preparing the System for Calibration
Pumping Down the Mass Spectrometer
 To bakeout the Orbitrap FT chamber
1. When the vacuum pressures become normal, place the mass spectrometer in Off mode
(see page 65).
2. Click the Diagnostics icon (lower left corner) to open the Diagnostics pane.
3. In the System - Vacuum list, select Bake Orbitrap Chamber (Figure 23), and then set
the following values in the parameters table:
• In the Bake Time (hours) box, enter 12.
• In the Cool Time (hours) box, enter 2.
Figure 23. Diagnostics pane showing the Bake Orbitrap Chamber parameter table
Select to bakeout the
Orbitrap chamber.
Search box
4. Click Start.
After the 14-hour period, you can turn on the LC system, if applicable.
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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 31.
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 “Orbitrap Fusion Series Chemicals Kit” in the Preface.
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 on page 21).
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 22.
b. Follow steps 2 and 3 in “To connect an infusion line between the LC union and the
grounding union” on page 23.
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.”
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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 and makes a
change record in the History pane.
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 65).
This completes the setup for calibrating the mass spectrometer. Go to Chapter 6,
“Establishing a Stable Ionization Spray.”
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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:
–
3 μL/min in the Current LC Flow box
–
3 μL/min flow rate and 500 μL syringe volume
–
Positive ion polarity mode
–
Profile data type
c. Go to step 4.
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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 65).
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 24).
, to open the Plot Chromatogram dialog
Figure 24. Plot Chromatogram dialog box with the TIC option selected
b. Select the Spray Stability check box to monitor the RSD of the desired ion current.
c. Select the TIC option.
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Establishing a Stable Ionization Spray
Optimizing the API Source Parameters
d. Click OK to plot the full 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.”
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.
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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 25).
Figure 25. 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 26).
Figure 26. Report Generation Options dialog box
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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” in Appendix A.
4. Optimize the remaining source parameters.
5. (Optional) Save the parameters’ state in the Favorites pane (see page 72). For additional
information about the Favorites pane, refer to the Tune Help.
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Establishing a Stable Ionization Spray
Optimizing the API Source Parameters
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7
Calibrating the Mass Spectrometer in H-ESI Mode
This chapter describes how to calibrate the Orbitrap Fusion Series MS in H-ESI mode by
having the syringe pump introduce the ESI calibration solution into the instrument at a
steady flow rate.
Calibration parameters are mass spectrometer parameters whose values do not vary with the
type of experiment. In positive mode, you can calibrate the ion optics, linear ion trap,
quadrupole, Orbitrap, and ETD source, if your instrument includes this option. In negative
mode, you can calibrate the ion optics, linear ion trap, and Orbitrap.
Note
• Calibrate the mass spectrometer in H-ESI mode before acquiring data in H-ESI, NSI,
APCI, or APPI 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 detector.
• The figures shown in this chapter exclude the features for the ETD and Internal
Calibration (IC) options. If the optional EASY-ETD or EASY-IC ion source is
installed in your mass spectrometer, refer to the EASY-ETD and EASY-IC Ion Sources
User Guide for the applicable figures.
Contents
• Running the Positive Ion Polarity Calibrations
• Running the Negative Ion Polarity Calibrations
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Calibrating the Mass Spectrometer in H-ESI Mode
Running the Positive Ion Polarity Calibrations
Running the Positive Ion Polarity Calibrations
Always run the positive ion polarity calibrations before the negative ion polarity calibrations.
IMPORTANT
• Before you continue, verify that you have set up the API source (Chapter 2), prepared
the system for calibrating in positive H-ESI mode (Chapter 5), and verified that the
infused calibration solution produces a stable ionization spray (Chapter 6).
• To minimize the possibility of cross-contamination, use a different syringe and length
of PEEK tubing for each type of calibration solution.
 To calibrate the mass spectrometer in positive polarity mode
1. Open the Tune window (see page 64).
2. Click Calibration to open the Calibration pane (Figure 27).
Figure 27. Calibration pane (example)
Calibration tab
Calibration pane
3. Verify that the Skip Spray Stability Evaluation check box is clear so that this test runs.
4. (Optional) Select the Set System to Standby on Completion check box.
5. Click the arrow next to the Positive check box to display the calibration categories (not
shown in the figure).
6. Select the Ion Optics check box.
7. Click Start, and then review the real-time plot of the mass spectrum.
After the Tune parameters reach their specified settings, the calibration process begins and
the status area appears in the Calibration pane. After completing the calibration, the Tune
application adds a change record to the History pane under History Logs.
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Calibrating the Mass Spectrometer in H-ESI Mode
Running the Negative Ion Polarity Calibrations
8. Run the calibration for each of the remaining categories—one at a time and in the order
specified in the Tune window.
IMPORTANT The Predictive AGC calibration depends on the other calibrations.
Therefore, you must run this as the last positive calibration.
Tip After the calibration, you see either a green check ( ) adjacent to the calibration
name to indicate a successful calibration or a red X mark ( ) to indicate a failed
calibration.
A date appears in the Last Calibrated column for each successful calibration test. A date
does not appear for failed calibrations.
This completes the positive polarity calibration process.
IMPORTANT Before you run the negative polarity calibrations, follow the procedure “To
flush the inlet components” in Appendix B.
Running the Negative Ion Polarity Calibrations
Make sure that you have completed the positive ion polarity calibrations and flushed the inlet
components before you start the negative ion polarity calibrations.
 To calibrate the mass spectrometer in negative polarity mode
1. Load another clean, 500 μL syringe with 500 μL of the ESI negative calibration solution.
2. In the Calibration pane, verify that the Skip Spray Stability Evaluation check box is
clear so that this test runs (Figure 27).
3. (Optional) Select the Set System to Standby on Completion check box.
4. Click the arrow next to the Negative check box to display the calibration categories.
5. Select the Ion Optics check box.
6. Click Start, and then review the real-time plot of the mass spectrum.
The status area appears in the pane. The Tune application adds change records to the
History pane under History Logs.
7. Run the calibration for each of the remaining categories—one at a time and in the order
specified in the Tune window.
This completes the negative polarity calibration process. 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” in Appendix B.
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Calibrating the Mass Spectrometer in H-ESI Mode
Running the Negative Ion Polarity Calibrations
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8
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.
You can follow these procedures with any suitable analyte. For demonstration purposes only,
the Thermo Fisher Scientific field service engineer infuses a 50 fg/μL reserpine sample
solution. See Appendix C, “Preparing the Reserpine Sample Solution.”
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
Follow these procedures:
1. Setting Up the LC/MS System for Analyte Optimization
2. Defining the Scan Parameters for Precursor Optimization
3. Optimizing the Fragmentation Parameters
4. (Optional) Defining the Scan Parameters for an MS3 Scan
5. Acquiring a Data File by Using the Tune Application
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Acquiring Sample Data
Using the Tune Application to Acquire Sample Data
Setting Up the LC/MS System for Analyte Optimization
Follow these procedures:
• To set up the inlet
• To configure the syringe pump
• To configure the LC pump
• To configure the API source for the reserpine example
• To set the instrument’s optimal pressure
 To set up the inlet
Do one of the following:
• To infuse an analyte, set up the inlet for high-flow infusion (see “Setting Up the Inlet
for High-Flow Infusion” on page 24).
• To infuse the reserpine sample solution, set up the inlet for manual-loop injection
(see “Setting Up the Inlet for Manual or Auto-Loop Injections” on page 27).
 To configure the syringe pump
1. In the Tune window, place the mass spectrometer in On mode.
The mass spectrometer begins scanning and applies high voltage to the spray insert. A
real-time mass spectrum appears in the Tune window.
2. Open the syringe parameters box, and then enter the following:
• 5 for the flow rate (μL/min)
• 500 for the Volume (μL)
The Tune application automatically saves the parameter values.
3. Click Syringe On (Off ) to start the syringe pump.
4. Verify that the inlet plumbing connections do not leak.
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8 Acquiring Sample Data
Using the Tune Application to Acquire Sample Data
When controlling the LC pump through the Xcalibur data system, use the Direct Control
dialog box to turn off the solvent flow. For example, to turn on the solvent flow from an
Accela™ pump, use the following procedure.
 To configure the LC pump
1. In the Xcalibur Instrument Setup window, click the icon for the LC pump.
2. In the menu bar, choose pump model > Direct Control to open the Direct Control
dialog box.
3. Click the tab for the LC pump, and then select the Take Pump Under Control check
box.
4. In the Flow box, type 0.4 (in mL/min).
5. Click the Start button.
6. Verify that the inlet plumbing connections do not leak.
 To configure the API source for the reserpine example
In the Tune window, open the Ion Source page in the Ion Source pane, and set the
parameters as listed in the following table.
Table 12. Parameter settings on the Ion Source page
Parameter
Setting
Positive Ion Spray Voltage (V)
3500
Sheath Gas (Arb)
50
Aux Gas (Arb)
20
Sweep Gas (Arb)
2
Ion Transfer Tube Temp (°C)
350
Vaporizer Temp (°C)
500
 To set the instrument’s optimal pressure
Do one of the following:
• For small molecules, bottom-up, and top-down protein experiments, click Standard
Pressure Mode (Intact Protein Mode) to select standard pressure mode.
Use this setting for the reserpine example.
• For intact protein experiments and top-down experiments with large fragment ions,
click Intact Protein Mode (Standard Pressure Mode) to select intact protein mode.
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Acquiring Sample Data
Using the Tune Application to Acquire Sample Data
Defining the Scan Parameters for Precursor Optimization
Before you perform the experiment, define the scan parameters, and then check the isolation
window for the analyte to ensure the effective isolation of the target ion.
Follow these procedures:
1. To define the MS/MS scan parameters for precursor optimization
2. To optimize the isolation window
3. To optimize the API source parameters on an analyte
 To define the MS/MS scan parameters for precursor optimization
1. In the Tune window, click the Define Scan tab, and then select the MS2 Scan type.
2. Set the scan parameters that are appropriate for your analyte.
See Table 13 for an example using the 50 fg/μL reserpine.
Table 13. MS/MS scan parameters (reserpine example)
Parameter
Value
Parameter
Value
Scan Type
MS2 Scan
Detector Type
Ion Trap
Isolation Mode
Ion Trap
Ion Trap Scan Rate
Normal
Isolation Width (m/z)
12
Mass Range
Normal
Activation Type
CID
Scan Range (m/z)
165–615
RF Lens (%)
70
AGC Target
1.0e4
Typea Collision Energy (%) 33
a
Type is the selected activation type.
3. In the MSn Setting Table, enter the analyte’s precursor ion (for example, m/z 609).
For information about the MSn Setting Table, see “Using the MSn Setting Table in the
Define Scan Pane” in Appendix A.
4. Click Apply (or press ENTER).
The MS/MS scan starts and the mass spectrum appears.
 To optimize the isolation window
1. Display the chromatogram for the analyte’s precursor ion as follows:
a. Click the Plot Chromatogram icon,
box.
, to open the Plot Chromatogram dialog
b. Clear the Spray Stability check box.
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8 Acquiring Sample Data
Using the Tune Application to Acquire Sample Data
c. Select the User Defined m/z option, and then enter the analyte’s m/z value (for
example, m/z 609).
d. Click OK to plot the chromatogram.
2. In the Define Scan pane, in the Isolation Window (m/z) box, enter a slightly lower value.
Note The isolation window setting is typically m/z 1–3. The optimum value for the
isolation window is the smallest m/z width (instrument minimum width = m/z 0.1)
that gives a mass spectrum of maximum intensity for only the target ions. A narrow
isolation window increases the specificity of the scan results while a wider width
increases the signal at the expense of specificity.
When you obtain the optimum isolation window, the normalization level (NL) and
ionization time (IT) values are stable and the mass peak for the precursor ion is at its
maximum intensity and appears symmetrical. An isolation window value that is less
than optimum causes a substantial drop in the NL reading. A significant drop in
sensitivity indicates that the ions are not effectively isolated.
3. Enter successively smaller values for the isolation window, until the intensity of the
chromatogram is acceptable for your needs.
4. After you optimize the isolation window, compensate for minor changes in stability by
increasing the isolation window by an amount not to exceed m/z = 1.
5. In the Collision Energy (%) box, enter an appropriate value for the analyte (for
example, 33).
6. Click Apply (or press ENTER) to start the fragmentation process.
Figure 28 shows a spectrum example of a CID-MS/MS scan with fragmentation. For
descriptions of the spectrum header information and controls, refer to the Spectrum View
topic in the Tune Help.
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Acquiring Sample Data
Using the Tune Application to Acquire Sample Data
Figure 28. CID-MS/MS scan spectrum with fragmentation (reserpine example)
33% CID collision energy
 To optimize the API source parameters on an analyte
1. Follow the procedure “To optimize the API source parameters” on page 43, except use
your analyte solution, select m/z in the Signal Type list, and then type the m/z value for
the analyte in the m/z box.
2. (Optional) If you need to increase the sensitivity, optimize the following:
• Vaporizer Temperature (Ion Source page of the Ion Source pane)
• RF Lens (Define Scan pane)
• Spray direction (see “Adjusting the Spray Direction” on page 15)
3. (Optional) Save the parameters’ state in the Favorites pane (see page 72). For additional
information about the Favorites pane, refer to the Tune Help.
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8 Acquiring Sample Data
Using the Tune Application to Acquire Sample Data
Optimizing the Fragmentation Parameters
After you optimize the isolation window and the API source parameters for an MS/MS scan,
optimize the collision energy for optimum fragmentation.
 To optimize the collision energy
1. In the Define Scan pane, select the MS2 Scan type.
2. In the Collision Energy (%) box, enter an appropriate value (for example, 33).
3. Click Apply (or press ENTER).
4. Observe the mass spectrum from the specified fragmentation method, which is CID for
this chapter’s example.
5. Repeat steps 2 through 4, entering new values in 5% increments until you are satisfied
with the spectrum.
The normal range for CID collision energy is 20–40 percent. The normal range for HCD
collision energy is 10–50 percent.
Defining the Scan Parameters for an MS3 Scan
This section is optional. If you want to further improve the sensitivity of the data acquisition
example, use the MSn scan type (n = 3).
 To define the MS3 scan parameters
1. In the Define Scan pane, select the MSn Scan type.
2. Set the scan parameters that are appropriate for your analyte.
See Table 14 for an example using the 50 fg/μL reserpine.
Table 14. MS3 scan parameters (reserpine example)
Parameter
Value
Parameter
Value
Scan Type
MSn
Detector Type
Ion Trap
Isolation Mode
Ion Trap
Ion Trap Scan Rate
Normal
Isolation Width (m/z)
1.2
Mass Range
Normal
Activation Type
CID
Rf Lens (%)
70
AGC Target
1.0e4
m/z (setting table)
397
a
Type Collision Energy (%) 33
a
Scan
Type is the selected activation type.
3. Click Apply (or press ENTER).
Figure 29 shows a spectrum example of a CID-MS3 scan with fragmentation.
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Acquiring Sample Data
Using the Tune Application to Acquire Sample Data
Figure 29. CID-MS3 scan spectrum with fragmentation (reserpine example)
Acquiring a Data File by Using the Tune Application
 To acquire a sample data file
1. Open the Data Acquisition pane (Figure 30), and then do the following:
a. (Optional) To change the destination folder for the raw data, click the Browse icon.
The default folder location is in drive:\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).
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8 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 30. Data Acquisition pane in Tune
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.
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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 31).
The Yes in the Start Instrument column indicates the default start instrument for the
sequence run.
Figure 31. Run Sequence dialog box (partial) showing the selected start instrument
The LC device is the start instrument.
Change Instruments
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8 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 32).
Figure 32. 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.
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Acquiring Sample Data
Using the Xcalibur Data System to Acquire Sample Data
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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 Instrument Pressure Mode
• Setting the Tune Preferences
• Using the MSn Setting Table in the Define Scan Pane
• Using the History Pane
• Using the Favorites Pane to Save System Settings
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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 > model x.x Tune (Figure 33).
For information about the buttons and icons in the Tune application and what they
control, refer to the Tune Help.
Figure 33. Tune window showing the Define Scan pane
Three power mode icons
(On/Standby/Off)
Define Scan pane
Manual data
acquisition
Instrument
readback status
Monitor Mass Accuracy
Plot Chromatogram tool
Controls for the graphs
Panes: Status, History, and Favorites
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Setting the Instrument Power Mode
Setting the Instrument Power Mode
Use the three power mode icons in the Tune window (Figure 33) to set the mass
spectrometer’s power mode (on, standby, and off ).
When you remove the API source housing or the spray insert, the mass spectrometer
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 mass
spectrometer components under varying power conditions, refer to Chapter 6 in the
Orbitrap Fusion Series Hardware Manual.
 To set the instrument power mode
Click the icon for the power mode that you want (Figure 34).
The center of the selected icon changes from white to green.
Figure 34. 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 15 lists
the various readback states.
Table 15. 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.
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Using Basic Tune Functions
Controlling the Syringe Pump
Table 15. 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:
• Using the Favorites Pane to Save System Settings
• 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 35).
Figure 35. Toggle button for the syringe modes
 To set the syringe pump parameters
1. Click the dropdown arrow,
parameter box (Figure 36).
, next to the Syringe On/Off button, to open the syringe
Figure 36. 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.
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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 37).
Figure 37. 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 38).
Figure 38. Toggle button for the instrument polarity modes
Positive polarity
Negative polarity
Setting the Instrument Pressure Mode
 To set the instrument pressure mode
Click Standard Pressure Mode (Intact Protein Mode) to select the pressure mode that
you want (Figure 39).
Figure 39. Toggle button for the instrument pressure modes
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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 40).
Figure 40. 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.
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Using the MSn Setting Table in the Define Scan Pane
Using the MSn Setting Table in the Define Scan Pane
The MSn Setting Table appears when you select the MSn Scan type in the Define Scan pane.
Use this table to specify one or more precursor ions. To set different scan parameters for the
precursor ions, add the parameters to the MSn Setting 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 import a mass list from a file
• To export a mass list to a file
 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.
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Using Basic Tune Functions
Using the MSn Setting Table in the Define Scan Pane
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.
 To add or remove scan parameters from the table
Click the Table icon once to add the adjacent scan parameter to the table. Click it again
to remove the parameter from the table.
Figure 41 shows an example with Activation Type added to the MSn Settings Table.
Figure 41. Activation Type selected and added to the MSn Setting Table (CID example)
Activation Type is
selected.
These two
parameters
appear when you
select Activation
Type.
Activation Type is
added.
The Edit icon appears
when you select a
cell.
 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.
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Using Basic Tune Functions
Using the History Pane
 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.
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. 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 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.
Using the Favorites Pane to Save System Settings
You can manually save the current settings for the ion 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
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Using Basic Tune Functions
Using the Favorites Pane to Save System Settings
 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 42).
Figure 42. Favorites pane
4. Click Save Current State, and then type a unique name in the box (Figure 43).
Figure 43. State name box
State name box
5. Click Save Current State again to save the state.
The new favorite state appears first in the Favorites list. You can enter up to 100 states.
 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.
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Using the Favorites Pane to Save System Settings
 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.
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Using the Favorites Pane to Save System Settings
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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 the section “API Source Interface Maintenance,” in Chapter 8 of the Orbitrap Fusion Series
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
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Flushing the Inlet Components
Supplies
Supplies
Table 16 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 16. 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
76
Methanol, LC/MS-grade
Fisher Scientific: A456-1
Water, LC/MS-grade
Fisher Scientific: W6-1
Multiple sizes are available.
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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 71).
2. Place the mass spectrometer in Standby mode (see page 65).
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 9 on page 22).
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.
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Flushing the Inlet Components
Flushing the Inlet Components After Calibration
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C
Preparing the Reserpine Sample Solution
This appendix, for use by the Thermo Fisher Scientific field service engineer, describes how to
prepare the 50 fg/μL reserpine sample solution for H-ESI, APCI, and APPI modes. The
procedure calls for potentially hazardous chemicals, including methanol and reserpine.
For a list of solvent recommendations, refer to the Orbitrap Fusion Series Preinstallation
Requirements Guide. For a complete selection of LC/MS-grade consumables from Thermo
Fisher Scientific, visit www.FisherLCMS.com.
IMPORTANT
• Do not filter solvents. Filtering solvents can introduce contamination.
• Do not use plastic pipettes to prepare the sample solution. Plastic products can release
phthalates that can interfere with the analyses.
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.
Ideally, prepare the reserpine sample solution just before using it. If you must store the
solutions, keep them in a light-resistant container in the refrigerator until needed.
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Preparing the Reserpine Sample Solution
 To prepare the reserpine sample solution
1. Transfer 900 μL of 1% acetic acid in 50:50 methanol/water into a clean, minimum
1.5 mL polypropylene tube.
2. Add 100 μL of the 100 pg/μL reserpine standard solution to the tube.
The Orbitrap Fusion Series Chemicals Kit contains the reserpine standard solution.
3. Mix the solution (10 pg/μL) thoroughly.
4. Transfer 100 μL of the 10 pg/μL solution into a clean, minimum 1.5 mL polypropylene
tube.
5. Add 900 μL of 1% acetic acid in 50:50 methanol/water to the tube.
6. Mix the solution (1 pg/μL) thoroughly.
7. Transfer 50 μL of the 1 pg/μL solution into a clean, minimum 1.5 mL polypropylene
tube.
8. Add 950 μL of 1% acetic acid in 50:50 methanol/water to the tube.
9. Mix the solution thoroughly.
10. Label the tube Reserpine Sample Solution (50 fg/μL).
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D
Preparing the High Mass Range Calibration
Solution
This appendix describes how to prepare the high mass range calibration solution for H-ESI
mode. The procedures call for potentially hazardous chemicals, including enfuvirtide, which
is supplied in the Orbitrap Fusion Series Chemicals Kit (P/N 80000-62049).
Contents
• Supplies
• Guidelines
• Preparing the Enfuvirtide Calibration Solution
Supplies
To prepare the high mass range calibration solution, see the list of required chemicals and
equipment in Table 17 and Table 18, respectively. For a complete selection of LC/MS-grade
consumables from Thermo Fisher Scientific, visit www.FisherScientific.com.
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.
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Preparing the High Mass Range Calibration Solution
Supplies
Table 17. Required chemicals
a
Description
Grade
Part number
Acetic acid, glacial
HPLC grade
A113
Acetonitrile (ACN)
Optima™ LC/MS
A955
Ammonium hydroxide, 28–30%
Certified ACSa Plus
A669-500
Enfuvirtide, 90 mg
–
HAZMAT-01-00083
Methanol
Optima LC/MS
A456
Water
Optima LC/MS
W7-4
American Chemical Society
IMPORTANT
• Do not filter solvents. Filtering solvents can introduce contamination.
• Do not use plastic pipettes to prepare these solutions. Plastic products can release
phthalates that can interfere with the analyses.
Table 18. Required equipment
Description
a
82
Quantity
Bottle, glass, 100 mL
1
Bottle, glass, 500 mL
1
Cylinder, glass, 100 mL
1
Cylinder, glass, 250 mL
1
Gloves, nitrilea
(Fisher Scientific 19-120-2947 or Unity Lab Services 23827)
–
Pipet tips, 100 μL
3
Pipetter, 100–1000 μL
1
Sonicator
1
Spatula, stainless steel
1
Syringes, glass, 500 μL
3
Vial, scintillation, glass, 20 mL
1
Vortex mixer
1
Weighing container
1
Weight scale
1
Multiple sizes are available.
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Preparing the High Mass Range Calibration Solution
Guidelines
Guidelines
For optimal results, follow these guidelines when performing the procedures in this appendix:
• Make sure that the surrounding area is neat, clean, and well ventilated.
• Have nearby the necessary chemicals and equipment.
• Always wear protective safety glasses and a new pair of nitrile gloves when handling
solvents and samples—never reuse gloves after you remove them.
• Clean all bottles and vials with methanol and let dry before use.
• Proceed methodically.
Preparing the Enfuvirtide Calibration Solution
Follow these procedures to prepare the enfuvirtide calibration solution:
1. To prepare the 0.15% ammonium hydroxide solution
2. To prepare the 0.15% ammonium hydroxide/ACN-50:50 solution
3. To prepare the enfuvirtide stock solution
4. To prepare the 0.1% acetic acid/ACN-50:50 solution
5. To prepare the enfuvirtide final solution
CAUTION When using solvents or corrosives, always wear protective gloves and safety
glasses, and use the appropriate vapor respiratory equipment. Prepare the solution under a
fume hood.
 To prepare the 0.15% ammonium hydroxide solution
1. Transfer 99.5 mL of the LC/MS-grade water into a clean 100 mL glass bottle.
2. Use a syringe to transfer 500 μL of the ammonium hydroxide to the bottle.
3. Put on the lid and vortex the bottle of the ammonium hydroxide (28–30%) solution for
2 minutes.
4. Label the bottle 0.15% Ammonium Hydroxide Solution.
 To prepare the 0.15% ammonium hydroxide/ACN-50:50 solution
1. Pipet 60 mL of the acetonitrile into a clean 500 mL glass bottle.
2. Pipet 60 mL of the “0.15% Ammonium Hydroxide Solution” into the bottle.
3. Put on the lid and vortex the bottle of solution for 2 minutes.
4. Label the bottle 0.15% Ammonium Hydroxide/ACN-50:50 Solution.
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Preparing the High Mass Range Calibration Solution
Preparing the Enfuvirtide Calibration Solution
 To prepare the enfuvirtide stock solution
1. Obtain the enfuvirtide from the lab freezer, and verify the chemical name and expiration
date before you continue.
2. Weigh out 22 ±0.5 mg of the enfuvirtide.
IMPORTANT Refrigerate the opened enfuvirtide container. For long-term storage,
keep frozen at –25 to –15 °C (–13 to 5 °F).
3. Carefully pour the measured enfuvirtide into a clean 20 mL scintillation vial.
Discard the used weighing container according to established lab practices.
4. Pipet 5 mL of the “0.15% Ammonium Hydroxide/ACN-50:50 Solution” into the vial.
5. Put on the lid and vortex the bottle for 2 minutes.
6. Label the bottle Enfuvirtide Stock Solution and include the prepared and expiration
dates (Figure 44).
IMPORTANT
• Freeze the stock solution at –20 °C (–4 °F) or colder for up to 6 months.
• (Optional) To store the stock solution up to 1 year, make 1 mL aliquots.
Figure 44. Example labeling for the Enfuvirtide stock solution
Solution label
Dates: prepared and expiration
 To prepare the 0.1% acetic acid/ACN-50:50 solution
1. Transfer 100 mL of the water/ACN-50:50 Solution into a clean 100 mL glass bottle.
2. Use a syringe to transfer 100 μL of the glacial acetic acid to the bottle.
3. Put on the lid and vortex the bottle of solution for 2 minutes.
4. Label the bottle 0.1% Acetic Acid/ACN-50:50 Solution.
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Preparing the High Mass Range Calibration Solution
Preparing the Enfuvirtide Calibration Solution
 To prepare the enfuvirtide final solution
1. Transfer 10 mL of the “0.1% Acetic Acid/ACN-50:50 Solution” to a clean 20 mL glass
scintillation vial.
2. Use a syringe to transfer 100 μL of the “Enfuvirtide Stock Solution” into the vial.
3. Put on the lid and vortex the bottle of solution for 2 minutes.
4. Loosen the lid slightly and sonicate the bottle for 5 minutes.
5. Label the bottle Enfuvirtide Final Solution and include the prepared and expiration
dates.
Note Refrigerate the final solution at 4 °C (39 °F) for up to 4 weeks.
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G
Glossary
A
B
C
D
E
F
G
H
I
J
K
L M N O
A
activation time The time in milliseconds that the rf
used for fragmentation is applied in an ion trap. The
activation time value is 10 ms (not a user variable). In
general, shorter activation time results in less
fragmentation and a longer activation time results in
more fragmentation.
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.
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).
P
Q
R
S
T
U
V W X
Y
Z
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.
C
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.
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.
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.
Thermo Scientific
Orbitrap Fusion Series Getting Started Guide
87
Glossary: collision-induced dissociation (CID)
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 helium for the
Orbitrap Fusion Series MS. The collisions break the
bonds and fragment the ions into smaller pieces.
F
conversion dynode A highly polished metal surface
that converts ions from the mass analyzer into
secondary particles, which enter the electron
multiplier.
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.
D
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.
damping gas Helium gas introduced into the ion trap
mass analyzer that slows the motion of ions entering
the mass analyzer so that the ions can be trapped by
the rf voltage fields in the mass analyzer.
divert/inject valve A valve on the mass spectrometer
that can be plumbed as a loop injector or as a divert
valve.
E
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.
H
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.
electron transfer dissociation (ETD) A method of
fragmenting peptides and proteins. In ETD, singly
charged reagent anions transfer an electron to
multiply protonated peptides within the linear ion
trap mass analyzer. This leads to a rich ladder of
sequence ions derived from cleavage at the amide
groups along the peptide backbone. Amino acid side
chains and important modifications such as
phosphorylation are left intact.
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).
88
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.
Orbitrap Fusion Series Getting Started Guide
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.
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.
higher energy collision-induced dissociation
(HCD) Collision-induced dissociation that occurs in
the ion routing multipole (IRM) of the Orbitrap
mass analyzer. The IRM consists of a straight
multipole mounted inside a collision gas-filled tube.
A voltage offset between the C-trap and IRM
accelerates parent ions into the collision gas inside
the IRM, which causes the ions to fragment into
Thermo Scientific
Glossary: ion detection system
product ions. The product ions are then returned to
the Orbitrap mass analyzer for mass analysis. HCD
produces triple quadrupole-like product ion mass
spectra.
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.
ion polarity mode The mass spectrometer can operate
in either of two ion polarity modes: positive or
negative.
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.
isolation window The baseline width of a window for
a mass peak (or peak cluster) of interest for an
MS/MS or MSn scan.
ion-routing multipole The collision cell where higher
energy collision-induced dissociation (HCD) takes
place.
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.
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.
MSn scan mode Scan modes where 2 to 10 stages of
mass analysis are performed. The scan power equals 2
to 10, where the scan power is the power n in the
expression MSn. MSn is the most general expression
for the scan mode, which can include the following:
• The scan mode corresponding to the two or more
stages of mass analysis in a two-stage full- or
narrow-scan experiment.
Thermo Scientific
Orbitrap Fusion Series Getting Started Guide
89
Glossary: nano liquid chromatography (nanoLC)
• The scan mode corresponding to the 3 to 10 stages
of mass analysis (n = 3 to n = 10) in a multistage
full-scan experiment.
N
nano liquid chromatography (nanoLC) Liquid
chromatography with typical flow rates of
10–1000 nL/min and 10–150 μm diameter
columns.
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.
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).
profile data Data representing mass spectral peaks as
point-to-point plots, with each point having an
associated intensity value.
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.
qualitative analysis Chemical analysis designed to
determine the identity of the components of a
substance.
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).
quantitative analysis Chemical analysis designed to
determine the quantity or concentration of a specific
substance in a sample.
R
reagent carrier gas Ultra-high-purity nitrogen gas
used to transfer the reagent to the reagent ion source
that is regulated by the backpressure regulator.
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.
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.
90
Q
Orbitrap Fusion Series Getting Started Guide
retention time (RT) The time after injection at which
a compound elutes. The total time that the compound
is retained on the chromatograph.
Thermo Scientific
Glossary: sample loop
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.
scan mode and scan type combinations A function
that coordinates the three processes in the MS
detector: ionization, mass analysis, and ion detection.
You can combine the various scan modes and scan
types to perform a wide variety of experiments.
scan power The power n in the expression MSn. The
number of stages of mass analysis, expressed as MSn,
where n is the scan power. For example, a scan power
of n = 1 corresponds to an MS1 (or MS) scan with
one stage of mass analysis. A scan power of n = 2
corresponds to an MS2 (or MS/MS) scan with two
stages of mass analysis. A scan power of n = 3
corresponds to an MS3 scan with three stages of mass
analysis, and so on.
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.
Z
Zoom scan type A scan type that provides
information about the charge state of one or more
ions of interest. Zoom scans are slower scans with
higher resolution than normal scans.
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.
Thermo Scientific
Orbitrap Fusion Series Getting Started Guide
91
Glossary: Zoom scan type
92
Orbitrap Fusion Series Getting Started Guide
Thermo Scientific
I
Index
A
APCI mode
description 3
plumbing connection, direct infusion 23
spray insert, installing 14
API source
cautions 11
high voltage connector 13
installing or removing 11
solvent waste container, connecting 11
spray insert 14
APPI mode
description 3
spray insert, selecting 14
auto-loop injection
schematic 20
setup 27
uses 17
autosampler injection 29
B
bakeout, Orbitrap chamber 38
buffers, description 6
C
calibration
negative polarity mode 49
positive polarity mode 48
Calibration pane
See panes, Calibration
calibration, delay 48
caution symbols, description xviii
Change Instruments In Use dialog box 61
chemicals, storage precautions xvii
chromatograph, displaying 54
cleaning
inlet components 77
Thermo Scientific
ion sweep cone 75
ion transfer tube 75
spray cone 75
syringe 77
compliance
FCC iv
regulatory iii
contacting us xix
contamination, preventing 21, 39
D
data acquisition
button 59
Tune 58
Xcalibur 61
See also panes, Data Acquisition
data acquisition, delay 59
data type, setting 67
Define Scan pane
See panes, Define Scan
diagnostics
See panes, Diagnostics
direct infusion
connecting the plumbing for 23
description 18
schematic 20
directive, WEEE v
divert/inject valve
configurations
as divert valve 33
as loop injector 33
schematic of 34
controlling 34
description 32
positions 33
schematic 20
valve position indicator 34
Orbitrap Fusion Series Getting Started Guide
93
Index: E
documentation
accessing xiv
additional xiv
downloading xiv
related xiv
E
electromagnetic compatibility iii
EMC compliance iii
enfuvirtide, preparing the stock solution 84
F
favorite states, using to set parameters 71
Favorites pane
See panes, Favorites
FCC compliance iv
figures, list of xi
flow rates, setting 4
flow-injection analysis, description 19
flushing inlet components 77
forepump, fume exhaust system cautions 11
G
gas flow rates, adjusting for LC flow rate 4
grounding (ZDV) union xvi, 27
H
H-ESI mode
description 2
plumbing connection, direct infusion 23
spray insert, installing 14
high mass range calibration, preparing the final solution 81
high-flow infusion
connecting the plumbing for 24
description 18
schematic 20
HPLC with autosampler injection
schematic 20
uses 19
I
infusion line, connecting to grounding union 23
instrument
See mass spectrometer
Instrument Setup window 7
ion polarity mode, setting 67
Ion Source pane
See panes, Ion Source
94
Orbitrap Fusion Series Getting Started Guide
ion sweep cone, cleaning 75
ion transfer tube
cleaning 75
temperature, adjusting for LC flow rate 4
ionization time (IT) 55
isolation window
optimization value 55
optimizing 54
K
kits
Calibration xv
Orbitrap Fusion Chemicals xvii
Performance Specification xvi
L
LC pump
configuring 53
connecting to the divert/inject valve 26
LC union xvi
LC with autosampler injection, schematic 20
LC/MS experiments, connecting the plumbing for 29
LC/MS operational guidelines
APCI mode 5
H-ESI mode 5
NSI mode 5
loop injection
connecting the plumbing for 27
liquid chromatography description, and 19
M
manual loop injection
schematic 20
setup 27
uses 17
mass list
exporting from Tune 71
importing into Tune 70
mass spectrometer
API source, installing or removing 11
calibrating 47
data acquisition, manual 51
flow rates, setting 4
plumbing connections 17
power modes, setting 65
pressure mode, setting 53, 67
pumping down the vacuum 35
readback status, checking 65
sample introduction techniques 17
spray insert, installing or removing 14
spray stability, evaluating 41
Thermo Scientific
Index: N
MSDS 6
MSn setting table, using 69
panes
Calibration 48
Data Acquisition 59
Define Scan 64
Diagnostics 38
Favorites 72
History 71
Ion Source - Optimization page 44
Status 37
PEEK tubing, special notice 21
Pierce calibration solutions, part numbers xvii
plumbing connections, inlet 17
polarity mode
See ion polarity mode, setting
Predictive AGC calibration, special notice 49
pump down, MS 35
scan parameter, defining
MS/MS scan 54
MS3 scan 57
SDS 6
sequence run, start instrument in Xcalibur 60
Skip Spray Stability Evaluation check box 48
solvents
description 6
waste 11
source
See API source
spray cone, cleaning 75
spray insert, API source
adjustments
front-to-back 16
rotational 16
installing or removing 14
spray stability, evaluating 41
start instrument
configuring with Xcalibur 60
description 60
Run Sequence dialog box 60
Status pane 37
stock solution, enfuvirtide 84
syringe
adapter assembly 21
avoid contamination 39, 48
cleaning 77
syringe adapter assembly, drawing 32
syringe parameter box 66
syringe pump
controlling 66
default flow rate 31
description 31
setting up 22
R
T
readback status, description 65
Record button 59
regulatory compliance iii
report options 68
reserpine sample solution, preparing 80
Run Sequence dialog box (acquisition options) 60
time stamp, raw data file 58
Tune application
basic functions 63
opening 64
preferences, setting 68
Tune Preferences dialog box 68
S
U
safety standards iii
sample data, acquire by using Tune 51
sample introduction techniques
schematic diagrams 20
summary of connections 17
union types, plumbing
grounding (ZDV) xvi
LC xvi
Tee xvii
N
normalization level (NL) 55
NSI mode, description 4
O
optimization
API source parameters
analyte, for 56
general procedure 43
increase sensitivity 56
signal type, list 44
special notice 43
P
Thermo Scientific
Orbitrap Fusion Series Getting Started Guide
95
Index: V
V
vaporizer temperature, adjusting for LC flow rate 4
W
waste container, solvent 13
WEEE directive v
X
Xcalibur file type, raw data (.raw) 58
Xcalibur file type, sequence (.sld) 60
Z
ZDV union xvi
96
Orbitrap Fusion Series Getting Started Guide
Thermo Scientific
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