LTQ Series Getting Started Guide Rev.C

LTQ Series Getting Started Guide Rev.C
LTQ Series
Getting Started Guide
97055-97073 Revision C
May 2011
© 2011 Thermo Fisher Scientific Inc. All rights reserved.
Ion Max, LTQ XL, Velos, Velos Pro, Wideband Activation, and ZoomScan are trademarks and Accela, LTQ,
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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.
Thermo Fisher Scientific Inc. makes no representations that this document is complete, accurate or errorfree and assumes no responsibility and will not be liable for any errors, omissions, damage or loss that might
result from any use of this document, even if the information in the document is followed properly.
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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: Revision A, July 2009; Revision B, August 2010; Revision C, May 2011
Software version: Thermo LTQ Tune Plus version 2.7.0 or later; Microsoft Windows 7 Professional SP1—
Thermo Foundation version 2.0 or later, and Thermo Xcalibur version 2.2 or later; Windows XP Workstation
SP3—Foundation version 1.0.2 SP2 or earlier, and Xcalibur version 2.1 SP1 or earlier
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:
• LXQ Mass Spectrometer (February 2005)
• LTQ XL Mass Spectrometer (September 2006)
• LTQ XL/ETD System (January 2007)
• MALDI LTQ XL System (August 2007)
• LTQ Velos Mass Spectrometer (August 2008)
• LTQ Velos/ETD System (November 2008)
• Velos Pro Mass Spectrometer (April 2011)
• Velos Pro/ETD System (April 2011)
LXQ Mass Spectrometer (February 2005)
EMC Directive 89/336/EEC as amended by 92/31/EEC and 93/68/EEC
EMC compliance has been evaluated by Underwriters Laboratories, Inc.
EN 55011: 1998
EN 61000-4-3: 2002, A1: 2002
EN 61000-3-2: 1995, A1: 1998, A2: 1998, A14: 2000
EN 61000-4-4: 1995, A1: 2001, A2: 2001
EN 61000-3-3: 1995
EN 61000-4-5: 1995, A1: 2001
EN 61326-1: 1997
EN 61000-4-6: 1996, A1: 2001
EN 61000-4-2: 1995, A1: 1998, A2: 2001
EN 61000-4-11: 1994, A1: 2001
FCC Class A, CFR 47 Part 15, Subpart B: 2004
CISPR 11: 1999, A1: 1999, A2: 2002
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001.
LTQ XL Mass Spectrometer (September 2006)
EMC Directive 89/336/EEC
EMC compliance has been evaluated by TUV Rheinland of North America, Inc.
EN 55011: 1998, A1: 1999, A2: 2002
EN 61000-4-3: 2002
EN 61000-3-2: 1995, A1: 1998, A2: 1998, A14: 2000
EN 61000-4-4: 1995, A1: 2001, A2: 2001
EN 61000-3-3: 1995, A1:2001
EN 61000-4-5:1995, A1: 2001
EN 61326-1: 1997, A1: 1998, A2: 2001, A3: 2003
EN 61000-4-6: 2003
EN 61000-4-2: 2001
EN 61000-4-11: 2001
FCC Class A, CFR 47 Part 15: 2005
CISPR 11: 1999, A1: 1999, A2: 2002
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001.
LTQ XL/ETD System (January 2007)
EMC Directive 89/336/EEC
EMC compliance has been evaluated by TUV Rheinland of North America, Inc.
EN 61000-3-2: 1995, A1: 1998, A2: 1998, A14: 2000
EN 61000-4-4:1995, A1: 2000, A2:2001
EN 61000-3-3: 1995, A1:2001
EN 61000-4-5: 1995, A1: 2001
EN 61326-1: 1997, A1:1998, A2:2001, A3:2003
EN 61000-4-6: 2003
EN 61000-4-2: 2001
EN 61000-4-11: 1994, A1: 2001
EN 61000-4-3: 2002
CISPR 11: 1999, A1: 1999, A2: 2002
FCC Class A, CFR 47 Part 15: 2005
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 73/23/EEC and harmonized standard EN 61010-1:2001.
MALDI LTQ XL System (August 2007)
EMC Directive 2004/108/EC
EMC compliance has been evaluated by TUV Rheinland of North America, Inc.
EN 55011: 1998, A1: 1999, A2: 2002
EN 61000-4-3: 2002
EN 61000-3-2: 2000
EN 61000-4-4: 1995, A1: 2000, A2: 2001
EN 61000-3-3: 1995, A1: 2001
EN 61000-4-5: 2001
EN 61326-1: 1998, A2: 2001, A3: 2003
EN 61000-4-6: 2003
EN 61000-4-2: 2001
EN 61000-4-11: 2001
FCC Class A, CFR 47 Part 15: 2006
CISPR 11: 1998, A1:1999, A2: 2002
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 2006/95/EC and harmonized standard EN 61010-1:2001.
Safety of Laser Products
Compliance with safety of laser products is declared under Thermo Fisher Scientific sole responsibility. This device
complies with the harmonized standard IEC/EN 60825-1/A2: 2001.
LTQ Velos Mass Spectrometer (August 2008)
EMC Directive 2004/108/EEC
EMC compliance has been evaluated by TUV Rheinland of North America, Inc.
EN 55011: 2007, A2: 2007
EN 61000-4-3: 2006
EN 61000-3-2: 2006
EN 61000-4-4: 2004
EN 61000-3-3: 1995, A1: 2001, A2: 2005
EN 61000-4-5: 2005
EN 61326-1: 2006
EN 61000-4-6: 2007
EN 61000-4-2: 1995, A1: 1999, A2: 2001
EN 61000-4-11: 2004
FCC Class A, CFR 47 Part 15: 2007
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 2006/95/EEC and harmonized standard EN 61010-1:2001.
LTQ Velos/ETD System (November 2008)
EMC Directive 2004/108/EEC
EMC compliance has been evaluated by TUV Rheinland of North America, Inc.
EN 61326-1: 2006
EN 61000-4-4: 2004
EN 55011: 2007
EN 61000-4-5: 2005
EN 61000-3-2: 2006
EN 61000-4-6: 2007
EN 61000-3-3: 2005
EN 61000-4-11: 2004
EN 61000-4-2: 2001
FCC Part 15: 2007
EN 61000-4-3: 2006
Velos Pro Mass Spectrometer (April 2011)
EMC Directive 2004/108/EEC
EMC compliance has been evaluated by TUV Rheinland of North America, Inc.
EN 61326-1: 2006
EN 61000-4-3: 2006
EN 55011: 2007, A2: 2007
EN 61000-4-4: 2004
CFR 47, FCC Part 15, Subpart B, Class A: 2009
EN 61000-4-5: 2005
EN 61000-3-2: 2006
EN 61000-4-6: 2007
EN 61000-3-3: 1995, A1: 2001, A2: 2005
EN 61000-4-11: 2004
EN 61000-4-2: 1995, A1: 1999, A2: 2001
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 2006/95/EEC and harmonized standard EN 61010-1:2001.
Velos Pro/ETD System (April 2011)
EMC Directive 2004/108/EEC
EMC compliance has been evaluated by TUV Rheinland of North America, Inc.
EN 61326-1: 2006
EN 61000-4-3: 2006
EN 55011: 2007, A2: 2007
EN 61000-4-4: 2004
CFR 47, FCC Part 15, Subpart B, Class A: 2009
EN 61000-4-5: 2005
EN 61000-3-2: 2006
EN 61000-4-6: 2007
EN 61000-3-3: 1995, A1: 2001, A2: 2005
EN 61000-4-11: 2004
EN 61000-4-2: 1995, A1: 1999, A2: 2001
Low Voltage Safety Compliance
This device complies with Low Voltage Directive 2006/95/EEC and harmonized standard EN 61010-1:2001.
FCC Compliance Statement
THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES. OPERATION IS SUBJECT TO
THE FOLLOWING TWO CONDITIONS: (1) THIS DEVICE MAY NOT CAUSE HARMFUL
INTERFERENCE, AND (2) THIS DEVICE MUST ACCEPT ANY INTERFERENCE RECEIVED,
INCLUDING INTERFERENCE THAT MAY CAUSE UNDESIRED OPERATION.
CAUTION Read and understand the various precautionary notes, signs, and symbols contained inside
this manual pertaining to the safe use and operation of this product before using the device.
Notice on Lifting and Handling of
Thermo Scientific Instruments
For your safety, and in compliance with international regulations, the physical handling of this Thermo Fisher
Scientific instrument requires a team effort to lift and/or move the instrument. This instrument is too heavy and/or
bulky for one person alone to handle safely.
Notice on the Proper Use of
Thermo Scientific Instruments
In compliance with international regulations: Use of this instrument in a manner not specified by Thermo Fisher
Scientific could impair any protection provided by the instrument.
Notice on the Susceptibility
to Electromagnetic Transmissions
Your instrument is designed to work in a controlled electromagnetic environment. Do not use radio frequency
transmitters, such as mobile phones, in close proximity to the instrument.
For manufacturing location, see the label on the instrument.
WEEE Compliance
This product is required to comply with the European Union’s Waste Electrical & Electronic Equipment (WEEE)
Directive 2002/96/EC. It is marked with the following symbol:
Thermo Fisher Scientific has contracted with one or more recycling or disposal companies in each European Union
(EU) Member State, and these companies should dispose of or recycle this product. See www.thermo.com/
WEEERoHS for further information on Thermo Fisher Scientific’s compliance with these Directives and the
recyclers in your country.
WEEE Konformität
Dieses Produkt muss die EU Waste Electrical & Electronic Equipment (WEEE) Richtlinie 2002/96/EC erfüllen.
Das Produkt ist durch folgendes Symbol gekennzeichnet:
Thermo Fisher Scientific hat Vereinbarungen mit Verwertungs-/Entsorgungsfirmen in allen EU-Mitgliedsstaaten
getroffen, damit dieses Produkt durch diese Firmen wiederverwertet oder entsorgt werden kann. Mehr Information
über die Einhaltung dieser Anweisungen durch Thermo Fisher Scientific, über die Verwerter, und weitere Hinweise,
die nützlich sind, um die Produkte zu identifizieren, die unter diese RoHS Anweisung fallen, finden sie unter
www.thermo.com/WEEERoHS.
Conformité DEEE
Ce produit doit être conforme à la directive européenne (2002/96/EC) des Déchets d'Equipements Electriques et
Electroniques (DEEE). Il est marqué par le symbole suivant:
Thermo Fisher Scientific s'est associé avec une ou plusieurs compagnies de recyclage dans chaque état membre de
l’union européenne et ce produit devrait être collecté ou recyclé par celles-ci. Davantage d'informations sur la
conformité de Thermo Fisher Scientific à ces directives, les recycleurs dans votre pays et les informations sur les
produits Thermo Fisher Scientific qui peuvent aider la détection des substances sujettes à la directive RoHS sont
disponibles sur www.thermo.com/WEEERoHS.
C
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix
Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx
Getting a Trap-HCD License . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxi
Getting a New License Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxi
Installing a New License Code. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii
Safety and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxiii
Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiv
Chapter 1
Thermo Scientific
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Features of the LTQ Series Mass Spectrometer . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Linear Ion Trap Mass Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
ZoomScan Analysis for Charge State Determination . . . . . . . . . . . . . . . . . . . . 3
Wideband Activation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Switchable Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Optional Ion Sweep Cone and Ion Sweep Gas for Complex Sample
Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Sheath, Auxiliary, and Sweep Gases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Ionization Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Using ESI or H-ESI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Using APCI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Sample Introduction Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Direct Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
High-Flow Infusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Loop Injection (Flow-Injection Analysis). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Types of Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
LC Flow Rate Ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Tuning and Calibrating the Mass Spectrometer. . . . . . . . . . . . . . . . . . . . . . . . . 13
Ion Polarity Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Data Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Scan Power and Scan Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Scan Rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
LTQ Series Getting Started Guide
xi
Contents
Scan Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Full Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Selected Ion Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Selected Reaction Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Consecutive Reaction Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
ZoomScan and UltraZoom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Types of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
General MS or MSn Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Data Dependent Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Ion Mapping Experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Ion Tree Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Mass/Charge Range. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
xii
Chapter 2
Setting Up the API Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Opening the Tune Plus Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Placing the Mass Spectrometer in Standby Mode . . . . . . . . . . . . . . . . . . . . . . . 33
API Source Housing Installation and Removal . . . . . . . . . . . . . . . . . . . . . . . . . 34
Installing the API Source Housing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Removing the API Source Housing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
API Source Housing Drain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
ESI or HESI-II Probe Installation and Removal . . . . . . . . . . . . . . . . . . . . . . . . 40
Installing the ESI or HESI-II Probe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Removing the ESI or HESI-II Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
APCI Probe Installation and Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Installing the Corona Needle and APCI Probe . . . . . . . . . . . . . . . . . . . . . . . 53
Removing the APCI Probe and the Corona Needle . . . . . . . . . . . . . . . . . . . . 59
Adjusting the Probe Position on the Ion Max API Source Housing. . . . . . . . . . 61
Chapter 3
Automatic Tuning and Calibration in ESI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . .63
Setting Up the Syringe Pump for Tuning and Calibration. . . . . . . . . . . . . . . . . 64
Setting Up the Mass Spectrometer for Tuning and Calibration . . . . . . . . . . . . . 65
Testing the Mass Spectrometer in ESI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Tuning the Mass Spectrometer Automatically in ESI Mode . . . . . . . . . . . . . . . 74
Saving the ESI Tune Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Calibrating Automatically in the Normal Mass Range . . . . . . . . . . . . . . . . . . . . 79
Chapter 4
Tuning with an Analyte in ESI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83
Setting Up the Inlet for High-Flow Infusion in ESI Mode . . . . . . . . . . . . . . . . 85
Setting Up the Syringe Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Connecting the Plumbing to Introduce Sample by High-Flow Infusion . . . . 87
Setting Up the Mass Spectrometer to Tune with an Analyte in
ESI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Tuning the Mass Spectrometer Automatically . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Saving the ESI Tune Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
LTQ Series Getting Started Guide
Thermo Scientific
Contents
Chapter 5
Acquiring ESI Sample Data by Using Tune Plus . . . . . . . . . . . . . . . . . . . . . . . . . . .99
Setting Up to Acquire Full-Scan MS/MS Data . . . . . . . . . . . . . . . . . . . . . . . . . 99
Optimizing the Isolation Width for an MS/MS Experiment . . . . . . . . . . . . 100
Optimizing the Collision Energy Manually for an MS/MS Experiment . . . 102
Optimizing the Collision Energy Automatically for an MS/MS
Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Setting Up the Inlet for Flow Injection Analysis in ESI Mode . . . . . . . . . . . . . 106
Acquiring ESI Data in the SIM Scan Type . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Chapter 6
Tuning with an Analyte in APCI Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .113
Setting Up the Inlet for High-Flow Infusion in APCI Mode . . . . . . . . . . . . . . 114
Setting Up the Mass Spectrometer to Tune with an Analyte in
APCI Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Tuning the Mass Spectrometer Automatically . . . . . . . . . . . . . . . . . . . . . . . . . 120
Saving the APCI Tune Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Chapter 7
Acquiring APCI Sample Data by Using Tune Plus . . . . . . . . . . . . . . . . . . . . . . . .125
Setting Up the Inlet for Flow Injection Analysis in APCI Mode . . . . . . . . . . . 126
Acquiring APCI Data in the SIM Scan Mode . . . . . . . . . . . . . . . . . . . . . . . . . 127
Chapter 8
Cleaning the Mass Spectrometer After Tuning and Calibrating . . . . . . . . . . . .131
Cleaning Supplies and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Flushing the Sample Transfer Line, Sample Tube, and API Probe. . . . . . . . . . 133
Removing and Cleaning the Syringe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Cleaning the Ion Sweep Cone, Spray Cone, and Ion Transfer Tube . . . . . . . . 135
Appendix A Sample Formulations for the LXQ and LTQ XL Mass Spectrometers. . . . . . . . .139
Preparing the Normal Mass Range Calibration Solution for ESI Mode . . . . . . 140
Caffeine Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Preparing the MRFA Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Preparing the Ultramark 1621 Stock Solution . . . . . . . . . . . . . . . . . . . . . . . 143
Preparing the Normal Mass Range Calibration Solution for ESI Mode . . . . 143
Preparing the Reserpine Tuning and Sample Solutions for the ESI and
APCI Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Preparing the High Mass Range Calibration Solution . . . . . . . . . . . . . . . . . . . 145
Appendix B Sample Formulations for the Velos Pro Mass Spectrometer . . . . . . . . . . . . . . .147
Preparing the Normal Mass Range Calibration Solution for ESI Mode . . . . . . 148
Caffeine Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Preparing the MRFA Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Preparing the Ultramark 1621 Stock Solution . . . . . . . . . . . . . . . . . . . . . . . 151
Preparing the N-butylamine Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . 151
Preparing the Normal Mass Range Calibration Solution for ESI Mode . . . . 151
Preparing the Reserpine Tuning and Sample Solutions for ESI and
APCI Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Thermo Scientific
LTQ Series Getting Started Guide
xiii
Contents
Preparing the High Mass Range Calibration Solution . . . . . . . . . . . . . . . . . . . 153
Appendix C High Mass Range Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155
Verifying Coarse Calibration for the High Mass Range by Using the
Normal Mass Range Calibration Solution . . . . . . . . . . . . . . . . . . . . . . . . 156
Calibrating the High Mass Range by Using the Normal Mass Range
Calibration Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Performing Two-Point Manual Coarse Calibration—High Mass Range
Mode by Using Normal Mass Range Calibration Solution. . . . . . . . . . . . 164
Calibrating the High Mass Range by Using the High Mass Range
Calibration Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Cleaning the System After Calibration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .173
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177
xiv
LTQ Series Getting Started Guide
Thermo Scientific
F
Figures
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.
Figure 11.
Figure 12.
Figure 13.
Figure 14.
Figure 15.
Figure 16.
Figure 17.
Figure 18.
Figure 19.
Figure 20.
Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Thermo Scientific
Sample introduction by using direct infusion with a syringe pump . . . . . . . . . . . 7
Sample introduction by using high-flow infusion in ESI mode . . . . . . . . . . . . . . 8
Sample introduction by using loop injection in APCI mode . . . . . . . . . . . . . . . . 9
Sample introduction by using direct injection with an autosampler in
ESI mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Thermo Xcalibur Instrument Setup window showing the New Method
page (Velos Pro with ETD experiment types) 20
General MS experiment template on the MS Detector Setup page . . . . . . . . . . . 22
Data Dependent Triple-Play experiment template on the MS Detector
Setup page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Ion Mapping experiment template on the Total Ion Map page . . . . . . . . . . . . . 27
Ion Tree experiment template on the Data Dependent Ion Tree page . . . . . . . . 29
Tune Plus window for the LTQ XL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Ion Max-S API source housing (back view). . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
API ion source mount assembly showing the guide pins . . . . . . . . . . . . . . . . . . 35
Ion Max-S API source housing showing the locking levers and drain
(front view) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
API source drain assembly and waste container . . . . . . . . . . . . . . . . . . . . . . . . . 38
Ion Max API source housing (left side view) . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
ESI probe showing the guide pin and locking ring . . . . . . . . . . . . . . . . . . . . . . . 42
HESI-II probe showing the guide pin and locking ring . . . . . . . . . . . . . . . . . . . 42
ESI probe showing the guide pin inserted into the interlock block slot . . . . . . . 43
HESI-II probe showing the guide pin inserted into the interlock block
slot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
ESI probe installed in the Ion Max API source housing . . . . . . . . . . . . . . . . . . . 45
HESI-II probe installed in the Ion Max API source housing . . . . . . . . . . . . . . . 46
HESI-II probe connections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Sample transfer line disconnected from the grounding union . . . . . . . . . . . . . . 48
8 kV cable connector removed from the ESI probe . . . . . . . . . . . . . . . . . . . . . . 49
8 kV cable connector removed from the HESI-II probe (enlarged view) . . . . . . 49
HESI-II probe showing the disconnected vaporizer and 8 kV cables
(top view) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
API source housing with the HESI-II probe (left side view) . . . . . . . . . . . . . . . 51
API source housing with ESI probe (left side view) . . . . . . . . . . . . . . . . . . . . . . 52
Corona needle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Ion Max API source housing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
APCI probe guide pin touching the locking ring . . . . . . . . . . . . . . . . . . . . . . . . 56
APCI probe guide pin inserted into the interlock block slot . . . . . . . . . . . . . . . . 56
LTQ Series Getting Started Guide
xv
Figures
Figure 33.
Figure 34.
Figure 35.
Figure 36.
Figure 37.
Figure 38.
Figure 39.
Figure 40.
Figure 41.
Figure 42.
Figure 43.
Figure 44.
Figure 45.
Figure 72.
APCI probe installed in the Ion Max API source housing . . . . . . . . . . . . . . . . . 58
APCI probe connections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Corona needle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Ion Max API source housing showing probe adjustment controls . . . . . . . . . . . 61
Plumbing connection for the syringe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Plumbing connection between the LC union and the grounding union . . . . . . . 65
Define Scan dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Syringe Pump dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Spectrum of the calibration solution for the LTQ XL . . . . . . . . . . . . . . . . . . . . 70
Spectrum of the calibration solution for the Velos Pro (lower range) . . . . . . . . . 71
Spectrum of the calibration solution for the Velos Pro (upper range) . . . . . . . . . 72
Tune dialog box showing the Automatic page. . . . . . . . . . . . . . . . . . . . . . . . . . 74
Tune Plus window and Tune dialog box to set the LTQ XL on
m/z 195.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Tune Plus window and Tune dialog box to set the Velos Pro on
m/z 524.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Calibrate dialog box showing the Automatic page. . . . . . . . . . . . . . . . . . . . . . . 79
Tune Plus window and Calibrate dialog box for the LTQ XL . . . . . . . . . . . . . . 81
Plumbing setup for LC/ESI/MS infusion into the solvent flow from an
LC pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Plumbing connections for the syringe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Plumbing connections between the LC tee union and the LC union . . . . . . . . 88
Six-port divert/inject valve connections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Connections between the LC tee union and the divert/inject valve . . . . . . . . . . 89
Connections between the LC tee union and the grounding union
(ESI probe) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Plumbing setup for ESI/MS sample introduction with high-flow infusion . . . . 90
Grounding union connected to the sample inlet of the ESI probe. . . . . . . . . . . 91
Define Scan dialog box for acquiring reserpine SIM data in ESI mode . . . . . . . 92
Tune dialog box showing the Automatic page. . . . . . . . . . . . . . . . . . . . . . . . . . 95
Divert/Inject Valve dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Tune Plus window and Tune dialog box for automatic tuning in ESI
mode of the LTQ XL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Define Scan dialog box for acquiring reserpine full-scan data . . . . . . . . . . . . . . 100
Syringe Pump dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Define Scan dialog box (full-scan data) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Tune dialog box showing the Collision Energy page . . . . . . . . . . . . . . . . . . . . 104
Tune Plus window and the Tune dialog box showing the Collision
Energy page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Accept Optimized Value dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Divert/inject valve setup for loop injection . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Plumbing for loop injection into the solvent flow from an LC into an
ESI probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Define Scan dialog box (reserpine SIM-scan data) . . . . . . . . . . . . . . . . . . . . . . 108
Acquire Data dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Qual Browser window showing an ESI SIM chromatogram and mass
spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Plumbing setup for APCI/MS sample introduction with high-flow infusion. . . 114
LTQ Series Getting Started Guide
Thermo Scientific
Figure 46.
Figure 47.
Figure 48.
Figure 49.
Figure 50.
Figure 51.
Figure 52.
Figure 53.
Figure 54.
Figure 55.
Figure 56.
Figure 57.
Figure 58.
Figure 59.
Figure 60.
Figure 61.
Figure 62.
Figure 63.
Figure 64.
Figure 65.
Figure 66.
Figure 67.
Figure 68.
Figure 69.
Figure 70.
Figure 71.
xvi
Figures
Figure 73.
Figure 74.
Figure 75.
Figure 76.
Figure 77.
Figure 78.
Figure 79.
Figure 80.
Figure 81.
Figure 82.
Figure 83.
Figure 84.
Figure 85.
Figure 86.
Figure 87.
Figure 88.
Figure 89.
Figure 90.
Figure 91.
Figure 92.
Figure 93.
Figure 94.
Figure 95.
Figure 96.
Figure 97.
Figure 98.
Figure 99.
Thermo Scientific
Plumbing connections for the syringe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
APCI/MS plumbing connections for the LC tee union . . . . . . . . . . . . . . . . . . 116
Define Scan dialog box (reserpine SIM type data in APCI mode) . . . . . . . . . . 119
Tune dialog box showing the Automatic page. . . . . . . . . . . . . . . . . . . . . . . . . 121
Divert/Inject Valve dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Tune Plus window and Tune dialog box for automatic tuning in APCI
mode for the LTQ XL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Divert/inject valve setup for loop injection . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
APCI probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Define Scan dialog box (reserpine SIM-scan data) . . . . . . . . . . . . . . . . . . . . . . 128
Acquire Data dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Qual Browser window showing an APCI SIM chromatogram and mass
spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
APCI Source dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
ESI Source dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Ion source interface components for the LXQ and LTQ XL
(exploded view) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Ion transfer tube removal tool. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Sweep gas supply port in the API cone seal . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Define Scan dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Calibration solution in the normal mass range spectrum (LTQ XL, coarse
calibration) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Calibration solution in the normal mass range spectrum (Velos Pro, coarse
calibration) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Calibrate dialog box showing the High Mass Range Calibration page. . . . . . . 162
Calibrate dialog box showing the Calmix calibration mix (LXQ and
LTQ XL only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Define Scan dialog box (default ESI settings) . . . . . . . . . . . . . . . . . . . . . . . . . 164
Diagnostics dialog box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Diagnostics dialog box showing the mass calibration page (LXQ and
LTQ XL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Diagnostics dialog box showing the mass calibration page (Velos Pro) . . . . . . 167
Calibrate dialog box showing the high mass range calibration page with
PPG 2700 (factory) (LXQ and LTQ XL) . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Calibrate dialog box showing the high mass range calibration page
(Velos Pro) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
LTQ Series Getting Started Guide
xvii
P
Preface
The LTQ Series Getting Started Guide describes how to set up, calibrate, and tune the
following LTQ™ Series mass spectrometers (MSs):
• LXQ™, a single-segment 2D linear ion trap mass spectrometer
• LTQ XL™, a three-segment 2D linear ion trap mass spectrometer
• LTQ Velos™, a dual-cell 2D linear ion trap mass spectrometer
• Velos Pro™, a dual-cell 2D linear ion trap mass spectrometer
Note Unless otherwise noted:
• For the LTQ mass spectrometer, follow the LTQ XL information.
• For the LTQ Velos mass spectrometer, follow the Velos Pro information.
Contents
• Related Documentation
• Getting a Trap-HCD License
• Safety and Special Notices
• Contacting Us
 To suggest changes to documentation or to Help
Complete a brief survey about this document by clicking the button below.
Thank you in advance for your help.
Thermo Scientific
LTQ Series Getting Started Guide
xix
Preface
Related Documentation
In addition to this guide, Thermo Fisher Scientific provides the documentation listed in
Table 1 for the LTQ Series mass spectrometers. These PDF files are accessible from the data
system computer.
Table 1. LTQ Series mass spectrometers documentation
Model
Related documents
LXQ, LTQ XL, Velos Pro
LTQ Series Hardware Manual
LXQ, LTQ XL, Velos Pro,
LTQ XL/ETD system,
Velos Pro/ETD system,
MALDI LTQ XL system
LTQ Series Preinstallation Requirements Guide
LTQ Series Getting Connected Guide
ETD module
ETD Module Getting Started Guide
ETD Module Hardware Manual
MALDI source
MALDI Source Getting Started Guide
MALDI Source Hardware Manual
To access the manuals for the mass spectrometer, from the Microsoft™ Windows™ taskbar,
choose Start > Programs > Thermo Instruments > Manuals > model, where model is your
specific model, and then click the PDF file you want to view.
Note For Xcalibur data system version 2.0.7 or earlier, choose Start > Programs >
Xcalibur > Manuals > LTQ > model.
The software also provides Help. To access the Help, choose Help from the menu bar.
xx
LTQ Series Getting Started Guide
Thermo Scientific
Preface
Getting a Trap-HCD License
Ion trap higher energy collision-induced dissociation (Trap-HCD) fragmentation is an
optional feature for the Velos Pro mass spectrometer. If you purchased this option, you must
obtain a new license from Thermo Fisher Scientific and install it in your system before you
can use this feature.
Getting a New License Code
You can request a license code through e-mail or fax (see page xxiv). These instructions cover
e-mail requests only that you send to a specific Thermo Fisher Scientific e-mail address.
 To get a Trap-HCD license code
1. Choose Start > Programs > Thermo Foundation > Instrument Configuration to open
the Thermo Foundation Instrument Configuration window.
2. Under Available Devices, select the Velos Pro MS icon and click Add.
3. Under Configured Devices, select the Velos Pro MS icon and click Configure to open
the Velos Pro Configuration dialog box.
4. Select License and click Change License to open the LTQ License dialog box.
5. Highlight the license key in the License box.
Example license key
6. Press CTRL+C to copy the license key to the Windows Clipboard.
Thermo Scientific
LTQ Series Getting Started Guide
xxi
Preface
7. Send an e-mail message to [email protected]:
• In the Subject line, type License Request.
• In the body of the e-mail message, paste the license key and type your name,
company name, and phone number.
• If you purchased the Trap-HCD option, locate the bar code on the License
Trap-HCD for Velos Pro card that came with the instrument. In the body of the e-mail
message, type the product key that appears below the bar code.
• If you did not purchase the Trap-HCD option with the instrument, contact your
local Thermo Fisher Scientific Technical Sales representative.
When Thermo Fisher Scientific Customer Support sends you a new license code, see
“Installing a New License Code.”
Installing a New License Code
After you receive your new license code, follow this procedure.
 To install the HCD license number
1. Open the LTQ License dialog box (see step 1 through step 4 on page xxi).
2. In the License box, paste a copy of the new license number from the e-mail message and
click Set.
Example
3. Click OK when the following message appears:
The new license number has been set.
xxii
LTQ Series Getting Started Guide
Thermo Scientific
Preface
4. In the Velos Pro Configuration dialog box, verify the addition of the Ion Trap HCD
(Full) feature and click OK.
5. Reboot the data system and then the Velos Pro mass spectrometer.
Safety and Special Notices
Ensure that you follow the precautionary statements presented in this guide. The safety and
other special notices appear in boxes. Safety and special notices include the following:
CAUTION Highlights hazards to humans, property, or the environment. Each CAUTION
notice is accompanied by an appropriate CAUTION symbol.
IMPORTANT Highlights information necessary to prevent damage to software, loss of
data, or invalid test results; or might contain information that is critical for optimal
performance of the system.
Note Highlights information of general interest.
Tip Highlights helpful information that can make a task easier.
Table 2 lists additional caution-specific symbols that appear in the LTQ Series Getting Started
Guide.
Thermo Scientific
LTQ Series Getting Started Guide
xxiii
Preface
Table 2. Caution-specific symbols and their meanings
Symbol
Meaning
Electric Shock: An electric shock hazard is present in the instrument.
Proceed with caution.
Hot Surface: Allow heated components to cool before touching or
servicing the instrument.
Sharp Object: A sharp object is present in the instrument. Proceed
with caution.
Chemical: Hazardous chemicals might be present in the instrument.
Wear gloves when handling carcinogenic, corrosive, irritant,
mutagenic, or toxic chemicals. Use only approved containers and
procedures for disposing of waste oil.
Eye Hazard: Eye damage could occur from splattered chemicals or
airborne particles. Wear safety glasses when handling chemicals or
servicing the instrument.
Contacting Us
There are several ways to contact Thermo Fisher Scientific for the information you need.
 To contact Technical Support
Phone
800-532-4752
Fax
561-688-8736
E-mail
[email protected]
Knowledge base
www.thermokb.com
Find software updates and utilities to download at mssupport.thermo.com.
 To contact Customer Service for ordering information
xxiv
Phone
800-532-4752
Fax
561-688-8731
E-mail
[email protected]
Web site
www.thermo.com/ms
LTQ Series Getting Started Guide
Thermo Scientific
Preface
 To get local contact information for sales or service
Go to www.thermoscientific.com/wps/portal/ts/contactus.
 To copy manuals from the Internet
Go to mssupport.thermo.com, agree to the Terms and Conditions, and then click
Customer Manuals in the left margin of the window.
 To suggest changes to documentation or to Help
• Fill out a reader survey online at www.surveymonkey.com/s/PQM6P62.
• Send an e-mail message to the Technical Publications Editor at
[email protected]
Thermo Scientific
LTQ Series Getting Started Guide
xxv
1
Introduction
This chapter provides general information about the LTQ Series mass spectrometer. For
additional information, such as procedures for daily operation, maintenance, and system
startup and shutdown, refer to the LTQ Series Hardware Manual.
Note Unless otherwise noted:
• For the LTQ mass spectrometer, follow the LTQ XL information.
• For the LTQ Velos mass spectrometer, follow the Velos Pro information.
The “Glossary” on page 173 defines some of the terms used in this guide.
Contents
• Features of the LTQ Series Mass Spectrometer
• Sheath, Auxiliary, and Sweep Gases
• Ionization Techniques
• Sample Introduction Techniques
• Types of Buffers
• LC Flow Rate Ranges
• Tuning and Calibrating the Mass Spectrometer
• Ion Polarity Modes
• Data Types
• Scan Power and Scan Modes
• Scan Rates
• Scan Types
• Types of Experiments
• Mass/Charge Range
Thermo Scientific
LTQ Series Getting Started Guide
1
1
Introduction
Features of the LTQ Series Mass Spectrometer
Features of the LTQ Series Mass Spectrometer
The LTQ Series mass spectrometer has the following features:
• Linear Ion Trap Mass Analyzer
• ZoomScan Analysis for Charge State Determination
• Wideband Activation
• Switchable Ion Source
• Optional Ion Sweep Cone and Ion Sweep Gas for Complex Sample Matrices
Linear Ion Trap Mass Analyzer
All high performance liquid chromatography (HPLC) systems can provide chromatographic
separation and compound detection based on retention time. Retention time alone, however,
does not positively identify a compound because many compounds can have the same
retention time under the same experimental conditions. In addition, other compounds in the
sample can coelute with the compound of interest. The coelution of compounds causes
quantitation errors.
The attribute that sets the LTQ Series mass spectrometer apart from other detectors used with
LC systems, such as UV-Vis detectors or single-stage mass analyzer mass spectrometers, is the
high level of analytical specificity that it provides. In addition to mass-to-charge ratio (m/z),
the LTQ Series mass spectrometer can provide multiple levels of mass analysis. Each level of
mass analysis adds a new dimension of specificity for unequivocal compound identification.
The LTQ Series mass spectrometer provides the following levels of mass analysis:
• Single-stage mass analysis (provides molecular mass information)
• Two-stage mass analysis, MS/MS (provides structural information)
• Multi-stage mass analysis, MSn (provides structural information)
Single-stage mass analysis identifies analytes based on molecular mass information. Typically,
atmospheric pressure ionization (API) produces mass spectra that provide molecular mass
information.
Two-stage mass analysis can provide even more certainty in compound identification. MS/MS
analysis monitors how a parent ion fragments when exposed to an additional stage of
excitation. There are two types of MS/MS analysis:
• Full-scan MS/MS monitors the production of all product ions from a specific parent ion.
• Selected reaction monitoring (SRM) MS/MS analysis monitors a specific reaction path,
which is the production of a specific product ion from a specific parent ion.
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Features of the LTQ Series Mass Spectrometer
Using either type of MS/MS analysis, you can quantify target analytes in complex matrices
such as plant or animal tissue, plasma, urine, groundwater, or soil. Because of the specificity of
MS/MS measurements and the ability to eliminate interferences by an initial mass selection
stage, the LTQ Series mass spectrometer easily accomplishes quantitative analysis of target
compounds.
Multi-stage mass analysis provides a unique capability to obtain structural information that
can be useful in structure elucidation of metabolites, natural products, and sugars. MSn
techniques on the LTQ Series mass spectrometer allow for stepwise fragmentation pathways,
making interpretation of MSn spectra relatively straightforward. The LTQ Series mass
spectrometer has several advanced features that make its MSn capabilities extremely powerful
for qualitative analysis.
For additional information, see “Types of Experiments” on page 20.
ZoomScan Analysis for Charge State Determination
The ZoomScan scan type analysis provides information about the charge state of one or more
mass ions. ZoomScan™ data is collected by using slower scan rates that give higher resolution.
This level of resolution can determine the charge state of an ion without ambiguity.
For additional information, see “ZoomScan and UltraZoom” on page 19.
Wideband Activation
Use the WideBand Activation™ option for the LTQ Series mass spectrometer to apply
collision energy to ions during MS/MS fragmentation over a fixed mass range of 20 Da. With
this option, you can apply collision energy to both the parent ion and the product ions created
as a result of non-specific losses of water (18 Da) or ammonia (17 Da), or to product ions
formed from the loss of fragments smaller than 20 Da. For enhanced structural information
without resorting to MS3 analysis, choose the Wideband Activation option for qualitative
MS/MS analysis. Because the fragmentation efficiency is somewhat reduced with Wideband
Activation, you must increase the collision energy.
For an example procedure, see “Optimizing the Collision Energy Automatically for an
MS/MS Experiment” on page 103.
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Sheath, Auxiliary, and Sweep Gases
Switchable Ion Source
You can configure the LTQ Series mass spectrometer with a standard electrospray ionization
(ESI) probe (see Figure 16 on page 42), a heated-electrospray ionization (H-ESI) probe (see
Figure 17 on page 42), or an atmospheric pressure chemical ionization (APCI) probe (see
Figure 31 on page 56). By using additional ion source kits, you can operate the mass
spectrometer in the atmospheric pressure photoionization (APPI) mode and the nanospray
ionization (NSI) mode.
For information about choosing the appropriate ionization source for the analytes, see
“Ionization Techniques” on page 5. For instructions about installing the ESI or APCI probe,
see Chapter 2, “Setting Up the API Source.”
Optional Ion Sweep Cone and Ion Sweep Gas for Complex Sample Matrices
The ion sweep cone is a metal cone installed over the ion transfer tube. The ion sweep cone
channels the sweep gas toward the entrance of the API ion transfer tube. This helps to keep
the entrance of the ion transfer tube free of contaminants. The net result is a significant
increase in the number of samples to analyze without a loss of signal intensity. In addition,
keeping the ion transfer tube entrance as clean as possible reduces the need for frequent
maintenance.
For instructions about installing the ion sweep cone, refer to the LTQ Series Hardware
Manual.
Sheath, Auxiliary, and Sweep Gases
The LTQ Series mass spectrometer uses nitrogen as the sheath, auxiliary, and sweep gases. The
ESI and APCI probes have inlets for the sheath (S) gas and auxiliary (A) gas. The optional ion
sweep cone has an inlet for the sweep gas. The NSI probe does not use desolvation gases.
The descriptions of these gases are as follows:
• Sheath gas—An inner-coaxial gas that helps nebulize the sample solution into a fine mist
as the solution exits the probe nozzle.
• Auxiliary gas—An outer-coaxial gas that helps the sheath gas in the nebulization and
evaporation of the sample solution by focusing the vapor plume and lowering the
humidity in the ion source.
• Sweep gas—An off-axis gas that flows out from behind the optional ion sweep cone.
Install the ion sweep cone to improve ruggedness when analyzing complex matrices such as
plasma or nonvolatile salt buffers.
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Ionization Techniques
Auxiliary gas is not required for low-solvent flow applications. For high-solvent flow
applications, first optimize the sheath gas flow, then the auxiliary gas flow, and then the sweep
gas flow, if provided.
Table 3 on page 12 and Table 4 on page 13 list the guidelines for the operating parameters.
Ionization Techniques
Typically, ESI is the preferred ionization mode for more polar compounds such as amines,
peptides, and proteins. APCI is the preferred ionization mode for non-polar compounds such
as steroids.
Using ESI or H-ESI
ESI is a soft ionization technique. The ESI source transfers ions in solution to the gas phase.
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 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 ESI, the range of molecular masses that the LTQ Series mass spectrometer can
analyze can be greater than 50000 Da if there is multiple charging.
The 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 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 ESI needle 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 0.1–1000 μL/min. See Table 3
on page 12 for guidelines.
In 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 ESI process. Conversely, ESI favors
small droplets with low surface tension, high volatility, high surface charge, weak ion
solvation, and low conductivity.
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Ionization Techniques
To obtain good ESI results, follow these guidelines:
• Keep nonvolatile salts and buffers out of the solvent system. For example, avoid the use of
salts containing phosphate, potassium, or sodium. Use acetate salts or ammonium
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).
Using APCI
Like 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
2000 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 LTQ Series mass spectrometer in APCI
mode is typically high (0.2–2 mL/min). See Table 4 on page 13 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.
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Sample Introduction Techniques
Sample Introduction Techniques
The LTQ Series mass spectrometer has a divert/inject valve and a syringe pump. The
following techniques are available to introduce samples into the API source:
• Direct Infusion
• High-Flow Infusion
• Loop Injection (Flow-Injection Analysis)
• Liquid Chromatography
Direct Infusion
The direct infusion technique uses the syringe pump to infuse sample directly into the ion
source, as shown in Figure 1. Use this technique to introduce a calibration solution for
automatic tuning and calibrating in ESI mode. You can also use this technique to introduce a
solution of pure analyte at a steady rate in ESI mode for qualitative analyses, and perform
experiments at a low flow rate with the syringe pump.
Figure 1.
Sample introduction by using direct infusion with a syringe pump
ESI probe
3
4
Load
Detector
Inject
Waste
2
5
1
6
Power
Vacuum
Communication
System
Scanning
Pump On
Infusion line
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Sample Introduction Techniques
High-Flow Infusion
The high-flow infusion technique uses an LC tee union 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 ion source. Use this technique to tune on an
analyte (create a tune method for an analyte) in either ESI or APCI mode, using the same flow
rate and mobile phase composition that you plan to use for the LC/MS experiments.
Use the high-flow infusion method for performing 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 API spray cone more
frequently.
Figure 2 shows the inlet connections for high-flow infusion in ESI mode. When the
divert/inject valve is in the Load position, solvent flow from the LC pump enters the valve
through port 2 and exits the valve through port 3, which connects to the ion source. When
the divert/inject valve is in the Inject position, solvent flow from the LC pump enters the valve
through port 2 and exits the valve through port 1 to waste.
Figure 2.
Sample introduction by using high-flow infusion in ESI mode
From port 3
to ion source
4
3
3
2 2
1 1
5
6
From LC pump
to port 2
From port 1
to waste
ESI probe
LC tee union
Infusion line
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Sample Introduction Techniques
For instructions about setting up the inlet to perform high-flow infusion, see “Setting Up the
Inlet for High-Flow Infusion in ESI Mode” on page 85.
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 ion source (Figure 3). 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 ion source. Use this technique in either ESI
or APCI mode.
Figure 3.
Sample introduction by using loop injection in APCI mode
APCI probe
3
4
Load
Detector
Inject
Waste
2
5
1
6
Injection port fitting
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Sample Introduction Techniques
Liquid Chromatography
To perform loop injection by using the liquid chromatography (LC) technique, install an LC
column between the sample inlet of the ion source and port 3 of the divert/inject valve, or
connect an LC system with an autosampler to the LTQ Series 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 ion source, as shown in Figure 4.
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 ion source. Use this technique in
either ESI or APCI mode.
Figure 4.
Sample introduction by using direct injection with an autosampler in ESI mode
From port 3
to ion source
3
4
Plug
(optional)
2
1
5
6
3
4
Load
Detector
Inject
Waste
From LC
to port 2
From port 1
to waste
2
5
1
6
Power
Vacuum
Communication
System
Scanning
Pump On
Pump
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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 ion source, such as the
transfer tube and spray nozzle of the ionization probe. Using nonvolatile buffers without also
cleaning the ion 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
• Triethylamine (TEA)
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). The MSDSs describe the chemicals and must be freely
available to lab personnel to examine at any time. MSDSs provide summarized
information on the hazard and toxicity of specific chemical compounds. MSDSs also
provide information on the proper handling of compounds, first aid for accidental
exposure, and procedures for the remedy of spills or leaks.
Read the MSDS 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.
LC Flow Rate Ranges
The ESI probe 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 probe 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.
1
The ESI probe can generate ions from liquid flows as low as 1 μL/min. However, flows below 5 μL/min require
more care, especially with the position of the fused silica sample tube within the ESI probe.
2 For the APCI probe, flows below 200 μL/min require more care to maintain a stable spray.
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LC Flow Rate Ranges
While changing the flow rate of solvents entering the mass spectrometer, adjust the following
parameters:
• For 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 adjust the
flow rates for the sheath, auxiliary, and sweep gases.
• For both ionization modes, increase the temperature of the ion transfer tube and the gas
flow rates while increasing the flow rate of liquid into the mass spectrometer.
Table 3 lists the guidelines for ESI operation for ion transfer tube temperatures and gas flow
rates for various LC solvent flow rates.
Table 4 lists the guidelines for APCI operation for the ion transfer tube temperature, vaporizer
temperature, and gas flow rate for a range of LC solvent flow rates.
Table 3. Guidelines for setting operating parameters for LC/ESI/MS a
LC flow rate
Suggested
column size
Ion transfer tube
temperature
(typical)
Infusion or LC,
< 10 μL/min
Capillary
(0.1–0.3 mm)
150–200 °C
(302–392 °F)
LC,
50–200 μL/min
1 mm ID (microbore 200–275 °C
column)
(392–527 °F)
LC,
100–500 μL/min
LC,
0.4–1 mL/min
a
2–3 mm ID (narrow
bore column)
250–350 °C
(482–662 °F)
4.6 mm ID (standard 300–400 °C
column)
(572–752 °F)
Sheath gas
Auxiliary and/or
sweep gas
Not required
Not required
5–15 units, typical
0 units, typical
Required
Not required, but might help
depending on conditions
20–40 units, typical
0–20 units, typical
Required
Not required, but usually
helps to reduce solvent
background ions
30–60 units, typical
0–20 units, typical
Required
Required
60–100 units, typical 10–40 units, typical
Choose auxiliary gas, sweep gas, or both, according to the suggestions in “Sheath, Auxiliary, and Sweep Gases” on page 4.
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Tuning and Calibrating the Mass Spectrometer
Table 4. Guidelines for setting operating parameters for LC/APCI/MSa
a
LC flow rate
Vaporizer
temperature (typical)
Ion transfer tube
temperature (typical)
Sheath gas
Auxiliary gas
Sweep gas
LC,
200–2000 μL/min
LXQ:
350–500 °C
LXQ:
150–350 °C
Required
40–100 units
5–20 units
0–5 units
LTQ XL and
Velos Pro:
350–500 °C
LTQ XL and
Velos Pro:
250–350°C
Choose auxiliary gas, sweep gas, or both, according to the suggestions in “Sheath, Auxiliary, and Sweep Gases” on page 4.
Tuning and Calibrating the Mass Spectrometer
Calibrate the LTQ Series mass spectrometer to ensure its mass accuracy. Before calibrating the
mass accuracy, tune the mass spectrometer with the ESI calibration solution to optimize the
transmission of ions.
IMPORTANT Thermo Fisher Scientific recommends that you check the calibration once
a week and calibrate as needed.
Calibration parameters are instrument parameters whose values do not vary with the type of
experiment. Automatic and semi-automatic calibration (including checking the calibration)
require introducing the calibration solution into the mass spectrometer at a steady flow rate
while the calibration is running. Introduce the solution directly from the syringe pump into
the mass spectrometer in ESI/MS mode.
Tune parameters are instrument parameters whose values can vary with the type of
experiment. For example, if the experiment requires quantitative data on one or more
particular ions, you must tune the mass spectrometer with the analyte. You must also tune the
mass spectrometer if you change any parameter that is specific to the experiment or analyte.
Create tune methods for analytes by using the same solvent composition and flow rate that
you plan to use in the chromatographic method for the LC/MS experiments. Tune on a
particular peak in the mass spectrum of the analyte, or choose an ion in the calibration
solution that is closest to the mass-to-charge ratio for the target ion.
Automatic and semi-automatic tuning procedures (including optimizing the collision energy)
require introducing the analyte into the mass spectrometer at a steady rate in one of two ways:
• Direct infusion (see page 7)
• High-flow infusion (see page 8)
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Ion Polarity Modes
In most cases, you can use the tune file obtained from the automatic or semi-automatic
tuning procedures for the analytical experiments. However, for some applications, you might
need to tune several parameters. In that case, tune manually. With manual tuning, introduce a
tuning solution at a steady flow rate.
Note For ESI operation, the most important parameters that affect the signal quality are
the ion transfer tube temperature, API tube lens offset voltage (except for the LTQ Velos
and Velos Pro mass spectrometers), gases, and solvent flow rate. For optimum sensitivity,
tune the mass spectrometer in the operational mode that you plan to use for the
experiments.
The LTQ Velos and Velos Pro mass spectrometers do not have a API tube lens.
After creating a tune method for a particular application, add the tune method to the
instrument method. You can add a different tune method for each segment of the instrument
method. For example, if analyte A and analyte B elute at 2 and 4 minutes, respectively, create
an instrument method with two segments: Specify a tune method optimized for analyte A in
segment one and a tune method optimized for analyte B in segment two.
Table 5 lists the methods of sample introduction for each of the calibration and tuning
procedures.
Table 5. Summary of methods of sample introduction for calibration and tuning
Calibration
Tuning
Check
Auto
Semi-auto
Auto
Semi-auto
Manual
Collision
energy







Your tune solution/ Direct infusion




Your tune solution/ High-flow infusion




Sample/ Sample introduction
Calibration solution/ Direct infusion
Ion Polarity Modes
The LTQ Series mass spectrometer can operate in either positive or negative ion polarity
modes. The mass spectrometer controls whether positive ions or negative ions are transmitted
to the mass analyzer for mass analysis by changing the polarity of the voltage potentials
applied to the API source, ion optics, and ion detection system. The ion optics are located
between the API source and the mass analyzer.
The information obtained from a positive-ion mass spectrum is different from and
complementary to that obtained from a negative-ion mass spectrum. “Ionization Techniques”
on page 5 describes the spectral characteristics for these two polarity modes. The ability to
obtain both positive-ion and negative-ion mass spectra during a single scan run reduces the
time required to obtain a complete qualitative analysis of the sample.
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Data Types
Data Types
With the LTQ Series mass spectrometer you can acquire and display mass spectral data
(intensity versus mass-to-charge ratio) in one of two data types:
• Profile data
With profile data you can see the inherent shape of the peaks in the mass spectrum. The
mass spectrum divides each atomic mass unit into several sampling intervals. The
intensity of the ion current is determined at each sampling interval. The intensity at each
sampling interval is displayed with the intensities connected by a continuous line. In
general, use the profile scan data type when you tune and calibrate the mass spectrometer
so that you can easily see and measure mass resolution.
• Centroid data
Centroid data displays the mass spectrum as a bar graph. This scan data type sums the
intensities of each set of sampling intervals. This sum is displayed versus the integral
center of mass of the many sampling intervals. The disk space requirements for centroid
data are about one-tenth of what is required for profile data. Consequently, data
processing for centroid data is faster than that for profile data.
Scan Power and Scan Modes
Ions produced in the ion source are often referred to as parent ions or precursor ions. To
produce a mass spectrum, the mass analyzer scans its dc and rf voltages to sequentially eject
ions from the trap based on their m/z values. Or, by varying the rf voltages and applying an
additional isolation waveform voltage to the mass analyzer, the LTQ Series mass spectrometer
can first eject all ions, except for several selected parent ions, and then collide these ions with
the helium that is present in the mass analyzer. This helium is known as a buffer gas. The
collisions can cause the selected parent or precursor ions to fragment into product ions. The
ion trap can then sequentially eject these product ions based on their m/z values to produce a
mass spectrum of the product ions.
MSn represents the number of stages of mass analysis where n is the scan power. Each stage of
mass analysis where n > 1 includes an ion selection step. The mass spectrometer supports scan
powers of n = 1 to n = 10. As you raise the scan power, you can obtain more structural
information about the analyte.
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Scan Rates
The standard configurations of the mass spectrometer support several scan powers.
• MS scan mode
The mass spectrometer (MS) scan mode corresponds to a single stage of mass
analysis—that is, a scan power of n = 1. The MS scan modes only involve parent ions,
and no fragmentation of the parent ions occurs. The MS scan mode can be a full-scan
experiment or a selected ion monitoring (SIM) scan type experiment (see “Selected Ion
Monitoring” on page 18).
• MS/MS scan mode
The MS/MS scan corresponds to two stages of mass analysis (scan power of n = 2). In an
MS/MS scan, parent ions fragment into product ions. An MS/MS scan can be a full- scan
experiment or a selected reaction monitoring (SRM) scan type experiment (see “Selected
Reaction Monitoring” on page 18).
• MSn scan mode
An MSn scan involves three to 10 stages of mass analysis (scan power of n = 3 to n = 10).
However, the term can also apply to one stage of mass analysis (with n = 1) or to two
stages of mass analysis (with n = 2). An MSn scan can be either a full-scan experiment or a
consecutive reaction monitoring (CRM) scan type experiment (see “Consecutive
Reaction Monitoring” on page 19).
Scan Rates
The LTQ Series mass spectrometer can operate in the following scan rates: Normal,
Rapid (for the Velos Pro only), Enhanced, Turbo, Zoom, and UltraZoom. To use the Rapid
scan rate on the Velos Pro mass spectrometer, you must activate the Trap-HCD license; see
“Getting a Trap-HCD License” on page xxi. For information about these scan rates, refer to
the data system Help.
Scan Types
The LTQ Series mass spectrometer can operate in the following scan types:
• Full Scan
• Selected Ion Monitoring
• Selected Reaction Monitoring
• Consecutive Reaction Monitoring
• ZoomScan and UltraZoom
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Scan Types
Full Scan
A full-scan type provides an entire or wide range mass spectrum of the analyte in a particular
scan time. With a full scan, the mass analyzer scans from the first mass to the last mass
without interruption in the last step of mass analysis (ion scan-out).
A full scan provides more information about an analyte than does SIM or SRM. But because a
full scan scans an entire mass range during a particular scan time, it does not provide the
sensitivity that the other scan types can achieve.
• Single-stage full scan (MS1)
The single-stage full scan has one stage of mass analysis (scan power of n = 1). With the
single-stage full scan, the mass analyzer stores ions formed in the ion source. These ions
are then sequentially scanned out of the mass analyzer to produce a full mass spectrum (a
mass spectrum of the observable ions in the specified mass range at a specific time point
in the analysis).
Single-stage full-scan analysis is a useful tool for qualitative analysis. Use single-stage
full-scan experiments to determine the molecular weight of unknown compounds or the
molecular weight of each component in a mixture of unknown compounds.
After you determine the molecular weight of a target compound, you can use the SIM or
SRM scan type to perform routine quantitative analyses of the compound (see “Selected
Ion Monitoring” on page 18 and “Selected Reaction Monitoring” on page 18).
• Two-stage full scan (MS/MS)
The two-stage full scan has two stages of mass analysis (scan power of n = 2). In the first
stage, the mass analyzer stores ions formed in the ion source. Then ions of one
mass-to-charge ratio (the parent ions) are selected, and all other ions are ejected from the
mass analyzer. The parent ions are excited and collide with background gas that is present
in the mass analyzer. The collisions of the parent ions cause them to fragment to produce
one or more product ions.
In the second stage of mass analysis, the mass analyzer stores the product ions. Then they
are sequentially scanned out of the mass analyzer to produce a full-product ion mass
spectrum.
A two-stage full scan gives you more information about a sample than SRM does but at a
lower speed.
To use the SRM scan you must know which parent and product ions to observe. To
obtain this information you could use a one-stage full scan to determine the parent mass
spectrum and a two-stage full scan to determine the product mass spectrum for the parent
ions of interest. For subsequent routine quantitative analysis you would use an SRM scan
type based on the one-stage and two-stage full-scan results.
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Scan Types
Selected Ion Monitoring
SIM is a single-stage (scan power of n = 1) technique that monitors a particular ion or set of
ions. In a SIM scan, the mass analyzer stores ions formed in the ion source. The mass
spectrometer then selects ions of one or more mass-to-charge ratios, and ejects all other ions
from the mass analyzer. The selected ions are then sequentially scanned out of the mass
analyzer to produce a SIM mass spectrum.
Use SIM experiments to detect small quantities of a target compound in a complex mixture
when you know the mass spectrum of the target compound. SIM is useful in trace analysis
and in the rapid screening of a large number of samples for a target compound.
Because a SIM scan monitors only a few ions, SIM provides lower detection limits and greater
speed than a single-stage full-scan analysis. SIM achieves lower detection limits because more
time is spent monitoring significant ions that are known to occur in the mass spectrum of the
target sample. SIM achieves greater speed because it only monitors a few ions of interest while
ignoring regions of the spectrum that are empty or have no ions of interest.
SIM can improve the detection limit and decrease analysis time, but it can also reduce
target compound specificity compared to an MS/MS scan. SIM analysis decreases the analysis
time because it only monitors particular ions. SIM analysis reduces specificity because any
compound that produces ions of the m/z being monitored would appear to be the target
compound. To avoid false positive results when using SIM for routine analyses, first verify
that SIM is monitoring ions from the target compound and nothing else.
Selected Reaction Monitoring
SRM is a two-stage (scan power of n = 2) technique that monitors parent ion and product ion
pairs.
In the first stage of mass analysis, the mass analyzer stores the ions formed in the ion source.
The mass spectrometer selects ions of one mass-to-charge ratio (the parent ions) and ejects all
other ions from the mass analyzer. The parent ions are excited and collide with background
gas that is present in the mass analyzer. The collisions of the parent ions cause them to
fragment to produce one or more product ions.
In the second stage of mass analysis, the mass analyzer stores the product ions. The selected
m/z range of ions are then sequentially scanned out of the mass analyzer to produce an SRM
product ion mass spectrum.
Like SIM, SRM provides very rapid analysis of trace components in complex mixtures.
However, because you are monitoring pairs of ions (one product ion for each parent ion), the
specificity obtained in SRM can be much greater than that obtained in SIM. You are very
unlikely to get a false positive result with SRM. To get a false positive result, the interfering
compound must form a parent ion of the same mass-to-charge ratio as the selected parent ion
from the target compound. The compound must also fragment to form a product ion of the
same mass-to-charge ratio as the selected product ion from the target compound.
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Scan Types
Consecutive Reaction Monitoring
CRM is the multi-stage (scan power of n = 3 to n = 10) analog of SIM (n = 1) and SRM
(n = 2) that monitors a multi-step reaction path. In the first stage of mass analysis, the mass
analyzer stores the ions formed in the ion source. The mass spectrometer selects ions of one
mass-to-charge ratio (the parent ions) and ejects all other ions from the mass analyzer. The
parent ions are excited and collide with background gas that is present in the mass analyzer.
The collisions of the parent ions cause them to fragment to produce one or more product
ions.
In the second stage of mass analysis, the mass analyzer stores the product ions. The mass
spectrometer selects product ions of one mass-to-charge ratio and ejects all other ions from the
mass analyzer. The selected product ions now become the new parent ions for the next stage
of mass analysis. The new parent ions are excited and collide with background gas. The
collisions of the new parent ions cause them to fragment to produce one or more new product
ions.
In the third stage of mass analysis, the mass analyzer stores the new product ions. This process
repeats up to seven more times until the mass spectrometer produces the final product ions of
interest.
In the nth stage of mass analysis, the mass analyzer stores the final product. The mass
spectrometer then sequentially scans the m/z range of the selected ions out of the mass
analyzer to produce a CRM final product ion mass spectrum.
In CRM, the specificity increases as you increase the number of monitored consecutive
reactions. However, the sensitivity decreases as you increase the number of monitored
consecutive reactions—especially if many fragmentation pathways are available to the
parent ion.
ZoomScan and UltraZoom
Determining the mass of an ion from its mass-to-charge ratio can be complicated if the charge
state of the ion is unknown. ZoomScan and UltraZoom are scan modes in which the mass
spectrometer achieves a higher resolution. The resolution is sufficient to determine the
12C/13C isotopic separation that then allows the system to determine the charge state by using
the following:
• If the isotopic peaks are 1 Da apart, the ion has a charge state of ±1.
• If the isotopic peaks are 0.5 Da apart, the ion has a charge state of ±2.
• If the isotopic peaks are 0.33 Da apart, the ion has a charge state of ±3.
• If the isotopic peaks are 0.25 Da apart, the ion has a charge state of ±4.
• If the isotopic peaks are 0.2 Da apart, the ion has a charge state of ±5.
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Types of Experiments
With the UltraZoom scan mode, the preceding list can continue up to a charge state of ±10.
You can then determine the molecular weight of the ion from the knowledge of the charge
state and mass-to-charge ratio of the ion.
Types of Experiments
The New Method page of the Thermo Xcalibur Instrument Setup window for the LTQ Series
mass spectrometer (Figure 5) contains links to templates for various types of experiments. To
save time entering the parameters for the instrument method, open the 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).
Figure 5.
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Thermo Xcalibur Instrument Setup window showing the New Method page (Velos Pro with ETD experiment types)
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Types of Experiments
This section describes the types of experiments for the LTQ Series mass spectrometer. The
experiments are grouped into the following categories:
• General MS or MSn Experiments
• Data Dependent Experiments
• Ion Mapping Experiments
• Ion Tree Experiments
General MS or MSn Experiments
A General MS or MSn experiment is best used to collect qualitative data for structural
analysis. However, you can also use a General experiment for the quantitative analysis of
known compounds.
The Xcalibur data system includes an Instrument Method template in the Instrument Setup
window for a General MS or MSn experiment; click General MS or MSn on the New
Method page in the Xcalibur application (Figure 5 on page 20). Figure 6 shows an example of
a General MS or MSn experiment template.
In a General MS quantitation experiment, specify the following:
• Mass range of the analyte or analytes
• Parent (precursor) ion that fragments into distinctive product ions
• Mass-to-charge ratios of all the product ions
The LTQ Series mass spectrometer can then collect data on the ions in the mass range or on
the product ions of the parent ion or ions that you specify.
If using a General experiment to collect data for qualitative (structural) analysis, specify the
scan mode (MS through MSn) that you want data for on the MS Detector Setup page under
Scan Event 1 Settings (Figure 6). If specifying MS/MS or MSn, choose the parent ion or ions
that you want data for on the MS Detector Setup page under MSn Settings. Under MSn
Settings, you can specify one of these fragmentation methods: collision-induced dissociation
(CID), pulsed Q collision-induced dissociation (PQD), and for the Velos Pro there is also
higher energy collision-induced dissociation (HCD) (requires an activated Trap-HCD license;
see “Getting a Trap-HCD License” on page xxi). The LTQ Series mass spectrometer then
collects distinct qualitative information for structural analysis or for spectral reference.
The mass spectrometer can generate reproducible, analyte-specific spectra. Consequently, you
can use the reference spectra generated with one LTQ Series mass spectrometer to confirm
structures of compounds generated with another LTQ Series mass spectrometer.
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Types of Experiments
Figure 6.
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General MS experiment template on the MS Detector Setup page
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Types of Experiments
Data Dependent Experiments
A Data Dependent™ experiment is best used for the qualitative analysis of unknown
compounds for structure elucidation or confirmation. These experiments maximize the data
obtained while requiring minimal user input. The LTQ Series mass spectrometer uses the
information in a Data Dependent experiment to make decisions automatically about
subsequent experiments. The Xcalibur Instrument Setup window (Figure 5 on page 20)
contains the Instrument Method templates for Data Dependent experiments.
A Data Dependent experiment produces a large amount of data from a single sample analysis.
In a Data Dependent experiment, specify parent ions for fragmentation or let the mass
spectrometer automatically select the ions for fragmentation. The mass spectrometer can
automatically collect the structural information for every parent ion in the sample, even if the
sample is a mixture of compounds.
Find useful structural information about a compound automatically with the simplest Data
Dependent experiment, Data Dependent MS/MS. In this experiment, specify only the MS
scan range—you do not need to specify a parent ion. The mass spectrometer then collects
full-scan MS data, picks the most intense parent ion in the spectrum, and then fragments the
ion to generate product ions.
A Data Dependent Triple-Play experiment is the same as Data Dependent MS/MS but
includes the identification of the charge state of the parent with the ZoomScan feature. A
Data Dependent Triple-Play experiment collects full-scan MS data, and then uses ZoomScan
to determine the charge state of the parent ion and calculate the molecular weight. The parent
ion is then fragmented into product ions (MS/MS). For example, if the mass spectrometer
determines a charge state equal to two, and if the mass-to-charge ratio of the parent ion is
m/z 500, then the mass-to-charge ratios of the product ions can be up to and including
m/z 1000 (or 2 × 500). Figure 7 shows an example of a Data Dependent Triple-Play
experiment template.
Ion Mapping experiments can be Data Dependent. However, the Total Ion Map, Neutral Loss
Ion Map, and Parent Ion Map experiments are not Data Dependent. The Data Dependent
Zoom Map experiment collects ZoomScan data on every scan interval in a specified mass
range. An MS/MS experiment on the largest ion in each ZoomScan interval is automatically
performed. For additional information, see “Ion Mapping Experiments” on page 26.
Ion Tree experiments are types of Data Dependent experiments. These experiments provide
methods for automatically interpreting MSn data and arranging the data in formats that are
easy to manipulate. For additional information, see “Ion Tree Experiments” on page 28.
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Types of Experiments
Set up the Data Dependent experiments in one of two ways:
• If you have some idea of the parent ion, or if you expect a certain kind of parent, set up a
list of possible parent ions. After detecting one of the specified parent ions, you can
acquire product spectra and analyze the information. Conversely, you can set up a list of
ions that you do not want to be selected for fragmentation.
• If you have little information about the compound, you can set up the parameters of a
Data Dependent experiment so that if the intensity of the ion signal is above a specified
threshold, the mass spectrometer generates product spectra. (Decide later if the
information is useful.) Parameters might include threshold values for the intensity of the
MS or MSn ion signal. Whatever threshold values you choose should isolate the parent
ions of interest.
Use a Data Dependent experiment to do the following:
• Identify low-level impurities in high-purity compounds (Data Dependent MS/MS).
A Data Dependent MSn experiment can identify process impurities. In the quality
assurance process for aspirin, for example, the mass spectrometer can identify impurities
of less than 0.1 percent.
• Identify metabolites in a complex mixture (chromatographic separation with Data
Dependent MS/MS).
A Data Dependent MS/MS experiment of a complex mixture can provide highly specific
structural information. For example, characteristic masses along the metabolic pathways
of a drug can produce MSn spectra that are specific to the structure of the drug and its
metabolites. These spectra are essential in metabolite identification.
• Build a custom library of composite MSn spectra (Ion Tree).
A Data Dependent experiment can produce a composite spectrum of, for example,
MS/MS, MS3, and MS4 data. The mass spectrometer can store the MSn fingerprint data
in a custom MSn library spectrum. The data is valuable for use in process control, quality
assurance, or research.
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Types of Experiments
Figure 7.
Data Dependent Triple-Play experiment template on the MS Detector Setup page
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Types of Experiments
Ion Mapping Experiments
An Ion Mapping experiment is best used to get full structural characterization of unknown
molecules in complex mixtures. In an Ion Mapping experiment, you can get product ion scans
on every parent ion over a specified mass range. An Ion Mapping experiment can help to
identify automatically which parent ions were fragmented to yield a specified product ion.
The experiment “maps” one or more parent ions by using the information from product ion
scans.
These experiments require that sample solution enter the mass spectrometer at a composition
that is constant throughout. Therefore, use an infusion technique to introduce the sample for
these experiments. Figure 8 shows an example of an Ion Mapping experiment template.
The LTQ Series mass spectrometer includes the following Ion Mapping templates on the
Instrument Setup window for an Ion Mapping experiment:
• Total (or Full-scan) Ion Map
A Total (or Full-scan) Ion Map experiment produces product ion scans for every possible
parent ion in a specified mass range to determine which parent ions lost a particular
fragment to yield a particular product ion (neutral loss scan information). Furthermore,
you can use the data results to determine which parent ions are related to specific product
ions (parent scan information). For example, map all masses from m/z 400–2000 and
specify all possible scans for MS/MS product ions in incremental steps of every
mass-to-charge ratio, every fifth mass-to-charge ratio, or every tenth mass-to-charge ratio.
• Neutral Loss Ion Map
A Neutral Loss Ion Map experiment collects scans for masses and looks for the possible
loss of a particular neutral fragment. The Neutral Loss Ion Map identifies which parent
ions lost a neutral fragment of a particular mass. For example, specify a neutral loss of
80 Da (as in the case of a phosphorylated peptide). A Neutral Loss Ion Map experiment
can step through every product mass in the mixture. The experiment searches for
evidence of the loss of a neutral moiety of mass 80 Da. The data produced by a Neutral
Loss Ion Map is a subset of the data produced by the Total Ion Map but can be obtained
in a shorter time.
• Parent Ion Map
A Parent Ion Map experiment identifies all parent ions that produce a particular product
ion that you specify. For example, if you specify a product ion mass of m/z 250, a Parent
Ion Map includes all the parent ions that yielded the specified product ion, m/z 250. The
data produced by a Parent Ion Map is a subset of the data produced by the Total Ion Map
but can be obtained in a shorter time.
• Data Dependent Zoom Map
A Data Dependent Zoom Map is an Ion Map experiment that collects ZoomScan data on
every scan interval in a specified mass range and Data Dependent MS/MS product
spectra on the most intense mass peak above an intensity threshold with each ZoomScan.
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Types of Experiments
You can view the results of any of the Ion Mapping experiments in the Xcalibur Qual Browser
window.
Figure 8.
Ion Mapping experiment template on the Total Ion Map page
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Ion Tree Experiments
In an Ion Tree experiment, the LTQ Series mass spectrometer can collect MSn data
automatically. To maximize the structural information acquired for a particular sample,
perform an Ion Tree experiment. Specify a particular parent ion for MSn fragmentation or let
the mass spectrometer find the parent ions automatically and fragment them to any level
between MS/MS and MS10. The mass spectrometer automates the collection of data by
deciding what actions must occur next for the experiment to progress. Figure 9 shows an
example of an Ion Tree experiment template.
In an Ion Tree experiment, to prioritize how the mass spectrometer gathers information in
the allowed time, choose one of two options:
• Depth focus
Characterizes an ion by performing a series of MSn-level fragmentations (for example,
MS/MS, MS3, MS4, and so on) before characterizing the next most intense ion in the
MSn series.
• Breadth focus
Characterizes all ions to the same MSn level before advancing to the next MSn level.
For example, if you specify a Maximum Depth of 3 and a Maximum Breadth of 2 in an
Ion Tree experiment, the following occurs:
In either case, the mass spectrometer first scans for parent ions (MS) over the specified mass
range. Next, it selects the first most intense ion of the MS spectrum for fragmentation
(MS/MS).
• For depth focus, after fragmenting the most intense ion of the MS spectrum—producing
an MS/MS spectrum—the mass spectrometer selects and fragments the most intense ion
of the MS/MS spectrum. This results in an MS3 spectrum, the level specified as the
maximum depth for this example. The mass spectrometer then backs up one level and
fragments the second most intense ion of the MS/MS spectrum, creating more product
ions on the level of MS3 from this parent ion. This process then repeats for the second
most intense ion in the MS spectrum.
• For breadth focus, after fragmenting the most intense ion of the MS
spectrum—producing an MS/MS spectrum—the mass spectrometer selects and
fragments the second-most intense ion in the same MS spectrum. The fragmentation of
parent ions continues to the Max Breadth level (2, for this example). After the two most
intense peaks on the MS level are fragmented, the mass spectrometer selects and
fragments the two most intense ions in the first MS/MS spectrum. This results in product
ions on the level of MS3, the level specified as the maximum depth for this example. This
process then repeats for the second most intense ions in the second MS/MS spectrum.
You can view the results of a Data Dependent Ion Tree experiment in the Xcalibur Qual
Browser window. The results appear as a structure tree that originates from a particular
parent ion.
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Mass/Charge Range
Figure 9.
Ion Tree experiment template on the Data Dependent Ion Tree page
Mass/Charge Range
The LTQ Series mass spectrometer has three mass/charge range modes:
• Low: 15 to 200 Da
• Normal: 50 to 2000 Da
• High: 100 to 4000 Da
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Setting Up the API Source
This chapter provides information about setting up the API source for the ESI, HESI-II, or
APCI modes. The API source consists of the Ion Max™ or Ion Max-S™ API source housing
and an ESI, HESI-II, or APCI probe.
Note Tune and calibrate the LTQ Series mass spectrometer in ESI or HESI-II mode
before acquiring data in ESI, HESI-II, or APCI modes.
Contents
• Opening the Tune Plus Window
• Placing the Mass Spectrometer in Standby Mode
• API Source Housing Installation and Removal
• API Source Housing Drain
• ESI or HESI-II Probe Installation and Removal
• APCI Probe Installation and Removal
• Adjusting the Probe Position on the Ion Max API Source Housing
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Setting Up the API Source
Opening the Tune Plus Window
Opening the Tune Plus Window
There are several methods for opening the Tune Plus window.
 To open the Tune Plus window
Do one of the following to open the Tune Plus window (Figure 10):
• On the Windows taskbar, choose Start > Programs > Thermo Instruments > LTQ >
model Tune, where model is your specific LTQ Series model.
Note For LTQ Series version 2.5.0 or earlier, choose Start > Programs >
Xcalibur > model Tune.
• In the Xcalibur application, choose Roadmap view > Instrument Setup >
model (left pane) > Tune Plus.
–or–
• In the Xcalibur application, choose Roadmap view > Instrument Setup >
model (menu toolbar) > Start Tune Plus.
Figure 10. Tune Plus window for the LTQ XL
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Placing the Mass Spectrometer in Standby Mode
Placing the Mass Spectrometer in Standby Mode
Always place the LTQ Series mass spectrometer in standby mode before removing the ion
source probe or API source housing.
Note When the mass spectrometer is in standby mode, the API gases, high voltage, and
syringe pump are off.
 To place the mass spectrometer in Standby mode
1. Complete all data acquisition, if any.
2. Open the Tune Plus window (see page 32).
3. If an LC pump is provided, turn off the solvent flow to the API source.
When controlling the LC pump through the Xcalibur data system, you can turn off the
solvent flow from the Inlet Direct Control dialog box. For example, to turn off the solvent
flow from an Accela™ pump, do the following:
a. Choose Setup > Inlet Direct Control, and then click the tab for the LC pump.
b. Select the Take Pump Under Control check box, and then click the Stop button.
4. In the Tune Plus window, do one of the following:
• If the mass spectrometer is off, choose Control > Standby.
–or–
On Standby
• If the mass spectrometer is on, click the On/Standby button to select the Standby
mode.
When clicked, this button cycles through the power modes shown in the left margin.
The LC/MS system is now in standby mode and you can safely remove the ion source probe
or API source housing after it has cooled to room temperature.
CAUTION If you are using APPI, do not leave the LC or other liquid delivery device on
while the mass spectrometer is in standby mode. The absence of sheath and auxiliary gas
can cause the hot VUV vacuum lamp to break upon contact with liquids.
The mass spectrometer turns off the electron multipliers, consecutive reaction monitoring
(CRM) scan types, 8 kV power to the API source, main rf voltage, and ion optic rf voltages.
The mass spectrometer also turns off the auxiliary and sheath gas flows.
Refer to Chapter 3 in the LTQ Series Hardware Manual for the On/Off status of the mass
spectrometer components when the mass spectrometer is in standby mode. The System LED
on the front panel turns yellow when the system is in standby mode.
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Setting Up the API Source
API Source Housing Installation and Removal
API Source Housing Installation and Removal
The Ion Max or Ion Max-S API source housing holds the ESI, HESI-II, or APCI probe. The
Ion Max has two features that the Ion Max-S does not have: an adjustable probe port and a
front door with a window. Aside from these two features, these two source housings have the
same functionality and mount to the LTQ Series mass spectrometer in the same way. No tools
are needed to remove or install the API source housing or source drain.
Note These instructions apply to both the Ion Max and Ion Max-S API source housing,
unless otherwise noted.
This section provides the following procedures:
• “Installing the API Source Housing” on page 34
• “Removing the API Source Housing” on page 36
Installing the API Source Housing
 To install the API source housing
1. Place the mass spectrometer in Standby mode (see page 33).
2. Place the locking levers on the API source housing in the unlocked position (Figure 11).
3. Align the two guide pin holes on the back of the API source housing (Figure 11) with the
API source housing guide pins on the API source mount on the front of the mass
spectrometer (Figure 12), and carefully press the housing until it is flush with the mount.
Note From the back view, the Ion Max looks the same as the Ion Max-S.
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API Source Housing Installation and Removal
Figure 11. Ion Max-S API source housing (back view)
API source housing
locking levers
Guide pin holes
Figure 12. API ion source mount assembly showing the guide pins
API source housing
guide pins
4. Rotate the API source housing locking levers 90 degrees toward the housing to lock them.
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Setting Up the API Source
API Source Housing Installation and Removal
5. Install the source drain assembly as follows:
CAUTION Prevent solvent waste from backing up into the ion source and mass
spectrometer. Always ensure that liquid in the drain tube is able to drain to a waste
container and that the outlet of the drain tube is above the level of liquid in the waste
container.
a. Connect the source drain assembly to the API source housing drain fitting (see
page 38).
b. Attach the free end of the hose to a waste container, and vent the waste container to a
fume exhaust system.
Removing the API Source Housing
To access the APCI corona needle or the ion source interface, you must remove the API source
housing.
CAUTION HOT SURFACE At operating temperatures above 350 °C (662 °F), the probe
and API source housing can severely burn you.
• Before removing the probe or API source housing, allow the part to cool to room
temperature (approximately 20 minutes) before touching it.
• If the mass spectrometer connects to an LC system, leave the solvent flow from the
LC pump on while the probe cools to room temperature.
 To remove the API source housing
1. Place the mass spectrometer in Standby mode (page 33) and let it cool to room
temperature. See the preceding CAUTION statement.
2. If there is a probe connected to the API source housing, disconnect the external liquid
lines before removing the API source housing.
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API Source Housing Installation and Removal
3. Remove the source drain tube from the API source housing drain (Figure 13).
Figure 13. Ion Max-S API source housing showing the locking levers and drain (front view)
API source housing
locking levers
API source housing
drain
4. Rotate the API source housing locking levers 90 degrees away from the housing to
unlock it.
5. Pull the API source housing straight off of the API source mount assembly.
6. Place the housing in a safe location for temporary storage.
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Setting Up the API Source
API Source Housing Drain
API Source Housing Drain
When installing the API source, connect the drain at the bottom of the API source housing to
the solvent waste container (Figure 14).
Figure 14. API source drain assembly and waste container
Source drain adapter
API source drain
Tygon tubing, 1 in. ID
[at least 1 m (3 ft)]
Reducing connector
Tygon tubing, 0.5 in. ID
To an external vent
Solvent waste container
Table 6 lists the components of the solvent waste system. During the initial installation of the
mass spectrometer, a Thermo Fisher Scientific field service engineer installs the solvent waste
system.
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Setting Up the API Source
API Source Housing Drain
Table 6. Solvent waste system parts
Description
Part Number
Kit
Cap, filling/venting
00301-57022
MS Ship Kit
Heavy-duty, 4 L Nalgene™ container
00301-57020
MS Ship Kit
Reducing connector, single barbed fitting,
1 in. ID to 0.5 in. ID
00101-03-00001
MS Ship Kit
Source drain adapter, Teflon™
70111-20971
MS Accessory Kit
Tubing, Tygon™, 0.5 in. ID, 3/4 in. OD
00301-22920
MS Ship Kit
Tubing, Tygon PVC, 1 in. ID, 1-3/8 in. OD
00301-22922
MS Ship Kit
When reconnecting the API source drain tubing to the drain at the bottom of the API source
housing, ensure that you first connect the Teflon source drain adapter. This adapter can
withstand the high temperatures produced by the H-ESI or APCI source.
CAUTION
Follow these guidelines for the API source drain:
• Do not connect silicone tubing to the API source drain. If silicone tubing connects to
the outlet drain, you might observe background ions at m/z 536, 610, and 684. Use
the PVC tubing provided with the solvent waste container to connect the solvent
waste container to a fume exhaust system.
• Do not connect Tygon tubing directly to the API source drain. At high temperatures,
Tygon releases volatile contaminates. Use the Teflon Source Drain adapter as
described in the LTQ Series Getting Connected Guide.
• Prevent solvent waste from backing up into the mass spectrometer. Always ensure that
the Tygon line from the mass spectrometer to the solvent waste container and the
PVC line from the waste container to the exhaust are above the level of liquid in the
waste container.
Equip your lab with at least two fume exhaust systems:
• The analyzer optics can become contaminated if the API source drain tube and the
(blue) exhaust tubing from the forepumps connect to the same fume exhaust system.
Route the (blue) exhaust tubing from the forepumps to a dedicated fume exhaust
system.
• Do not vent the PVC drain tube (or any vent tubing connected to the waste
container) to the same fume exhaust system that the forepumps connect to. Vent the
waste container to a dedicated fume exhaust system. The exhaust system for the
Ion Max or Ion Max-S API source must accommodate a flow rate of up to 30 L/min
(64 ft3/h).
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ESI or HESI-II Probe Installation and Removal
ESI or HESI-II Probe Installation and Removal
The Ion Max and Ion Max-S API source housings have the same interlock socket, probe
interlock block, probe locking knob, and probe locking ring. No tools are needed to remove
or install the probe.
CAUTION HOT SURFACE At operating temperatures above 350 °C (662 °F), the probe
and API source housing can severely burn you.
• Before removing the probe or API source housing, allow the part to cool to room
temperature (approximately 20 minutes) before touching it.
• If the mass spectrometer connects to an LC system, leave the solvent flow from the
LC pump on while the probe cools to room temperature.
This section provides the following procedures:
• “Installing the ESI or HESI-II Probe” on page 40
• “Removing the ESI or HESI-II Probe” on page 47
Installing the ESI or HESI-II Probe
These instructions are for the ESI and HESI-II probes. All steps are for both probe types,
unless otherwise noted.
 To install the ESI or HESI-II probe
1. Place the mass spectrometer in Standby mode (see page 33) and let it cool to room
temperature. See the preceding CAUTION statement.
2. If the mass spectrometer is set up for APCI mode, follow the procedure “Removing the
APCI Probe and the Corona Needle” on page 59.
3. If necessary, inspect and clean the selected probe before installing it.
4. If installing the ESI probe, ensure that it has a fused-silica or metal needle sample tube
installed (Figure 23 on page 48).
Refer to the LTQ Series Hardware Manual or the installation guide provided in the Metal
Needle Kit.
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2 Setting Up the API Source
ESI or HESI-II Probe Installation and Removal
5. Turn the probe locking knob counterclockwise until the probe locking ring opens to its
widest position (Figure 15).
Figure 15. Ion Max API source housing (left side view)
Vaporizer cable connected
to the interlock socket
8 kV cable connected to the
source housing high-voltage socket
Probe interlock block
Probe locking knob
Probe locking ring
Probe port
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ESI or HESI-II Probe Installation and Removal
6. Position the probe in the API source housing probe port as follows:
a. Hold the probe with the nozzle facing down and the guide pin facing toward the left,
and then slowly insert the probe into the port until the guide pin meets the locking
ring on the source housing.
Figure 16 shows the ESI probe, and Figure 17 shows the HESI-II probe.
Figure 16. ESI probe showing the guide pin and locking ring
Slot on the left side of
the interlock block
Guide pin
Locking ring
Figure 17. HESI-II probe showing the guide pin and locking ring
Slot on the left side of
the interlock block
Guide pin
Locking ring
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ESI or HESI-II Probe Installation and Removal
b. Pull the probe slightly upward until the guide pin is level with the slot on the left side
of the interlock block, and then turn the probe clockwise until the guide pin meets
resistance from the interlock block.
Figure 18 shows the ESI probe, and Figure 19 shows the HESI-II probe.
Figure 18. ESI probe showing the guide pin inserted into the interlock block slot
Guide pin inserted into the slot on
the left side of the interlock block
A
C B
D
A, B, C, and D
probe depth
markers
Figure 19. HESI-II probe showing the guide pin inserted into the interlock block slot
Guide pin inserted into the slot on
the left side of the interlock block
A
C B
D
Thermo Scientific
A, B, C, and D
probe depth markers
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Setting Up the API Source
ESI or HESI-II Probe Installation and Removal
c. Push the probe down into the port to the appropriate depth indicated by the A, B, C,
and D depth markers on the probe.
Insert the probe to a depth of B, C, or D. For high solvent flow rates, adjust the probe
depth so that the nozzle is farther away from the ion interface (depth C or D).
Conversely, for low solvent flow rates, adjust the probe depth so that the nozzle is
closer to the ion interface (depth B or C).
Note Probe position A is used only for instrument calibration (flow rates less
than 10 μL/min).
7. Lock the probe in place by turning the probe locking knob clockwise until you feel
resistance.
8. Connect the nitrogen gas lines to the probe as follows:
• Connect the sheath gas fitting (blue) to the sheath gas inlet (S).
• Connect the auxiliary gas fitting (green) to the auxiliary gas inlet (A).
9. Connect the 8 kV cable connector to the 8 kV cable socket on the probe, and then
tighten the locking ring by turning it clockwise.
CAUTION HIGH VOLTAGE The mass spectrometer must be in standby mode before
you disconnect or connect the 8 kV cable.
10. If installing the HESI-II probe, do the following:
• Unplug the vaporizer cable connector from the interlock socket and connect it to the
vaporizer cable connector socket on the probe (Figure 28 on page 52).
• Align the socket pins with the socket by aligning the red dot on the vaporizer cable
connector with the red dot on the interlock socket.
11. Ensure that the grounding union is positioned in the grounding union bar.
12. Use two fingertight fittings to connect a length of red PEEK™ tubing to the LC outlet and
to the left side of the grounding union.
13. If installing the HESI-II probe, use two fingertight fittings to connect a short length of
red PEEK tubing to the probe sample inlet and to the right side of the grounding union.
Figure 20 shows the ESI probe installed in the Ion Max API source housing. Figure 21 on
page 46 shows the HESI-II probe installed in the Ion Max API source housing.
Note The LTQ Series mass spectrometer auto-senses the installed probe type.
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2 Setting Up the API Source
ESI or HESI-II Probe Installation and Removal
Figure 20. ESI probe installed in the Ion Max API source housing
Vaporizer cable
8 kV cable
Sheath gas line
(blue fitting)
A
S
Fused-silica sample tube
with safety sleeve
Auxiliary gas line
(green fitting)
Sample inlet
Stainless steel
grounding union
Connection to LC inlet
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Setting Up the API Source
ESI or HESI-II Probe Installation and Removal
Figure 21. HESI-II probe installed in the Ion Max API source housing
Vaporizer
cable
8 kV cable
A
S
Auxiliary gas line
(green fitting)
Sheath gas line
(blue fitting)
Sample inlet
To the LC system
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Grounding union holder with a
stainless steel grounding union
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2 Setting Up the API Source
ESI or HESI-II Probe Installation and Removal
Removing the ESI or HESI-II Probe
These instructions are for the ESI and HESI-II probes. All steps are for both probe types,
unless otherwise noted.
 To remove the probe from the API source housing
1. Place the mass spectrometer in Standby mode (see page 33) and let it cool to room
temperature. See the CAUTION statement on page 40.
2. If provided, turn off the solvent flow from the optional LC pump to the API source
(see step 3 on page 33).
3. If removing the HESI-II probe, disconnect the tubing from the left side of the HESI-II
probe grounding union (Figure 22).
Figure 22. HESI-II probe connections
8 kV cable
Vaporizer
cable
Sheath gas line
Auxiliary gas line
Connection between
grounding union and
sample inlet
To LC outlet
Probe locking knob
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Setting Up the API Source
ESI or HESI-II Probe Installation and Removal
4. Disconnect the sample transfer line from the grounding union, which is positioned in the
grounding bar (Figure 23).
Tip Unless you want to replace the sample tube, do not disconnect it from the probe
or the stainless steel grounding union when removing the probe from the API source
housing.
Figure 23. Sample transfer line disconnected from the grounding union
8 kV cable
Fused-silica sample tube
with natural PEEK safety
sleeve
Stainless steel grounding
union (seated in the
grounding bar)
Sample transfer line
(disconnected from
the grounding
union)
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2 Setting Up the API Source
ESI or HESI-II Probe Installation and Removal
5. Disconnect the 8 kV cable from the probe (Figure 24 and Figure 25) as follows:
CAUTION HIGH VOLTAGE The mass spectrometer must be in standby mode before
you disconnect or connect the 8 kV cable.
a. Unlock the cable connector by turning the probe locking ring counterclockwise.
b. Pull the 8 kV cable connector from the probe high-voltage socket.
Figure 24. 8 kV cable connector removed from the ESI probe
Locking ring
High-voltage socket
Figure 25. 8 kV cable connector removed from the HESI-II probe (enlarged view)
Locking ring
High-voltage socket
(for the 8 kV cable
connector)
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Setting Up the API Source
ESI or HESI-II Probe Installation and Removal
6. Disconnect the vaporizer cable from the probe vaporizer cable socket (Figure 26).
Figure 26. HESI-II probe showing the disconnected vaporizer and 8 kV cables (top view)
Vaporizer cable
(disconnected)
8 kV cable
(disconnected)
Sheath gas line
Auxiliary
gas line
A
Interlock socket
S
Vaporizer cable
socket
Cover over
high-voltage
socket
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ESI or HESI-II Probe Installation and Removal
7. Connect the vaporizer cable connector to the interlock socket on the interlock block, and
align the socket pins by aligning the red dot on the vaporizer cable connector with the red
dot on the interlock socket (Figure 27).
Figure 27. API source housing with the HESI-II probe (left side view)
Vaporizer cable
Vaporizer cable
socket
Sheath gas inlet
Red dot on the
vaporizer cable
Interlock socket
8. Disconnect the nitrogen gas lines from the probe as follows:
• Disconnect the auxiliary gas fitting (green) from the auxiliary gas inlet (A).
• Disconnect the sheath gas fitting (blue) from the sheath gas inlet (S).
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ESI or HESI-II Probe Installation and Removal
9. If removing the ESI probe, remove the grounding union from the grounding bar.
Figure 28 shows the nitrogen lines and 8 kV cable disconnected from the ESI probe, and
the grounding union removed from the grounding union bar.
Figure 28. API source housing with ESI probe (left side view)
Fused-silica sample tube with
natural PEEK safety sleeve
A
C B
D
Stainless steel
grounding union
10. Unlock the probe locking ring by turning the probe locking knob counterclockwise.
11. Remove the probe as follows:
a. Slowly pull the probe out of the port until you feel the resistance caused by the probe
guide pin meeting the interlock block.
b. Turn the probe counterclockwise until the guide pin is free of the interlock block.
c. When the guide pin is free of the interlock block, pull the probe out of the port.
12. If needed, clean the probe before storing it in its original shipping container.
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2 Setting Up the API Source
APCI Probe Installation and Removal
APCI Probe Installation and Removal
The only tools needed to remove or install the corona needle is a set of pliers. This section
provides the following procedures:
• “Installing the Corona Needle and APCI Probe” on page 53
• “Removing the APCI Probe and the Corona Needle” on page 59
CAUTION HOT SURFACE At operating temperatures above 350 °C (662 °F), the probe
and API source housing can severely burn you.
• Before removing the probe or API source housing, allow the part to cool to room
temperature (approximately 20 minutes) before touching it.
• If the mass spectrometer connects to an LC system, leave the solvent flow from the
LC pump on while the probe cools to room temperature.
Note The figures in this section show the Ion Max API source housing.
Installing the Corona Needle and APCI Probe
To operate the system in APCI mode, install the corona needle and APCI probe.
 To install the corona needle
1. Place the mass spectrometer in Standby mode (see page 33) and let it cool to room
temperature. See the preceding CAUTION statement.
2. If the mass spectrometer is set up for ESI or H-ESI mode, follow the procedure
“Removing the ESI or HESI-II Probe” on page 47.
3. Remove the API source housing (see page 36).
4. Grasp the clean corona needle by using the pliers and carefully push the larger end
straight into the corona needle contact (Figure 29).
CAUTION SHARP OBJECT The corona needle is very sharp and can puncture your
skin. Handle it with care.
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Setting Up the API Source
APCI Probe Installation and Removal
Figure 29. Corona needle
Gold-plated
corona needle contact
Corona needle
5. Ensure that the needle tip aligns with the travel path between the APCI probe and the ion
source interface.
6. Reinstall the API source housing (see page 34).
 To install the APCI probe
1. Connect the 8 kV cable connector to the high-voltage socket on the API source housing
as follows:
a. On the right side of the API source housing, remove the cover over the high-voltage
socket, which is for the corona needle.
b. Connect the 8 kV cable connector into the high-voltage socket on the API source
housing (Figure 30).
c. Lock the 8 kV cable by turning the locking ring clockwise.
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2 Setting Up the API Source
APCI Probe Installation and Removal
Figure 30. Ion Max API source housing
Vaporizer cable plugged into
the interlock socket
Interlock block
8 kV cable plugged into the corona
needle high-voltage socket
Probe locking knob
Probe locking ring
Probe port
2. Turn the probe locking knob counterclockwise until the probe locking ring opens to its
widest position.
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Setting Up the API Source
APCI Probe Installation and Removal
3. Position the probe in the API source housing probe port as follows:
a. Hold the probe with the nozzle facing down and the guide pin facing toward the left,
and then slowly insert the probe into the port until the guide pin meets the locking
ring on the API source housing (Figure 31).
Figure 31. APCI probe guide pin touching the locking ring
Slot on the left side of
the interlock block
Probe guide pin
Probe locking ring
b. Pull the probe slightly upward until the guide pin is level with the slot on the left side
of the interlock block, and then turn the probe clockwise until the guide pin meets
resistance from the interlock block (Figure 32).
Figure 32. APCI probe guide pin inserted into the interlock block slot
Guide pin inserted into the slot on
the left side of the interlock block
A
C B
D
A, B, C, and D
probe depth markers
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APCI Probe Installation and Removal
c. Push the probe down into the port to the appropriate depth indicated by the A, B, C,
and D depth markers on the probe.
Insert the probe to a depth of B, C, or D. For high solvent flow rates, adjust the probe
depth so that the nozzle is farther away from the ion interface (depth C or D).
Conversely, for low solvent flow rates, adjust the probe depth so that the nozzle is
closer to the ion interface (depth B or C).
Note Probe position A is used only for instrument calibration (flow rates less
than 10 μL/min).
4. Lock the probe in place by turning the probe locking knob clockwise until you feel
resistance.
5. Connect the nitrogen gas lines to the probe as follows:
• Connect the sheath gas fitting (blue) to the sheath gas inlet (S).
• Connect the auxiliary gas fitting (green) to the auxiliary gas inlet (A).
6. Unplug the vaporizer cable connector from the interlock socket and connect it to the
vaporizer cable connector socket on the APCI probe (Figure 33).
7. Align the socket pins with the socket by aligning the red dot on the vaporizer cable
connector with the red dot on the interlock socket.
8. Connect the sample transfer line to the probe sample inlet.
Figure 33 shows the APCI probe installed in the Ion Max API source housing.
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APCI Probe Installation and Removal
Figure 33. APCI probe installed in the Ion Max API source housing
Vaporizer cable connected
to the APCI probe
A
Sheath gas line
connected to the
sheath gas inlet
S
Auxiliary gas line
connected to the
auxiliary gas inlet
8 kV cable connected to
the corona needle
high-voltage socket
Sample transfer line
connected to the
APCI probe sample inlet
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High-voltage socket
cover
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2 Setting Up the API Source
APCI Probe Installation and Removal
Removing the APCI Probe and the Corona Needle
This section describes how to remove the APCI probe and the corona needle.
 To remove the APCI probe
1. Place the mass spectrometer in Standby mode (see page 33) and let it cool to room
temperature. See the CAUTION statement on page 53.
2. If an LC pump is provided, turn off the solvent flow to the API source (see step 3 on
page 33).
3. Unplug the vaporizer cable from the probe vaporizer cable socket (Figure 34).
Figure 34. APCI probe connections
Vaporizer cable
A
S
Probe vaporizer
cable socket
4. Disconnect the nitrogen gas lines from the probe as follows:
• Disconnect the auxiliary gas fitting (green) from the auxiliary gas inlet (A).
• Disconnect the sheath gas fitting (blue) from the sheath gas inlet (S).
5. Connect the vaporizer cable connector to the interlock socket on the interlock block.
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Setting Up the API Source
APCI Probe Installation and Removal
6. Align the socket pins with the socket by aligning the red dot on the vaporizer cable
connector with the red dot on the interlock socket (Figure 30 on page 55).
7. Unlock the probe locking ring by turning the probe locking knob counterclockwise.
8. Remove the probe from the probe port in the API source housing as follows:
a. Slowly pull the probe out of the port until you feel the resistance caused by the probe
guide pin meeting the interlock block.
b. Turn the probe counterclockwise until the guide pin is free of the interlock block.
c. When the guide pin is free of the interlock block, pull the probe out of the port.
9. If needed, clean the probe before storing it in its original shipping container.
 To remove the corona needle
CAUTION SHARP OBJECT The corona needle is very sharp and can puncture your
skin. Handle it with care.
1. Place the mass spectrometer in Standby mode (see page 33) and let it cool to room
temperature. See the CAUTION statement on page 53.
2. Remove the APCI probe (see page 59).
3. Disconnect the 8 kV cable from the high-voltage socket on the API source housing.
4. Insert the high-voltage socket cover into the high-voltage socket on the API source
housing.
5. Remove the API source housing (see page 36).
6. Grasp the corona needle by using the pliers and carefully pull it straight out of the corona
needle contact (Figure 35).
The corona needle is in the corona assembly inside of the API source housing across from
the window.
Figure 35. Corona needle
Gold-plated
corona needle contact
Corona needle
(grasp this with pliers
to remove it)
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Adjusting the Probe Position on the Ion Max API Source Housing
7. Reinstall the API source housing or place it in a safe location for temporary storage.
8. If needed, clean the corona needle before storing it in its original shipping container.
Adjusting the Probe Position on the Ion Max API Source Housing
If the LTQ Series mass spectrometer has an Ion Max API source housing, you can maximize
sensitivity by adjusting the side-to-side and front-to-back probe position by a few millimeters
(Figure 36).
Note The probe position on the Ion Max-S API source housing is not adjustable.
 To adjust the probe position
• To adjust the front-to-back probe position, use the micrometer on the front of the Ion
Max source housing.
• To adjust the side-to-side probe position, use the knurled nut on the left side of the
housing and the –1 to +1 markers on the top front of the Ion Max source housing.
• To adjust the probe depth, use the A, B, C, and D markers on the probe as a guide.
Figure 36. Ion Max API source housing showing probe adjustment controls
Side-to-side
S
Front-to-back
CAUTION - HOT SURFACE
1
+
0
0 1
1
Knurled nut
(below grounding union)
Side-to-side
position set to 0
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Automatic Tuning and Calibration in ESI Mode
This chapter provides information about how to tune and calibrate the LTQ Series mass
spectrometer. First, tune and calibrate the mass spectrometer in ESI mode—that is, set up the
mass spectrometer for ESI mode (see “Installing the ESI or HESI-II Probe” on page 40).
Then, set up the scan parameters for a full-scan, single-stage mass analysis. For most
applications, you infuse a calibration solution directly into the ion source while running the
automatic tune and automatic calibration options provided within the data system.
You must calibrate the mass spectrometer every one to three months of operation for
optimum performance over the entire mass range of the detector.
Contents
• Setting Up the Syringe Pump for Tuning and Calibration
• Setting Up the Mass Spectrometer for Tuning and Calibration
• Testing the Mass Spectrometer in ESI Mode
• Tuning the Mass Spectrometer Automatically in ESI Mode
• Saving the ESI Tune Method
• Calibrating Automatically in the Normal Mass Range
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Automatic Tuning and Calibration in ESI Mode
Setting Up the Syringe Pump for Tuning and Calibration
Setting Up the Syringe Pump for Tuning and Calibration
Use the syringe pump, located on the front of the LTQ Series mass spectrometer, to infuse
solution for tuning and calibration.
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 infusion
1. Load a clean, 500 μL Unimetrics™ syringe with 450 μL of the ESI calibration solution.
To prepare the ESI calibration solution, follow one of these procedures:
• For the LXQ and LTQ XL, see “Preparing the Normal Mass Range Calibration
Solution for ESI Mode” on page 140.
• For the Velos Pro, see “Preparing the Normal Mass Range Calibration Solution for
ESI Mode” on page 148.
Note To minimize the possibility of cross-contamination of the assembly, be sure to
wipe off the needle tip with a clean, lint-free tissue before reinserting it into the
syringe adapter assembly.
2. Hold the plunger of the syringe in place and carefully insert the tip of the syringe needle
into the end of a 4 cm (1.5 in.) Teflon tube with a fingertight fitting and a ferrule to the
(black) LC union (Figure 37).
The LC union has a 10-32, coned-bottom receiving port.
Figure 37. Plumbing connection for the syringe
Fingertight fitting
LC Union
Ferrule
Teflon tube
3. Place the syringe into the syringe holder of the syringe pump.
4. Use a fingertight fitting and ferrule to connect an infusion line of red PEEK tubing
(0.005 in. ID, 1/16 in. OD) between the LC union and the grounding union held by the
grounding bar of the ion source.
Figure 38 shows the suggested fittings and ferrules for connecting the LC union to the
grounding union. Both the LC union and the grounding union have 10-32,
coned-bottom receiving ports.
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Setting Up the Mass Spectrometer for Tuning and Calibration
Figure 38. Plumbing connection between the LC union and the grounding union
Grounding bar of the ion source
Grounding union, 10-32 internal ports, 0.010 in. thru-hole
Ferrule
Fingertight fitting
Red PEEK tubing
LC union
5. While squeezing the blue release button on the syringe pump handle, push the handle
forward until it just contacts the syringe plunger.
Setting Up the Mass Spectrometer for Tuning and Calibration
Before running the automatic calibration procedure on the LTQ Series mass spectrometer,
manually tune with calibration solution to establish a stable spray of solution and to ensure
that the mass analyzer is transmitting a sufficient level of ions to the mass detector. After
establishing these conditions, calibrate the mass spectrometer automatically to optimize the
parameters that affect ion detection.
CAUTION Before beginning normal operation each day, ensure that there is sufficient
nitrogen for the ESI source. If you run out of nitrogen, the mass spectrometer
automatically turns off to prevent atmospheric oxygen from damaging the ion source. The
presence of oxygen in the ion source when the mass spectrometer is on could be unsafe. In
addition, if the mass spectrometer turns off during an analytical run, you could lose data.
To set up the mass spectrometer for tuning and calibration in ESI mode, follow these
procedures:
1. To open a tune method for ESI mode
2. To view the pre-tune ESI source settings
3. To define the scan parameters
4. To set the data type and ion polarity mode on page 67
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Automatic Tuning and Calibration in ESI Mode
Setting Up the Mass Spectrometer for Tuning and Calibration
 To open a tune method for ESI mode
1. Open the Tune Plus window (see page 32).
2. Click the Open button.
3. Browse to the drive:\Thermo\Instruments\LTQ\methods folder, and then select either a
recently saved tune file that worked on the instrument (preferred), or use the default tune
file, Default_ESI.
4. Click Open.
The tune method parameters download to the mass spectrometer.
 To view the pre-tune ESI source settings
1. In the Tune Plus window, choose Setup > ESI Source.
2. Observe the settings.
3. Click OK when finished.
 To define the scan parameters
1. In the Tune Plus window, click the Define Scan button to open the Define Scan dialog
box (Figure 39).
Figure 39. Define Scan dialog box
2. Under Scan Description, do the following:
• In the Mass Range list, select Normal.
• In the Scan Rate list, select Normal.
• In the Scan Type list, select Full.
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Setting Up the Mass Spectrometer for Tuning and Calibration
3. Under Scan Time, do the following:
• In the Microscans box, enter 1.
• In the Max. Inject Time (ms) box, enter 10.000.
4. Under Source Fragmentation, do one of the following:
• For the LXQ and LTQ XL, clear the On check box.
Note Selecting the Source Fragmentation check box allows collision-induced
fragmentation to occur in the ion source. Because collision-induced
fragmentation performed in the ion source is not very specific, this feature is
rarely used.
• For the Velos Pro, select the On check box.
Note You must select the Source Fragmentation check box to produce the
calibration peak at m/z 138, which is a fragment of caffeine m/z 195.
5. Under Scan Ranges, do the following:
a. In the Input list, select From/To.
b. In the First Mass (m/z) column, type 150.00.
c. In the Last Mass (m/z) column, type 2000.00.
6. Ensure the settings in the Define Scan dialog box are the same as those shown in
Figure 39 on page 66.
7. Click OK.
 To set the data type and ion polarity mode
Centroid
Profile
1. In the Tune Plus window, click the Centroid/Profile button to select the profile
data type.
2. Click the Positive/Negative button to select the positive ion polarity mode.
Positive
polarity
Negative
polarity
Thermo Scientific
This completes the setup to tune with the calibration solution in ESI mode.
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Automatic Tuning and Calibration in ESI Mode
Testing the Mass Spectrometer in ESI Mode
Testing the Mass Spectrometer in ESI Mode
To test the operation of the LTQ Series mass spectrometer, infuse the calibration solution into
the ESI source and monitor the real-time display of the mass spectrum of the calibration
solution.
CAUTION Do not infuse calibration solution at syringe pump flow rates above
10 μL/min. You can contaminate the sample with high concentrations of the
Ultramark 1621 component of the calibration solution.
To test the operation of the mass spectrometer in ESI mode, follow these procedures:
1. To infuse the calibration solution into the ion source
2. To monitor the mass spectrum and the spray current
 To infuse the calibration solution into the ion source
1. In the Tune Plus window, choose Setup > Syringe Pump to open the Syringe Pump
dialog box (Figure 40).
Figure 40. Syringe Pump dialog box
2. Under Flow Control, do the following:
a. Select the On option.
b. In the Flow Rate (μL/min) box, enter 5.00.
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Testing the Mass Spectrometer in ESI Mode
3. Under Type, do the following:
a. Select the type of syringe.
A 500 μL Unimetrics syringe is supplied with the LTQ Series mass spectrometer. If
you select the Hamilton™ or Unimetrics option, the Syringe ID (mm) box remains
unavailable; the data system uses a predefined syringe ID for these syringes. If you
select the Other option, the Syringe ID (mm) box becomes available and the
Volume (μL) list becomes unavailable.
b. Do one of the following:
• For a Hamilton or Unimetrics syringe, in the Volume (μL) list, select the volume
of the syringe.
• For another type of syringe, in the Syringe ID (mm) box, enter the ID of the
syringe.
4. Click OK.
The syringe pump starts. Or, use the On/Off syringe pump button.
Before starting the next procedure, ensure that you have set up the syringe pump to infuse the
ESI calibration mixture as described in “To infuse the calibration solution into the ion source”
on page 68.
 To monitor the mass spectrum and the spray current
1. In the Tune Plus window, click the On/Standby button to select the On mode.
On Standby
After you turn on the mass spectrometer, it begins scanning, nitrogen flows into the ESI
ion source, high voltage is applied to the ESI ion source, and a real-time mass spectrum
display appears in the Spectrum view of the Tune window. For information about
spectrums, refer to the topic “Spectrum View” in the data system Help.
2. Click the Display Spectrum View button.
3. Observe the mass spectra of the singly-charged ions in the calibration solution (Figure 41,
Figure 42, and Figure 43 on pages 70 to 72).
The ions are as follows:
• Caffeine: m/z 195 for the LXQ and LTQ XL; m/z 138.1 and 195 for Velos Pro
• MRFA: m/z 524
• Ultramark 1621: m/z 1022, 1122, 1222, 1322, 1422, 1522, 1622, 1722, 1822,
and 1922
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Automatic Tuning and Calibration in ESI Mode
Testing the Mass Spectrometer in ESI Mode
Figure 41. Spectrum of the calibration solution for the LTQ XL
Ultramark peaks
IT=0.093
NL=1.48E9
Caffeine peak
MRFA peak
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Testing the Mass Spectrometer in ESI Mode
Figure 42. Spectrum of the calibration solution for the Velos Pro (lower range)
N-butylamine peak
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Testing the Mass Spectrometer in ESI Mode
Figure 43. Spectrum of the calibration solution for the Velos Pro (upper range)
Caffeine peak
Ultramark peaks
MRFA peak
4. At the top of the Spectrum view, check the values for the ionization time (IT) and
normalization level (NL) (Figure 41 on page 70).
5. Choose Setup > ESI Source.
6. Check the Spray Current readback.
7. Observe the values for NL and IT in the Spectrum view.
As calibration solution infuses, and the readback values fluctuate, consider the following
questions about the ion current signal:
• Is the signal present?
• Is the signal stable, varying by less than about 15% from scan to scan?
To assist in assessing the signal stability, a diagnostic tool is available. In Tune Plus,
choose Diagnostics > Diagnostics. In the Tools list, select System Evaluation, and
then click the API Stability Evaluation check box.
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Testing the Mass Spectrometer in ESI Mode
If you answered “yes” to the questions in step 7, then the mass spectrometer is operating
properly.
If you answered “no” to either of these questions, try the following troubleshooting measures:
• Ensure that the fused-silica sample tube on a ESI probe does not extend beyond the tip of
the ESI needle. For instructions about inserting the fused-silica sample tube, refer to the
LTQ Series Hardware Manual.
• Ensure that the entrance to the ion transfer tube is clean and is not blocked.
• Ensure that the solution entering the probe is free of air bubbles and that the tubing and
connectors are free of leaks.
• Ensure that the flow rate of the sheath gas is 5–8 units.
If you have demonstrated that the mass spectrometer is operating properly in ESI mode, you
are now ready to tune and calibrate the mass spectrometer. Leave the mass spectrometer as it
is, and go to the next topic, “Tuning the Mass Spectrometer Automatically in ESI Mode.”
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Tuning the Mass Spectrometer Automatically in ESI Mode
Tuning the Mass Spectrometer Automatically in ESI Mode
To optimize important parameters, tune the LTQ Series mass spectrometer automatically in
ESI mode.
 To automatically tune the mass spectrometer in ESI mode
1. In the Tune Plus window, click the Tune button to open the Tune dialog box (Figure 44).
Figure 44. Tune dialog box showing the Automatic page
2. Under What to Optimize On, select the Mass (m/z) option.
3. To optimize the transmission of ions for a specific peak in the mass spectrum of the ESI
calibration solution, enter, for example, 195.1 in the Mass (m/z) box.
The mass spectrometer optimizes the tune on the peak at m/z 195.1. However, you can
optimize the tune on any peak in the mass spectrum of the ESI calibration solution.
4. Click Start.
A message appears:
Please ensure that the 500 microliter syringe is full.
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Tuning the Mass Spectrometer Automatically in ESI Mode
5. Ensure that the syringe contains at least 450 μL of calibration solution.
To prepare the ESI calibration solution, follow these procedures:
• For the LXQ and LTQ XL, see “Preparing the Normal Mass Range Calibration
Solution for ESI Mode” on page 140.
• For the Velos Pro, see “Preparing the Normal Mass Range Calibration Solution for
ESI Mode” on page 148.
6. Click OK.
7. In the Tune Plus window, click the Graph View button.
8. Observe the Tune Plus window and the Tune dialog box.
While automatic tuning is in progress, the mass spectrometer displays the results of
various adjustments in the Spectrum and Graph views in the Tune Plus window and
displays various messages under Status in the Tune dialog box.
For the LXQ and LTQ XL, check that the Tune Plus window looks similar to the one
shown in Figure 45. Figure 46 shows the Velos Pro spectrum.
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Tuning the Mass Spectrometer Automatically in ESI Mode
Figure 45. Tune Plus window and Tune dialog box to set the LTQ XL on m/z 195.1
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Tuning the Mass Spectrometer Automatically in ESI Mode
Figure 46. Tune Plus window and Tune dialog box to set the Velos Pro on m/z 524.3
The Optimization Complete message in the Status box of the Tune dialog box should
show a positive increase in the signal.
9. After the automatic tuning process ends, choose Setup > ESI Source.
10. Observe the post-tune settings for the ESI source.
11. Choose Setup > Ion Optics.
12. Observe the post-tune settings for the ion optics.
This completes the tuning in ESI mode by using the calibration solution.
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Saving the ESI Tune Method
Saving the ESI Tune Method
You can save the parameters that you just set in a tune method specific to the particular
analyte and solvent flow rate. Recall the tune method and use it as a starting point for
optimizing the mass spectrometer on a different analyte or at a different flow rate.
Note You must save the tune method while the mass spectrometer is on.
 To save the ESI tune method
1. In the Tune Plus window, click the Save button.
2. Browse to the drive:\Thermo\Instruments\LTQ\methods folder.
3. In the File Name box, type a name, such as ESImyTune, to identify the tune method.
4. Click Save.
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Calibrating Automatically in the Normal Mass Range
Calibrating Automatically in the Normal Mass Range
Note To calibrate the mass spectrometer in the high mass range, see Appendix C, “High
Mass Range Calibration.”
 To automatically calibrate the mass spectrometer in the normal mass range
1. In the Tune Plus window, click the Calibrate button to open the Calibrate dialog box
(Figure 47).
Figure 47. Calibrate dialog box showing the Automatic page
2. Specify the instrument state when the calibration is completed:
• To have the mass spectrometer switch to Standby mode after the calibration, click the
Set Instrument to Standby when Finished check box.
• To have the mass spectrometer remain in the On mode after the calibration, clear the
Set Instrument to Standby when Finished check box.
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Calibrating Automatically in the Normal Mass Range
3. Click Start.
A message appears:
Please ensure that the 500 microliter syringe is full.
4. Ensure that the syringe contains at least 450 μL of the calibration solution.
To prepare the ESI calibration solution, follow these procedures:
• For the LXQ and LTQ XL, see “Preparing the Normal Mass Range Calibration
Solution for ESI Mode” on page 140.
• For the Velos Pro, see “Preparing the Normal Mass Range Calibration Solution for
ESI Mode” on page 148.
5. Click OK.
6. Observe the Tune Plus window and the Calibrate dialog box.
While the automatic calibration is in progress, test results appear in the Spectrum and
Graph views and messages appear in the Status box of the Calibrate dialog box
(Figure 48).
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Calibrating Automatically in the Normal Mass Range
Figure 48. Tune Plus window and Calibrate dialog box for the LTQ XL
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Automatic Tuning and Calibration in ESI Mode
Calibrating Automatically in the Normal Mass Range
The automatic calibration procedure typically takes 20–30 minutes.
After the calibration procedure ends, the full-scan ESI mass spectrum appears in the
Spectrum view (Figure 48 on page 81).
• If a calibration item is successful, the mass spectrometer saves the new calibration
values automatically to the master calibration file on the hard disk drive.
• If a calibration item fails, try calibrating on that item again after ensuring that the
spray is stable, the solution flow rate is sufficient, and all the ions in the calibration
solution are present with adequate signal-to-noise ratios. Use the semi-automatic
calibration page to calibrate the specific item that failed.
Tip After performing an automatic calibration, green checks , red X marks ,
or both appear in the results column of the Semi-Automatic page of the
Calibration dialog box. A green check indicates a successful calibration, and a red
X mark indicates a failed calibration for the corresponding item. Additional
information is available in the Status box at the bottom of the Calibration dialog
box.
• If repeated failures occur, consider clearing the ZoomScan Mode option.
After all the calibration items are successful, the mass spectrometer is properly tuned and
calibrated for low-flow experiments. A successful calibration exhibits adequate intensities of
the following calibrant ions at the correct masses:
• For the LXQ and LTQ XL: m/z 195, 524, 1222, 1522, and 1822
• For the Velos Pro: m/z 74, 138, 195, 524, 1222, 1522, and 1822
In many cases, fine tuning on the particular analyte is not necessary if the intensity of these
ions is sufficient.
Note To calibrate the mass spectrometer in the high mass range, see Appendix C, “High
Mass Range Calibration.”
Before tuning with an analyte, you must clean the mass spectrometer as described in
Chapter 8, “Cleaning the Mass Spectrometer After Tuning and Calibrating.”
Tip If you plan to run analytical samples in high-flow ESI mode (by using flow rates of
50–1000 μL/min), optimize the tune further by following the procedures in Chapter 4,
“Tuning with an Analyte in ESI Mode,” on page 83.
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Tuning with an Analyte in ESI Mode
This chapter describes how to tune the LTQ Series mass spectrometer in ESI mode by using
an analyte. An automated procedure optimizes the sensitivity of the mass spectrometer to the
specific analyte. You can often use the customized tune methods available for each instrument
type without further tuning of the mass spectrometer, as they are optimized for a wide range
of applications.
Contents
• Setting Up the Inlet for High-Flow Infusion in ESI Mode
• Setting Up the Mass Spectrometer to Tune with an Analyte in ESI Mode
• Tuning the Mass Spectrometer Automatically
• Saving the ESI Tune Method
The following mass spectrometer parameters have the most important impact on signal
quality in ESI mode:
• Ion transfer tube voltage (LXQ and LTQ XL only)
• Electrospray voltage
• Tube lens voltage (LXQ and LTQ XL only)
• Heated ion transfer tube temperature (voltage)
• Gas flow rates for the API gases
• S-lens rf level (Velos Pro only)
The settings for these parameters depend on the solvent flow rate and target analyte
composition. In general, fine-tune the mass spectrometer whenever you change the solvent
flow rate conditions of the particular application. In this procedure, you use a low-flow tune
method (or the tune file that you created for the analyte by using direct infusion) as a starting
point, and then further optimize the parameters by using an automated procedure. The
automatic procedure adjusts the voltages applied to the ion optics until the ion transmission
of the analyte is maximized.
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Tuning with an Analyte in ESI Mode
Note Based on the LC flow rate of the experiment, specify the value of each of the
following tuning parameters: the mass spectrometer’s sheath, auxiliary, and sweep gas
pressures; ESI needle (or “spray”) voltage; ion transfer tube temperature; and probe
position. Table 3 on page 12 lists the recommended tuning parameters, with the exception
of the ESI needle voltage and probe position. Automatic tuning sets the values of the
parameters not mentioned here.
The ion transfer tube is heated to maximize the ion transmission to the mass spectrometer.
For ESI only, set the ion transfer tube temperature proportional to the flow rate of the
solution. See Table 3 on page 12 for guidelines on setting the operating parameters for ESI
mode.
Note If performing the experiment at a flow rate less than 10 μL/min, and you can obtain
the results without optimizing the mass spectrometer on the particular analyte, see
Chapter 5, “Acquiring ESI Sample Data by Using Tune Plus,” for instructions about
acquiring sample data.
Before optimizing the tune for the analyte, ensure that the mass spectrometer has been
calibrated within the last three months. If the system needs to be calibrated, follow the
procedures in Chapter 3, “Automatic Tuning and Calibration in ESI Mode.”
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Setting Up the Inlet for High-Flow Infusion in ESI Mode
Setting Up the Inlet for High-Flow Infusion in ESI Mode
This section describes how to set up the LTQ Series mass spectrometer to infuse a sample
solution with the syringe pump into the solvent flow from an LC pump. Figure 49 shows the
plumbing connections to introduce sample by high-flow infusion.
Figure 49. Plumbing setup for LC/ESI/MS infusion into the solvent flow from an LC pump
From LC pump
outlet to port 2
From port 1 to waste container
From port 3
to LC tee union
3
4
Load
Detector
Inject
Waste
2
5
1
6
Power
Vacuum
Communication
System
Scanning
From LC tee union
to grounding
union
Pump On
From syringe
to LC tee union
To connect the inlet plumbing for high-flow infusion analyses, use the parts listed in Table 7,
which are in the instrument accessory kit.
Table 7. Inlet plumbing parts for high-flow infusion analysis (Sheet 1 of 2)
Description
Part number
Grounding union (ESI mode)
00101-18182
LC tee union
00101-18204
LC union
00101-18202
PEEK fingertight fittings and ferrules or stainless steel fittings and
ferrules for conical 10-32 receiving ports and 1/16 in. OD tubing:
•
•
•
•
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PEEK ferrule
PEEK fitting
Stainless steel nut
Stainless steel ferrule
00101-18196
00101-18081
2522-0066
2522-3830
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Setting Up the Inlet for High-Flow Infusion in ESI Mode
Table 7. Inlet plumbing parts for high-flow infusion analysis (Sheet 2 of 2)
Description
Part number
Red PEEK tubing (0.005 in. ID, 1/16 in. OD)
00301-22912
Teflon tubing or
Injection Port filler
00301-22915
00101-18206
To make the plumbing connections for ESI sample introduction from the syringe pump into
solvent flow from an LC pump, follow these procedures:
• Setting Up the Syringe Pump
• Connecting the Plumbing to Introduce Sample by High-Flow Infusion
Setting Up the Syringe Pump
 To set up the syringe pump
CAUTION SHARP OBJECT The syringe needle can puncture your skin. Handle it
with care.
1. Load a clean, 500 μL Unimetrics syringe with 100 pg/μL solution of reserpine or the
analyte.
To prepare the reserpine tuning solution, follow one of these procedures:
• For the LXQ and LTQ XL, see “Preparing the Reserpine Tuning and Sample
Solutions for the ESI and APCI Modes” on page 144.
• For the Velos Pro, see “Preparing the Reserpine Tuning and Sample Solutions for ESI
and APCI Modes” on page 152.
2. Hold the plunger of the syringe in place and carefully insert the tip of the syringe needle
into the end of a 4 cm (1.5 in.) Teflon tube with a fingertight fitting and a ferrule to the
(black) LC union (Figure 50).
Figure 50. Plumbing connections for the syringe
Fingertight fitting
LC union
Ferrule
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Setting Up the Inlet for High-Flow Infusion in ESI Mode
3. Place the syringe into the syringe holder of the syringe pump.
4. Use a fingertight fitting and ferrule to connect an infusion line of red PEEK tubing
between the LC union and the grounding union held by the grounding bar of the ion
source (Figure 38 on page 65).
5. While squeezing the blue release buttons on the syringe pump handle, push the handle
forward until it just contacts the syringe plunger.
Connecting the Plumbing to Introduce Sample by High-Flow Infusion
To introduce sample by high-flow infusion, follow these procedures to connect solvent flow
from the syringe pump and the LC pump to the ion source:
1. To connect the syringe to the LC tee union
2. To connect the LC tee union to the divert/Inject valve
3. To connect the LC pump to the divert/inject valve on page 89
4. To connect the divert/ inject valve to a waste container on page 89
5. To connect the LC tee union to the grounding union on page 90
6. To connect the Grounding Union to the ESI Probe Sample Inlet on page 91
 To connect the syringe to the LC tee union
1. Set up the syringe pump (see page 86).
2. Use a fingertight fitting and a ferrule to connect the red PEEK infusion line to the free
end of the LC union that connects to the syringe.
3. Use a fingertight fitting and a ferrule to connect the other end of the red PEEK infusion
line to the LC tee union.
Figure 51 shows the fittings required to connect the LC union to the union tee.
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Setting Up the Inlet for High-Flow Infusion in ESI Mode
Figure 51. Plumbing connections between the LC tee union and the LC union
LC tee union
Ferrule
Fingertight fitting
Red PEEK tubing
Ferrule
LC union
Syringe
Fingertight fitting
 To connect the LC tee union to the divert/Inject valve
1. Use a fingertight fitting and a ferrule to connect a length of red PEEK tubing to port 3 of
the divert/inject valve. Or, use a stainless steel nut and ferrule to connect the tubing.
2. Use a fingertight fitting and a ferrule to connect the other end of the tubing to the free
end of the LC tee union (Figure 52 and Figure 53).
Figure 52. Six-port divert/inject valve connections
To LC tee union
To LC tee union
From
LC
3
Plug
(Optional)
4
2
5
1
Plug
(Optional)
6
From
LC
3
4
2
5
1
6
To waste
To waste
Detector position
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4 Tuning with an Analyte in ESI Mode
Setting Up the Inlet for High-Flow Infusion in ESI Mode
Figure 53. Connections between the LC tee union and the divert/inject valve
Stainless steel nut
3
Stainless steel ferrule
4
2
5
1
6
PEEK tubing
LC tee union
Infusion line
LC union
Syringe
 To connect the LC pump to the divert/inject valve
1. Use a fingertight fitting and a ferrule to connect a length of PEEK tubing to port 2 of the
divert/inject valve (Figure 52 on page 88).
2. Use an appropriate fitting and ferrule to connect the other end of the tubing to the outlet
of the LC.
 To connect the divert/ inject valve to a waste container
1. Use a fingertight fitting and a ferrule to connect a length of red PEEK tubing to port 1 of
the divert/inject valve (Figure 52 on page 88).
2. Insert the other end of the tubing into a suitable waste container.
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Tuning with an Analyte in ESI Mode
Setting Up the Inlet for High-Flow Infusion in ESI Mode
 To connect the LC tee union to the grounding union
1. Use a fingertight fitting and a ferrule to connect one end of a length of red PEEK tubing
to the LC tee union (Figure 55).
The grounding union slides into the grounding bar on the ion source as shown in
Figure 54. For instructions about connecting the grounding union to the ESI probe
sample inlet, refer to the LTQ Series Hardware Manual. Figure 56 shows the complete
connection.
Figure 54. Connections between the LC tee union and the grounding union (ESI probe)
Ferrules
Grounding union
Fingertight fittings
PEEK tubing
Syringe
2. Use a fingertight fitting and a ferrule to connect the other end of the tubing to the
grounding union that is held by the grounding bar of the API source (Figure 54 on
page 90).
Figure 55. Plumbing setup for ESI/MS sample introduction with high-flow infusion
From LC pump
outlet to port 2
From port 1 to waste container
From port 3 to
LC tee union
3
4
Load
Detector
Inject
Waste
2
5
1
6
Power
Vacuum
Communication
System
Scanning
From LC tee union
to grounding
union
Pump On
From syringe to
LC tee union
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4 Tuning with an Analyte in ESI Mode
Setting Up the Mass Spectrometer to Tune with an Analyte in ESI Mode
 To connect the Grounding Union to the ESI Probe Sample Inlet
For instructions about connecting the PEEK safety sleeve and fused-silica sample tube
from the grounding union to the ESI probe sample inlet (Figure 56), refer to the
LTQ Series Hardware Manual.
Figure 56. Grounding union connected to the sample inlet of the ESI probe
Sample inlet
Grounding
union
Grounding bar
Setting Up the Mass Spectrometer to Tune with an Analyte in
ESI Mode
You can tune the LTQ Series mass spectrometer in ESI mode by using a high solvent flow:
a 100 pg/μL solution of reserpine or a solution of the analyte. To prepare the 100 pg/μL
reserpine tuning solution, follow one of these procedures:
• For the LXQ and LTQ XL, see “Preparing the Reserpine Tuning and Sample Solutions
for the ESI and APCI Modes” on page 144.
• For the Velos Pro, see “Preparing the Reserpine Tuning and Sample Solutions for ESI and
APCI Modes” on page 152.
To set up the mass spectrometer for tuning at the solvent flow rate for the experiments, follow
these procedures:
1. To open a stored tune method
2. To define the scan parameters, scan type, and scan polarity for the analyte
3. To set the data type and ion polarity mode on page 93
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Setting Up the Mass Spectrometer to Tune with an Analyte in ESI Mode
 To open a stored tune method
1. Open the Tune Plus window (see page 32).
2. Click the On/Standby button to select the On mode.
On Standby
The mass spectrometer applies high voltage to the ESI probe and begins scanning. A
real-time display begins in the Spectrum view.
3. Open ESImyTune, or the file name used for your saved tune method from Chapter 3, as
follows:
a. Click the Open button.
b. Browse to the drive:\Thermo\Instruments\LTQ\methods folder, and then select
the tune method you saved.
c. Click Open.
Tune Plus downloads the tune method parameters to the mass spectrometer, and the
title bar in the Tune Plus window lists the name of the current tune method, for
example, C:\Thermo\Instruments\LTQ\ESImyTune.
 To define the scan parameters, scan type, and scan polarity for the analyte
1. In the Tune Plus window, click the Define Scan button to open the Define Scan
dialog box.
Figure 57 shows the typical settings for acquiring reserpine data by using the selected ion
monitoring (SIM) scan type.
Figure 57. Define Scan dialog box for acquiring reserpine SIM data in ESI mode
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Setting Up the Mass Spectrometer to Tune with an Analyte in ESI Mode
2. Under Scan Description, do the following:
• In the Mass Range list, select Normal.
• In the Scan Rate list, select Normal.
• In the Scan Type list, select SIM.
3. Under Scan Time, do the following:
• In the Microscans box, enter 2.
• In the Max. Inject Time (ms) box, enter 200.000.
4. Under Source Fragmentation, clear the On check box.
Note Selecting the Source Fragmentation check box allows collision-induced
fragmentation to occur in the ion source. Because collision-induced fragmentation
performed in the ion source is not very specific, this feature is rarely used.
5. Under Scan Ranges, do the following:
a. In the Input list, select Center/Width.
b. In the Center Mass (m/z) column, type the mass of the analyte to set the center mass
for the scan range. If tuning on reserpine, type 609.20 for the center mass.
c. In the Width (m/z) column, type 2.00.
6. Verify the scan settings:
• If tuning on reserpine, ensure that the settings in the Define Scan dialog box match
those shown in Figure 57 on page 92.
• If using another analyte, ensure that the settings in the Define Scan dialog box are
appropriate for the analyte.
7. Click OK.
 To set the data type and ion polarity mode
Centroid
Profile
1. In the Tune Plus window, click the Centroid/Profile button to select the profile
data type.
2. Click the Positive/Negative button to select the positive ion polarity mode.
Positive
polarity
Negative
polarity
Note In the positive ion polarity mode, the mass spectrometer scans for positive ions. For
basic compounds, such as amines, low pH solutions favor the formation of positive ions.
Reserpine can form a positive ion [(M + H)+ m/z = 609] in low pH solutions.
This completes the mass spectrometer setup with an analyte in ESI mode.
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Tuning the Mass Spectrometer Automatically
Tuning the Mass Spectrometer Automatically
To maximize the transmission of ions for the analyte at a specified solvent flow rate, run the
automatic tuning procedure and optimize on the target mass-to-charge ratio.
Before optimizing the tune of the LTQ Series mass spectrometer on a particular
mass-to-charge ratio and creating a tune method for the analyte, for best results, first calibrate
the mass accuracy of the mass spectrometer. The automated calibration procedure takes
approximately 20 minutes. Or, for best results in a limited amount of time, infuse the ESI
calibration mixture (or an analyte that you have mass spectral data for), and visually check the
mass spectrum for mass accuracy. For information about calibrating the mass accuracy, see
“Calibrating Automatically in the Normal Mass Range” on page 79.
Note The most important parameters that affect the signal quality during ESI operation
for the LXQ and LTQ XL mass spectrometers are the electrospray voltage, ion transfer
tube temperature, heated tube voltage, tube lens voltage, gases, and solution flow rate. For
the Velos Pro mass spectrometer, the important parameters are the electrospray voltage,
transfer tube voltage, S-lens rf level, and solution flow rate.
If any of these parameters change, you must reoptimize the mass spectrometer parameters.
Use the Semi-Automatic tune procedure to tune the mass spectrometer on individual
parameters.
The following procedure uses a 100 pg/mL solution of reserpine to optimize the LTQ Series
automatically on the reserpine peak at m/z 609.2 at a flow rate of 0.4 μm/min. You can
substitute the reserpine solution with a sample solution that is suitable for analysis in ESI
mode. For guidelines on setting an appropriate flow rate and temperatures, see Table 3 on
page 12.
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Tuning the Mass Spectrometer Automatically
 To automatically optimize the tune of the mass spectrometer for the analyte
1. In the Tune Plus window, click the Tune button to open the Tune dialog box (Figure 58).
Figure 58. Tune dialog box showing the Automatic page
2. Under What to Optimize On, select the Mass (m/z) option.
3. In the Mass (m/z) box, enter 609.2 (or the appropriate mass of the analyte).
4. Click the Divert/Inject Valve button to open the Divert/Inject Valve dialog box
(Figure 59).
Figure 59. Divert/Inject Valve dialog box
5. Select the Load Detector option, and then click Close.
Note When the divert/inject valve is in the Detector position, the solvent flow from
the LC pump enters and exits the divert/inject valve through ports 2 and 3,
respectively (Figure 52 on page 88). Port 3 is connected to the ion source.
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Tuning the Mass Spectrometer Automatically
6. In the Tune dialog box, start the automatic tuning procedure as follows:
a. Click Start.
A message appears:
Please ensure that the 500 microliter syringe is full.
b. Ensure that the syringe pump contains at least 450 μL of the 100 pg/μL reserpine
tuning solution or the analyte.
c. Click OK.
7. In the Tune Plus window, click the Graph View button.
8. Observe the Tune Plus window and the Tune dialog box.
During automatic tuning, the mass spectrometer displays the results of various tests in the
Spectrum and Graph views in the Tune Plus window and displays various messages under
Status in the Tune dialog box.
Check that the Tune Plus window looks similar to the one shown in Figure 60.
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Tuning the Mass Spectrometer Automatically
Figure 60. Tune Plus window and Tune dialog box for automatic tuning in ESI mode of the LTQ XL
This completes the tuning of the mass spectrometer in ESI mode for the compound reserpine
(or the analyte).
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Tuning with an Analyte in ESI Mode
Saving the ESI Tune Method
Saving the ESI Tune Method
Tip You must save the tune method while the mass spectrometer is on.
 To save the ESI tune method
1. In the Tune Plus window, click the Save button.
2. Browse to the drive:\Thermo\Instruments\LTQ\methods folder.
3. In the File Name box, type reserpine (or the name of the analyte).
4. Click Save.
If you tuned the mass spectrometer by using reserpine, clean the mass spectrometer as
described in Chapter 8, “Cleaning the Mass Spectrometer After Tuning and Calibrating.” If
you tuned with an analyte, as described in Chapter 5, “Acquiring ESI Sample Data by Using
Tune Plus,” you are ready to acquire data on the analyte.
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5
Acquiring ESI Sample Data by Using Tune Plus
This chapter describes how to optimize the isolation width and collision energy for MS/MS
experiments and how to acquire sample data in the selected ion monitoring (SIM) mode. In
addition, this chapter describes how to set up the inlet for loop injections into the solvent flow
from an LC pump.
For demonstration purposes only, the procedures in this chapter use a 1 μg/μL reserpine
solution. You can use any analyte that is suitable for analysis in ESI mode.
Note Before beginning the analysis of the sample solution, verify that the mass
spectrometer has been tuned and calibrated in ESI mode and that you have created and
saved a tune method for the particular analyte by using a suitable flow rate.
Contents
• Setting Up to Acquire Full-Scan MS/MS Data
• Setting Up the Inlet for Flow Injection Analysis in ESI Mode
• Acquiring ESI Data in the SIM Scan Type
Setting Up to Acquire Full-Scan MS/MS Data
Before performing full-scan MS/MS experiments, optimize the isolation width to ensure the
effective isolation of the ion, and then optimize the relative collision energy parameters to
ensure efficient fragmentation of the parent ion. The relative collision energy for a particular
analysis can depend on the type of sample being analyzed. Optimize the collision energy
manually or use the automated process provided by the software.
The information in this section applies to both the ESI and APCI modes. The following
procedures use reserpine for ESI mode. For APCI mode, use a suitable analyte.
• Optimizing the Isolation Width for an MS/MS Experiment
• Optimizing the Collision Energy Manually for an MS/MS Experiment
• Optimizing the Collision Energy Automatically for an MS/MS Experiment
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Setting Up to Acquire Full-Scan MS/MS Data
Optimizing the Isolation Width for an MS/MS Experiment
To optimize the isolation width for the analyte under the same or similar high-flow conditions
intended for experiments, set up the inlet for high-flow infusion. Then, as you apply minor
changes to the collision energy, observe the mass spectrum of the analyte. For this experiment,
and for most applications, leave the Activation Q and activation time parameters set to their
default values. For more information about these parameters, refer to the data system Help.
 To optimize the isolation width
1. Set up the inlet for high-flow infusion.
For ESI mode, see page 85. For APCI mode, see page 114.
2. Open the Tune Plus window (see page 32).
3. Click the On/Standby button to select the On mode.
On Standby
Centroid
Profile
4. Click the Centroid/Profile button to select the centroid data type.
5. Ensure that the scan parameters are defined to acquire full-scan MS/MS data for reserpine
(or the analyte) as follows:
a. Click the Define Scan button to open the Define Scan dialog box (Figure 61).
The initial settings optimize the isolation width of an MS/MS experiment for
reserpine.
Figure 61. Define Scan dialog box for acquiring reserpine full-scan data
b. If infusing reserpine, verify that the values in the dialog box match those shown in
Figure 61. If infusing a different analyte, ensure that the Parent Mass (m/z) box
contains the correct value and that the scan range is appropriate.
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Setting Up to Acquire Full-Scan MS/MS Data
c. Under MSn Settings, in the Isolation Width (m/z) column, start with a relatively wide
isolation width of 2.0 Da.
d. After entering the values, click Apply and leave the Define Scan dialog box open.
6. Turn on the LC pump and specify a flow rate of 0.4 mL/min.
7. Verify that the inlet plumbing connections do not leak.
8. Choose Setup > Syringe Pump to open the Syringe Pump dialog box (Figure 62).
Figure 62. Syringe Pump dialog box
9. Turn on the syringe pump and set an infusion flow rate of 5 μL/min as follows:
a. Under Flow Control, select the On option.
b. In the Flow Rate (μL/min) box, enter 5.00.
c. Under Type, select the type of syringe, and do one of the following:
• For a Hamilton or Unimetrics syringe, in the Volume (μL) list, select the volume
of the syringe.
• For another type of syringe, in the Syringe ID (mm) box, enter the ID of the
syringe.
d. Click Apply to apply the syringe parameters and start the syringe pump.
10. Keep the Syringe Pump dialog box open and move it out of the way to the top of the
screen.
11. Optimize the isolation width as follows:
a. In the Tune Plus window, observe the mass spectrum for the parent ion of reserpine,
m/z 609.2 or the parent ion of the analyte. Ensure that the readback values for NL
and IT are relatively stable.
b. In the Define Scan dialog box, under MSn Settings, type 2.0 in the Isolation Width
(m/z) column.
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Setting Up to Acquire Full-Scan MS/MS Data
c. Click Apply.
Note The optimum value for the Isolation Width 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. When the optimum Isolation Width is
obtained, the values for NL and IT are stable and the mass peak for the parent ion
is at its maximum intensity and appears symmetrical. An Isolation Width value
that is less than the optimum value causes a substantial drop in the NL reading. A
significant drop in sensitivity indicates that the ions are not effectively isolated.
d. Repeat step 11a and step 11b, entering successively smaller values for the isolation
width. Continue to observe the intensity of the mass spectrum of the parent ion, and
ensure that the values for NL and IT are stable with each change made to the
Isolation Width setting.
Note The Isolation Width setting is typically m/z 1–3. After optimizing the
isolation width, compensate for minor changes in tune stability by increasing the
isolation width value a small amount. The adjustment should not exceed m/z = 1.
Optimizing the Collision Energy Manually for an MS/MS Experiment
 To optimize the collision energy manually
1. In the Tune Plus window, click the Define Scan button to open the Define Scan dialog
box (Figure 63).
2. Under MSn Settings, in the Normalized Collision Energy column, type 20.0.
3. Click Apply.
4. In the Tune Plus window, observe the mass spectrum of the product ions of reserpine (or
the analyte).
5. If necessary, increase the value for the Normalized Collision Energy in increments of 5%
until the intensity of the precursor ion is less than 5% of the intensity of the product ions.
Note After each change to the value in the Normalized Collision Energy column, you
must click Apply to implement the change.
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Acquiring ESI Sample Data by Using Tune Plus
Setting Up to Acquire Full-Scan MS/MS Data
Figure 63 shows typical settings for acquiring full-scan MS/MS data on reserpine.
Figure 63. Define Scan dialog box (full-scan data)
Optimizing the Collision Energy Automatically for an MS/MS Experiment
 To optimize the relative collision energy automatically
1. Identify the m/z of a product ion for the analyte.
2. In the Tune Plus window, click the Tune button to open the Tune dialog box.
3. Click the Collision Energy tab (Figure 64).
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Setting Up to Acquire Full-Scan MS/MS Data
Figure 64. Tune dialog box showing the Collision Energy page
4. Under What to Optimize On, select the Product Ion Mass (m/z) option and type
397.20 or a value for one of the product ions of the analyte.
The mass spectrometer can optimize the collision energy required to analyze the analyte
automatically by using the m/z value of one of its product ions.
5. Click Start.
A message appears:
Ensure that the 500 microliter syringe is full.
6. Ensure that the syringe contains at least 450 μL of the 100 pg/μL reserpine tuning
solution or the analyte.
7. Click OK.
8. Check that the Tune Plus window looks similar to the one shown in Figure 65.
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Setting Up to Acquire Full-Scan MS/MS Data
Figure 65. Tune Plus window and the Tune dialog box showing the Collision Energy page
9. In the Spectrum view of Tune Plus, observe the full-scan MS/MS spectrum of reserpine
or the analyte.
After optimizing the collision energy, the Accept Optimized Value dialog box opens
(Figure 66).
Figure 66. Accept Optimized Value dialog box
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Setting Up the Inlet for Flow Injection Analysis in ESI Mode
10. Click Accept.
The new value appears in the Define Scan dialog box.
11. In the Syringe Pump dialog box, select the Off option and click Apply to turn off the
syringe pump. Click OK to close the dialog box.
12. Click Cancel.
After optimizing the relative collision energy, the mass spectrometer is ready to acquire
MS/MS data on the analyte.
Setting Up the Inlet for Flow Injection Analysis in ESI Mode
This section provides information about how to introduce sample by loop injection (flow
injection analysis) into the solvent flow from an LC pump.
 To set up the inlet for loop injections
1. Connect a loop filler to port 5 of the divert/inject valve (Figure 67).
Figure 67. Divert/inject valve setup for loop injection
3
LC pump to port 2
Ion source to port 3
Loop filler to port 5
5
1
Sample loop to ports 1 and 4
Waste container to port 6
2. Connect a sample loop of the desired volume to ports 1 and 4.
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Setting Up the Inlet for Flow Injection Analysis in ESI Mode
3. Connect the LC pump to port 2 of the divert/inject valve as follows:
• Use an appropriate fitting and a ferrule to connect one end of a length of red PEEK
tubing to the outlet of the LC pump.
To produce a stable solvent flow, the Accela 1250 Pump requires a minimum
backpressure of 40 bar (580 psi). To connect the LC pump, use a length of
0.005 in. ID PEEK tubing sufficient to exert a backpressure of 40 bar (580 psi), or
connect an inline backpressure regulator between the LC pump outlet and the
divert/inject valve.
• Use a fingertight fitting and a ferrule to connect the other end of the tubing to port 2.
4. For the ESI probe, use two fingertight fittings and two ferrules to connect a length of red
PEEK tubing between port 3 of the divert/inject valve and the grounding union
(Figure 68).
5. Connect the other end of the grounding union to the ESI probe sample inlet (see
“Installing the ESI or HESI-II Probe” on page 40).
Figure 68. Plumbing for loop injection into the solvent flow from an LC into an ESI probe
From LC pump
to port 2
From port 6
to waste container
3
4
From port 3
to ion source
Load
Detector
Inject
Waste
2
5
1
6
6. Connect the divert/inject valve to a waste container as follows:
• Use a fingertight fitting and a ferrule to connect one end of a length of red PEEK
tubing to port 6.
• Connect the other end of the tubing to an appropriate waste container.
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Acquiring ESI Data in the SIM Scan Type
Acquiring ESI Data in the SIM Scan Type
Note The data system computer automatically saves the acquired data to its hard disk
drive.
 To acquire a data file containing SIM data
1. In the Tune Plus window, click the On/Standby button to select the On mode.
On Standby
Centroid
The mass spectrometer begins scanning and applies high voltage to the ESI probe. A
real-time display appears in the Spectrum view.
2. Click the Centroid/Profile button to select the centroid data type.
3. Ensure that the scan parameters are defined to acquire SIM data for reserpine (or the
analyte) as follows:
a. Click the Define Scan button.
Figure 69 shows the Define Scan dialog box with typical settings for acquiring SIM
data for reserpine.
Figure 69. Define Scan dialog box (reserpine SIM-scan data)
b. Verify that the values in the dialog box match those in Figure 69, and then click OK.
4. Turn on the LC pump, and specify a flow rate of 0.4 mL/min.
Ensure that the system is free of leaks.
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Acquiring ESI Data in the SIM Scan Type
5. In the Tune Plus window, click the Acquire Data button.
Figure 70 shows the acquisition status of the raw data file in the Acquire Data dialog box.
Figure 70. Acquire Data dialog box
6. Specify the acquisition parameters as follows:
a. In the File Name box, type reserpine (or the name of the analyte).
b. In the Sample Name box, type reserpine (or the name of the analyte).
c. In the Comment box, type a comment about the experiment.
For example, describe the scan mode, scan type, ionization mode, sample amount, or
method of sample introduction. The Xcalibur 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 XReport reporting
software. To open the XReport application, choose Start > Programs >
Thermo Xcalibur > XReport.
d. Under Acquire Time, select the Continuously option (acquires data until you stop
the acquisition).
7. Click Start.
8. Leave the Acquire Data dialog box open during data acquisition and move it out of
the way.
9. In the Tune Plus window, click the Divert/Inject Valve button to open the Divert/Inject
Valve dialog box.
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Acquiring ESI Data in the SIM Scan Type
10. Inject the sample into the ESI source a total of four times as follows:
a. Select the Load Detector option.
When the valve is in the Load position, the solvent flow from the LC pump bypasses
the sample loop.
b. Overfill the injector loop with a sample solution or a 1 pg/μL reserpine solution.
To prepare the reserpine tuning solution, follow one of these procedures:
• For the LXQ and LTQ XL, see “Preparing the Reserpine Tuning and Sample
Solutions for the ESI and APCI Modes” on page 144.
• For the Velos Pro, see “Preparing the Reserpine Tuning and Sample Solutions for
ESI and APCI Modes” on page 152.
c. Select the Inject Waste option.
When the valve is in the Inject position, the solvent flow from the LC pump
backflushes the contents of the sample loop into the ion source.
d. In the Spectrum view, observe the reserpine peak (m/z 609.2) or the peak of the
analyte.
e. Repeat step a through step d three more times.
11. In the Divert/Inject Valve dialog box, click Close.
12. In the Acquire Data dialog box, click Stop, and then click Cancel.
Review the mass spectrum and chromatogram in the raw file just acquired by using the
Xcalibur Qual Browser window. Figure 71 shows a chromatogram (left side) containing the
results from the four loop injections of reserpine and a mass spectrum of reserpine (right side).
For more information about reviewing the acquired data, refer to the Thermo Xcalibur
Qualitative Analysis User Guide or the Qual Browser Help.
Note To acquire full-scan MS/MS data, see “Setting Up to Acquire Full-Scan MS/MS
Data” on page 99.
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Acquiring ESI Data in the SIM Scan Type
Figure 71. Qual Browser window showing an ESI SIM chromatogram and mass spectrum
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Tuning with an Analyte in APCI Mode
This chapter describes how to create a tune method for an analyte in the APCI high-flow
mode.
Contents
• Setting Up the Inlet for High-Flow Infusion in APCI Mode
• Setting Up the Mass Spectrometer to Tune with an Analyte in
APCI Mode
• Tuning the Mass Spectrometer Automatically
• Saving the APCI Tune Method
You do not have to recalibrate the LTQ Series mass spectrometer when switching to APCI
operation. You can use the calibration settings obtained from the successful automatic
calibration procedure performed in ESI mode.
The following mass spectrometer parameters have the most impact on signal quality in APCI
mode:
• Electrospray voltage
• Tube lens voltage (LXQ and LTQ XL only)
• Heated ion transfer tube temperature (voltage)
• Ion transfer tube voltage (LXQ and LTQ XL only)
• Gas flow rates for the API gases
• S-lens rf level (Velos Pro only)
• Solution flow rate
• Vaporizer temperature
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Setting Up the Inlet for High-Flow Infusion in APCI Mode
Note
• These procedures assume that you are familiar with the LTQ Series mass
spectrometers and the Tune Plus window. For more information, refer to the data
system Help, LTQ Series Getting Connected Guide, and LTQ Series Hardware Manual,
as needed.
• Before setting up the inlet plumbing, ensure that the ion source is set up for APCI
mode and that you have tuned and calibrated the mass spectrometer in ESI mode.
• This chapter refers to a 100 pg/mL solution of reserpine as the sample analyte. You
can substitute an analyte of your choice that is suitable for analysis in APCI mode.
Setting Up the Inlet for High-Flow Infusion in APCI Mode
This section describes how to set up the LTQ Series mass spectrometer to infuse a sample
solution with the syringe pump into the solvent flow from an LC pump. Figure 72 shows the
plumbing connections to introduce sample by high-flow infusion.
Table 7 on page 85 lists the inlet plumbing parts for high-flow infusion analysis.
Figure 72. Plumbing setup for APCI/MS sample introduction with high-flow infusion
From LC pump
outlet to port 2
From port 3
to LC tee union
From port 1 to waste container
3
4
Load
Detector
Inject
Waste
2
5
1
6
Power
Vacuum
Communication
System
Scanning
From LC tee union
to sample inlet
of APCI probe
Pump On
From syringe
to LC tee union
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Setting Up the Inlet for High-Flow Infusion in APCI Mode
 To set up the syringe pump for infusion
CAUTION SHARP OBJECT The syringe needle can puncture your skin. Handle it
with care.
1. Load a clean, 500 μL Unimetrics syringe with 450 μL of a 100 pg/μL solution of
reserpine or the analyte.
To prepare the reserpine tuning solution, follow one of these procedures:
• For the LXQ and LTQ XL, see “Preparing the Reserpine Tuning and Sample
Solutions for the ESI and APCI Modes” on page 144.
• For the Velos Pro, see “Preparing the Reserpine Tuning and Sample Solutions for ESI
and APCI Modes” on page 152.
Note To minimize the possibility of cross-contamination of the assembly, be sure to
wipe off the needle tip with a clean, lint-free tissue before reinserting it into the
syringe adapter assembly.
2. Hold the plunger of the syringe in place and carefully insert the tip of the syringe needle
into the end of a 4 cm (1.5 in.) Teflon tube with a fingertight fitting and a ferrule to the
(black) LC union (Figure 73).
Figure 73. Plumbing connections for the syringe
Fingertight fitting
LC union
Ferrule
Syringe
Teflon tube
3. Place the syringe into the syringe holder of the syringe pump.
4. Connect a red PEEK infusion line from the LC union to the LC tee union as follows:
a. Use a fingertight fitting and ferrule to connect the infusion line to the free end of the
LC union.
b. Use a fingertight fitting and ferrule to connect the other end of the infusion line to a
leg of the LC tee union.
Figure 74 shows the connections to the LC tee union.
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Setting Up the Inlet for High-Flow Infusion in APCI Mode
Figure 74. APCI/MS plumbing connections for the LC tee union
LC tee union
From the divert/inject
valve
To the sample inlet of the
APCI probe
Ferrule
Infusion line PEEK tubing
Fingertight fitting
From LC union
Tip Use a PEEK tubing cutter to cut the PEEK tubing. Use a PEEK cutter to ensure
that the tubing is cut straight. To prevent adding dead volume to the inlet plumbing,
ensure that the LC fittings, ferrules, and PEEK tubing are properly installed. Dead
volume in the plumbing connections can broaden the chromatographic peaks and
increase carryover.
5. Use a fingertight fitting and a ferrule to connect one end of a length of red PEEK tubing
to the LC tee union (Figure 74).
6. Use a fingertight fitting and a ferrule to connect the other end of the Teflon tubing
directly to the sample inlet of the APCI probe.
Figure 72 shows the connection between the LC tee union and the sample inlet of the
APCI probe.
Note Do not use the grounding bar of the API source housing for the APCI probe. A
knurled nut secures the grounding bar to the ion source. You do not need to remove
the grounding bar to run the system in APCI mode.
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Setting Up the Mass Spectrometer to Tune with an Analyte in APCI Mode
7. Connect the LC pump to port 2 of the divert/inject valve as follows:
• Use an appropriate fitting and a ferrule to connect one end of a length of tubing to
the outlet of the LC pump.
• Use a fingertight fitting and a ferrule to connect one end of the tubing to port 2 of
the divert/inject valve.
8. Connect the divert/inject valve to a waste container as follows:
• Use a fingertight fitting and a ferrule to connect one end of a length of tubing to
port 1.
• Connect the other end of the tubing to an appropriate waste container.
Setting Up the Mass Spectrometer to Tune with an Analyte in
APCI Mode
You can tune the LTQ Series mass spectrometer in APCI mode by using a high solvent flow:
a solution of reserpine or a solution of the analyte. To prepare the reserpine tuning solution,
follow one of these procedures:
• For the LXQ and LTQ XL, see “Preparing the Reserpine Tuning and Sample Solutions
for the ESI and APCI Modes” on page 144.
• For theVelos Pro, see “Preparing the Reserpine Tuning and Sample Solutions for ESI and
APCI Modes” on page 152.
To set up the mass spectrometer for tuning at the solvent flow rate for the experiments, follow
these procedures:
• To open a stored tune method with preset conditions for APCI mode
• To view the pre-tune APCI source settings
• To define the scan parameters for an analyte in APCI mode on page 119
• To set the scan type and ion polarity mode on page 120
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Tuning with an Analyte in APCI Mode
Setting Up the Mass Spectrometer to Tune with an Analyte in APCI Mode
 To open a stored tune method with preset conditions for APCI mode
1. Open the Tune Plus window (see page 32).
2. Click the On/Standby button to select the On mode.
On Standby
The mass spectrometer begins scanning and applies high voltage to the corona needle. A
real-time display appears in the Spectrum view.
3. Open the tune method file that stores the factory default tune settings for APCI mode as
follows:
a. In the Tune Plus window, click the Open button.
b. Browse to the drive:\Thermo\Instruments\LTQ\methods folder, and then select
Default_APCI.LTQTune.
This file contains preset conditions for the APCI mode.
c. Click Open.
Tune Plus downloads the tune method parameters to the mass spectrometer.
 To view the pre-tune APCI source settings
1. In the Tune Plus window, choose Setup > APCI Source.
2. Observe the settings.
3. Click OK.
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Setting Up the Mass Spectrometer to Tune with an Analyte in APCI Mode
 To define the scan parameters for an analyte in APCI mode
1. In the Tune Plus window, click the Define Scan button to open the Define Scan dialog
box (Figure 75).
If the dialog box looks different from the one shown in Figure 75, the display is probably
missing the advanced settings. If the advanced settings do not appear, in Tune Plus choose
ScanMode > Advanced Scan Features.
Figure 75. Define Scan dialog box (reserpine SIM type data in APCI mode)
2. Under Scan Description, do the following:
• In the Mass Range list, select Normal.
• In the Scan Rate list, select Normal.
• In the Scan Type list, select SIM.
3. Under Scan Time, do the following:
• In the Microscans box, enter 2.
• In the Max. Inject Time (ms) box, enter 200.000.
4. Under Source Fragmentation, clear the On check box.
Note Selecting the Source Fragmentation check box allows collision-induced
fragmentation to occur in the ion source. Because collision-induced fragmentation
performed in the ion source is not very specific, this feature is rarely used.
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Tuning the Mass Spectrometer Automatically
5. Under Scan Ranges, do the following:
a. In the Input list, select Center/Width.
b. In the Center Mass (m/z) column, type the m/z value for the parent ion of the analyte,
or type 609.20 for reserpine.
c. In the Width (m/z) column, type 2.00.
6. Verify that the settings in the Define Scan dialog box match those in Figure 75 on
page 119.
7. Click OK.
 To set the scan type and ion polarity mode
Centroid
Profile
1. In the Tune Plus window, click the Centroid/Profile button to select the profile data
type.
2. Click the Positive/Negative button to select the positive ion polarity mode.
Positive
polarity
Negative
polarity
This completes the mass spectrometer set up to tune with an analyte in APCI mode.
Tuning the Mass Spectrometer Automatically
Tune the LTQ Series mass spectrometer automatically in APCI mode to optimize the
parameters listed on page 113.
Note The most important parameters that affect the signal quality during APCI
operation for the LXQ and LTQ XL mass spectrometers are the ion transfer tube
temperature, vaporizer temperature, tube lens voltage, gases, and solution flow rate. For
the Velos Pro mass spectrometer, the important parameters are the spray current, S-lens rf
level, and solution flow rate.
If any of these parameters change, you must reoptimize the mass spectrometer parameters.
Use the Semi-Automatic tune procedure to tune the mass spectrometer on individual
parameters.
The following procedure uses a 100 pg/mL reserpine tuning solution to optimize the mass
spectrometer automatically on the reserpine peak at m/z 609.2 at a flow rate of 0.4 μm/min.
You can substitute the reserpine solution with a sample solution that is suitable for analysis in
APCI mode. For guidelines on setting an appropriate flow rate, tube temperature, and
vaporizer temperature for the application, see Table 4 on page 13.
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Tuning the Mass Spectrometer Automatically
 To automatically optimize the tune of the mass spectrometer for the analyte
1. In the Tune Plus window, click the Tune button to open the Tune dialog box (Figure 76).
Figure 76. Tune dialog box showing the Automatic page
2. Under What to Optimize On, select the Mass (m/z) option.
3. In the Mass (m/z) box, enter 609.20 (or the appropriate mass of the analyte).
4. Click the Divert/Inject Valve button to open the Divert/Inject dialog box (Figure 77).
Figure 77. Divert/Inject Valve dialog box
5. Select the Load Detector option, and then click Close.
Note When the divert/inject valve is in the Detector position, the solvent flow from
the LC pump enters and exits the divert/inject valve through ports 2 and 3,
respectively (Figure 52 on page 88). Port 3 is connected to the ion source.
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Tuning the Mass Spectrometer Automatically
6. In the Tune dialog box, start the automatic tuning procedure as follows:
a. Click Start.
A message appears:
Please ensure that the 500 microliter syringe is full.
b. Ensure that the syringe pump contains at least 450 μL of a 100 pg/μL reserpine
tuning solution or the analyte.
To prepare the reserpine tuning solution, follow one of these instructions:
• For the LXQ and LTQ XL, see “Preparing the Reserpine Tuning and Sample
Solutions for the ESI and APCI Modes” on page 144.
• For the Velos Pro, see “Preparing the Reserpine Tuning and Sample Solutions for
ESI and APCI Modes” on page 152.
c. Click OK.
7. In the Tune Plus window, click the Graph View button.
8. Observe the Tune Plus window and the Tune dialog box.
During automatic tuning, the mass spectrometer displays the results of various tests in the
Spectrum and Graph views in the Tune Plus window and displays various messages under
Status in the Tune dialog box.
Check that the Tune Plus window looks similar to the one shown in Figure 78.
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6 Tuning with an Analyte in APCI Mode
Tuning the Mass Spectrometer Automatically
Figure 78. Tune Plus window and Tune dialog box for automatic tuning in APCI mode for the LTQ XL
9. After the automatic tuning process ends, choose Setup > APCI Source to observe the
post-tune settings for the APCI source.
10. Choose Setup > Ion Optics to observe the post-tune settings for the ion optics.
This completes the tuning of the mass spectrometer in APCI mode for the compound
reserpine (or the analyte).
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6
Tuning with an Analyte in APCI Mode
Saving the APCI Tune Method
Saving the APCI Tune Method
You can save the parameters that you just set in a tune method specific to the particular
analyte and solvent flow rate. Recall the tune method and use it as a starting point for
optimizing the mass spectrometer on a different analyte or at a different flow rate.
Tip You must save the tune method while the mass spectrometer is on.
 To save the APCI tune method
1. In the Tune Plus window, click the Save button.
2. Browse to the drive:\Thermo\Instruments\LTQ\methods folder.
3. In the File Name box, type a name, such as APCImyTune, to identify the tune method.
4. Click Save.
If you tuned the mass spectrometer by using reserpine, clean the mass spectrometer as
described in Chapter 8, “Cleaning the Mass Spectrometer After Tuning and Calibrating.” If
you tuned with an analyte, as described in Chapter 7, “Acquiring APCI Sample Data by Using
Tune Plus,” you are ready to acquire data on the analyte.
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7
Acquiring APCI Sample Data by Using Tune Plus
This chapter describes how to set up the inlet to make loop injections in APCI mode and how
to acquire sample data in the selected ion monitoring (SIM) mode.
For demonstration purposes only, the procedures in this chapter use a 1 pg/mL reserpine
solution. You can use any analyte that is suitable for analysis in APCI mode. If you do not
have a sample suitable for analysis in APCI mode, use the reserpine provided in the
instrument’s chemicals kit to prepare a 1 pg/mL reserpine solution as described in one of the
following:
• For the LXQ and LTQ XL, see “Preparing the Reserpine Tuning and Sample Solutions
for the ESI and APCI Modes” on page 144.
• For the Velos Pro see“Preparing the Reserpine Tuning and Sample Solutions for ESI and
APCI Modes” on page 152.
Note
• Before beginning the analysis of the sample solution, verify that the mass spectrometer
has been tuned and calibrated in ESI mode and that you have created and saved a
tune method for the particular analyte by using a suitable flow rate.
• Before performing full-scan MS/MS experiments, follow the procedures in “Setting
Up to Acquire Full-Scan MS/MS Data” on page 99.
Contents
• Setting Up the Inlet for Flow Injection Analysis in APCI Mode
• Acquiring APCI Data in the SIM Scan Mode
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7 Acquiring APCI Sample Data by Using Tune Plus
Setting Up the Inlet for Flow Injection Analysis in APCI Mode
Setting Up the Inlet for Flow Injection Analysis in APCI Mode
This section provides information about how to introduce sample by loop injection (flow
injection analysis) into the solvent flow from an LC pump.
 To set up the inlet for loop injection
1. Connect a 2 μL sample loop to ports 1 and 4 of the divert/inject valve.
2. Connect the LC pump to port 2 of the divert/inject valve as follows:
• Use an appropriate fingertight fitting and a ferrule to connect one end of a length of
red PEEK tubing to the outlet of the LC pump.
To produce a stable solvent flow, the Accela 1250 Pump requires a minimum
backpressure of 40 bar (580 psi). To connect the LC pump, use a length of
0.005 in. ID PEEK tubing sufficient to exert a backpressure of 40 bar (580 psi), or
connect an inline backpressure regulator between the LC pump outlet and the
divert/inject valve.
• Use a fitting and a ferrule to connect the other end of the tubing to port 2 of the
divert/inject valve.
3. For the APCI probe, use fingertight fittings to connect an appropriate length of red PEEK
tubing between port 3 of the divert/inject valve to the sample inlet fitting on the APCI
probe (Figure 79 and Figure 80).
Figure 79. Divert/inject valve setup for loop injection
To API source
To API source
3
Inject port
4
2
5
1
6
From
LC
3
Inject port
4
2
5
1
6
To
waste
Load position
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From
LC
To
waste
Inject position
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7
Acquiring APCI Sample Data by Using Tune Plus
Acquiring APCI Data in the SIM Scan Mode
Figure 80. APCI probe
Guide pin
Vaporizer
heater cable
socket
Sheath gas inlet (S)
Auxiliary gas inlet (A)
Sample inlet fitting
4. Connect the divert/inject valve to a waste container as follows:
• Use a fingertight fitting and a ferrule to connect one end of a length of red PEEK
tubing to port 6.
• Connect the other end of the tubing to an appropriate waste container.
The mass spectrometer is now set up to introduce sample by loop injection into the solvent
flow from an LC pump.
Acquiring APCI Data in the SIM Scan Mode
Note The data system automatically saves the acquired data to its hard disk drive.
 To acquire a data file containing SIM data
1. Open the Tune Plus window (see page 32).
2. Click the On/Standby button to select the On mode.
On Standby
The mass spectrometer begins scanning and applies high voltage to the APCI corona
needle. A real-time display appears in the Spectrum view.
3. Click the Centroid/Profile button to select the centroid data type.
Centroid
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Profile
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7 Acquiring APCI Sample Data by Using Tune Plus
Acquiring APCI Data in the SIM Scan Mode
4. Ensure that the scan parameters are defined to acquire SIM data for reserpine (or the
analyte) as follows:
a. Click the Define Scan button.
Figure 81 shows the Define Scan dialog box with typical settings for acquiring SIM
data for reserpine.
Figure 81. Define Scan dialog box (reserpine SIM-scan data)
b. Verify that the values in the dialog box are the same as those in Figure 81, and then
click OK.
5. Turn on the LC pump and specify a flow rate of 0.4 mL/min.
Ensure that the system is free of leaks.
6. In the Tune Plus window, click the Acquire Data button.
Figure 82 shows the acquisition status of the raw data file in the Acquire Data dialog box.
Figure 82. Acquire Data dialog box
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Acquiring APCI Sample Data by Using Tune Plus
Acquiring APCI Data in the SIM Scan Mode
7. Specify the acquisition parameters as follows:
• In the File Name box, type reserpine (or the name of the analyte).
• In the Sample Name box, type reserpine (or the name of the analyte).
• In the Comment box, type a comment about the experiment.
For example, describe the scan mode, scan type, ionization mode, sample amount, or
method of sample introduction. The Xcalibur 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 XReport reporting
software. To open the XReport application, choose Start > Programs > Thermo
Xcalibur > XReport.
• Under Acquire Time, select the Continuously option (acquires data until you stop
the acquisition).
8. Click Start.
9. Leave the Acquire Data dialog box open during data acquisition and move it out of
the way.
10. Click the Divert/Inject Valve button.
11. Inject the sample into the APCI source a total of four times as follows:
a. Select the Load Detector option.
When the valve is in the Load position, the solvent flow from the LC pump bypasses
the sample loop.
b. Overfill the 2 μL injector loop with a sample solution or a 1 pg/μL reserpine solution.
To prepare the reserpine tuning solution, follow these procedures:
• For the LXQ and LTQ XL, see “Preparing the Reserpine Tuning and Sample
Solutions for the ESI and APCI Modes” on page 144.
• For the Velos Pro, see “Preparing the Reserpine Tuning and Sample Solutions for
ESI and APCI Modes” on page 152.
c. Select the Inject Waste option.
When the valve is in the Inject position, the solvent flow from the LC pump
backflushes the contents of the sample loop into the ion source.
d. In the Spectrum view, observe the reserpine peak (m/z 609.2) or the peak of the
analyte.
e. Wait approximately one minute before the next injection.
f.
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Repeat step 11a to step 11e three more times.
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7 Acquiring APCI Sample Data by Using Tune Plus
Acquiring APCI Data in the SIM Scan Mode
12. In the Divert/Inject Valve dialog box, click Close.
13. In the Acquire Data dialog box, click Stop, and then click Cancel.
Review the mass spectrum and chromatogram in the raw file just acquired by using the
Xcalibur Qual Browser window. Figure 83 shows a chromatogram (left side) containing the
results from the four loop injections of reserpine and a mass spectrum of reserpine (right side).
For more information about reviewing the acquired data, refer to the Thermo Xcalibur
Qualitative Analysis User Guide or the Qual Browser Help.
Note To acquire full-scan MS/MS data, see “Setting Up to Acquire Full-Scan MS/MS
Data” on page 99.
Figure 83. Qual Browser window showing an APCI SIM chromatogram and mass spectrum
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8
Cleaning the Mass Spectrometer After Tuning and
Calibrating
After infusing the calibration solution, you must clean the system before tuning with an
analyte. Complete all data acquisition before proceeding.
Contents
• Cleaning Supplies and Chemicals
• Flushing the Sample Transfer Line, Sample Tube, and API Probe
• Removing and Cleaning the Syringe
• Cleaning the Ion Sweep Cone, Spray Cone, and Ion Transfer Tube
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8
Cleaning the Mass Spectrometer After Tuning and Calibrating
Cleaning Supplies and Chemicals
Cleaning Supplies and Chemicals
You need the following supplies and chemicals.
Supplies
Chemicals
Gloves, lint-free and powder-free
Acetone
Graduated cylinder or beaker
(for use with methanol)
Formic acid
Kimwipes or lint-free industrial tissues
Methanol, LCMS-grade
Sonicator
Water, LCMS-grade
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). The MSDSs describe the chemicals and must be freely
available to lab personnel to examine at any time. MSDSs provide summarized
information on the hazard and toxicity of specific chemical compounds. MSDSs also
provide information on the proper handling of compounds, first aid for accidental
exposure, and procedures for the remedy of spills or leaks.
Read the MSDS 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.
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Cleaning the Mass Spectrometer After Tuning and Calibrating
Flushing the Sample Transfer Line, Sample Tube, and API Probe
Flushing the Sample Transfer Line, Sample Tube, and API Probe
Flush the sample transfer line, sample tube, and API probe at the end of each work day (or
more often if you suspect they are contaminated) with a 50:50 methanol/water solution from
the LC system through the API source at a flow rate of 200–400 μL/min for approximately
15 minutes to remove contamination.
 To flush the sample transfer line, sample tube, and API probe
1. Open the Tune Plus window (see page 32).
On Standby
2. In the Tune Plus window, ensure that the On/Standby button indicates the On mode,
and then do one of the following:
• If operating in APCI or APPI mode, go to step 3.
• If operating in ESI mode, go to step 4.
3. To flush the APCI source:
a. Choose Setup > APCI Source to open the APCI Source dialog box (Figure 84).
Figure 84. APCI Source dialog box
b. In the Vaporizer Temp (°C) box, enter 500.00.
c. In the Sheath Gas Flow Rate (arb) box, enter 30.
d. In the Aux Gas Flow Rate (arb) box, enter 5.
e. In the Sweep Gas Flow Rate (arb) box, enter 0.
f.
In the Discharge Current box, enter 0.
g. Click OK.
h. Go to step 5.
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Cleaning the Mass Spectrometer After Tuning and Calibrating
Flushing the Sample Transfer Line, Sample Tube, and API Probe
4. To flush the ESI source:
a. Choose Setup > ESI Source to open the ESI Source dialog box (Figure 85).
Figure 85. ESI Source dialog box
b. In the Sheath Gas Flow Rate (arb) box, enter 30.
c. In the Aux Gas Flow Rate (arb) box, enter 5.
d. In the Sweep Gas Flow Rate (arb) box, enter 0.
e. In the Spray Voltage (kV) box, enter 0.
f.
Click OK.
5. To set up and start a flow of 50:50 methanol/water solution from the LC system to the
API source:
a. Choose Setup > Inlet Direct Control to open the Inlet Direct Control dialog box.
Note The Xcalibur data system controls the LC pumps from several
manufacturers including Thermo Fisher Scientific Inc., Agilent™ Technologies,
and Waters™ Corporation. Contact your Thermo Fisher Scientific sales
representative for information about the liquid chromatography systems
compatible with the LTQ Series mass spectrometer.
b. Click the LC Pump tab.
c. Set the solvent proportions to 50% methanol and 50% water.
d. Start the solvent flow.
6. Let the solution flow through the sample transfer line, sample tube, and API probe for
15 minutes.
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Cleaning the Mass Spectrometer After Tuning and Calibrating
Removing and Cleaning the Syringe
7. After 15 minutes, turn off the flow of liquid from the LC to the API source as follows:
a. Leave the API source (including the APCI vaporizer, sheath gas, and auxiliary gas) on
for an additional 5 minutes.
b. Click the Pump Off or Stop Pump button.
8. After another 5 minutes, place the mass spectrometer in Standby mode (see page 33).
Removing and Cleaning the Syringe
 To remove and clean the syringe
1. Squeeze the blue pusher blocks and pull back on the syringe pump handle to free the
syringe.
2. Remove the syringe from the holder.
3. Disconnect the tip of the syringe needle from the Teflon tubing.
4. Clean the syringe with a solution of 5% formic acid in water.
5. Rinse the syringe with a solution of 50:50 methanol/water.
6. Rinse the syringe with acetone several times.
Cleaning the Ion Sweep Cone, Spray Cone, and Ion Transfer Tube
IMPORTANT
• Prepare a clean work surface by covering the area with lint-free paper or a sheet of
aluminum foil.
• Put on a new pair of lint- and powder-free gloves before starting each cleaning and
component installation procedure.
CAUTION HOT SURFACE At operating temperatures above 350 °C (662 °F), the probe
and API source housing can severely burn you.
• Before removing the probe or API source housing, allow the part to cool to room
temperature (approximately 20 minutes) before touching it.
• If the mass spectrometer connects to an LC system, leave the solvent flow from the
LC pump on while the probe cools to room temperature.
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Cleaning the Mass Spectrometer After Tuning and Calibrating
Cleaning the Ion Sweep Cone, Spray Cone, and Ion Transfer Tube
 To clean the ion sweep cone, spray cone, and ion transfer tube
1. Remove the API source housing (see page 36).
2. Remove the ion sweep cone by grasping the outer ridges of the ion sweep cone and
pulling it off of the API cone seal.
You might need to loosen the ball plungers on the ion sweep cone. There are two versions
of the ion sweep cone. Figure 86 shows the version with a nipple. Figure 88 on page 138
shows the version with an offset orifice.
CAUTION To avoid contaminating the ion transfer tube, do not touch its exposed
entrance.
Figure 86. Ion source interface components for the LXQ and LTQ XL (exploded view)
Spray cone
Ion transfer tube
Ion sweep cone
3. Remove the ion transfer tube by turning its exposed spray cone counterclockwise with the
custom removal tool provided in the MS Accessory Kit (Figure 87). When the tube is free
of the spray cone, pull it straight out of the ion source interface.
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Cleaning the Mass Spectrometer After Tuning and Calibrating
Cleaning the Ion Sweep Cone, Spray Cone, and Ion Transfer Tube
Figure 87. Ion transfer tube removal tool
Custom removal
tool
Spray cone
(of the ion transfer tube)
4. Clean the ion sweep cone and optional spray cone by wiping the insides and outsides with
Kimwipe tissues soaked in methanol.
5. Clean the ion transfer tube as follows:
a. Place it into a graduated cylinder containing 50:50 methanol/water.
b. Sonicate the component for 15 minutes.
CAUTION Take these precautions when reinstalling the ion transfer tube:
• Ensure that everything is properly aligned to prevent stripping the threads on
the ion transfer tube.
• Do not bend the ion transfer tube. Rotate it as you insert it.
6. Reinstall the ion transfer tube as follows:
a. Rotate the ion transfer tube while reinserting it into the heater block.
b. Use the custom tool to turn the tube clockwise until tight.
7. Reinstall the ion sweep cone as follows:
a. Put on a new pair of lint- and powder-free gloves.
b. Carefully align the gas inlet on the ion sweep cone with the sweep gas supply port
(Figure 88). Firmly press the ion sweep cone into position.
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Cleaning the Mass Spectrometer After Tuning and Calibrating
Cleaning the Ion Sweep Cone, Spray Cone, and Ion Transfer Tube
c. If necessary to achieve a proper ion sweep cone installation, adjust the ball plungers
around the perimeter of the ion sweep cone (Figure 88).
Figure 88. Sweep gas supply port in the API cone seal
Ball plungers
Sweep gas
support port
Ion sweep cone with
offset orifice
The ion sweep cone is now properly installed on the mass spectrometer.
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A
Sample Formulations for the LXQ and LTQ XL Mass
Spectrometers
This appendix describes how to prepare the tuning and calibration solutions for the LXQ and
LTQ XL mass spectrometers.
CAUTION NEVER use the solutions described in this appendix to calibrate the Velos Pro
mass spectrometer. Instead, use the solutions described in Appendix B, “Sample
Formulations for the Velos Pro Mass Spectrometer.”
Contents
• Preparing the Normal Mass Range Calibration Solution for ESI Mode
• Preparing the Reserpine Tuning and Sample Solutions for the ESI and
APCI Modes
• Preparing the High Mass Range Calibration Solution
The Chemical Accessory Kit provides the caffeine, Met-Arg-Phe-Ala acetate salt (MRFA),
Ultramark 1621, and reserpine needed to make the solutions. You can order replacement
Chemical Accessory Kits (P/N 97455-62045) from Thermo Fisher Scientific.
You can also order specific chemicals from Thermo Fisher Scientific, which are sold under its
Fisher Chemical brand. As specified in Table 8, use only LCMS-grade chemicals for
calibrating the mass spectrometers.
Table 8. Recommended chemicals
Thermo Scientific
Solvent/reagent
Specifications
Acetic acid (modifier)
LCMS-grade
Acetonitrile
LCMS-grade
Formic acid (modifier)
99–100%
(Must be supplied in a glass bottle.)
Isopropyl alcohol
LCMS-grade
Methanol
LCMS-grade
Water
LCMS-grade
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Sample Formulations for the LXQ and LTQ XL Mass Spectrometers
Preparing the Normal Mass Range Calibration Solution for ESI Mode
For a complete selection of LCMS-grade consumables from Thermo Fisher Scientific, visit
www.FisherLCMS.com.
Note Do not filter solvents. Filtering solvents can introduce contamination.
Potentially hazardous chemicals used in procedures throughout this appendix include the
following:
•
•
•
•
•
Acetonitrile
Formic acid
Glacial acetic acid
Methanol
Reserpine
IMPORTANT Do not use plastic pipettes to prepare the tuning and calibration standards.
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). The MSDSs describe the chemicals and must be freely
available to lab personnel to examine at any time. MSDSs provide summarized
information on the hazard and toxicity of specific chemical compounds. MSDSs also
provide information on the proper handling of compounds, first aid for accidental
exposure, and procedures for the remedy of spills or leaks.
Read the MSDS 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.
Preparing the Normal Mass Range Calibration Solution for ESI Mode
For tuning and calibrating the LXQ and LTQ XL in ESI mode, use a calibration solution of
caffeine, MRFA, and Ultramark 1621 in an acetonitrile/methanol/water solution containing
1% acetic acid. To prepare the ESI calibration solution, follow these procedures:
• “Caffeine Stock Solution” on page 142
• “Preparing the MRFA Stock Solution” on page 142
• “Preparing the Ultramark 1621 Stock Solution” on page 143
• “Preparing the Normal Mass Range Calibration Solution for ESI Mode” on page 143
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Sample Formulations for the LXQ and LTQ XL Mass Spectrometers
Preparing the Normal Mass Range Calibration Solution for ESI Mode
Note The Chemical Accessory Kit contains vials of caffeine and MRFA. The following
compound part numbers are available from Sigma Chemical Co.:
• P/N C6035 is for caffeine at a concentration of 1 mg/mL in methanol.
• P/N M1170 is for MRFA.
To order these compounds from Sigma, write or call:
Sigma Chemical Company
P. O. Box 14508
St. Louis, Missouri U.S. 63178-9916
(800) 325-3010 (U.S.)
(905) 829-9500 (Canada)
(314) 771-3750 (outside the U.S. or Canada)
www.sigmaaldrich.com
Note The Chemical Accessory Kit contains a vial of Ultramark 1621 (neat liquid). This
compound is available from Lancaster Synthesis.
The structure of Ultramark 1621 follows (x is 1, 2, or 3):
H(CF2CF2)x H2C O
H(CF2CF2)x H2C O
P
N
N
P
O CH2(CF2CF2)xH
O CH2(CF2CF2)xH
N
P
H(CF2CF2)x H2C O
O CH2(CF2CF2)xH
The Lancaster product number for Ultramark 1621 is L16698 (Ultramark 1621,
Mass Spec Std.). To order this compound from Lancaster Synthesis, write or call:
Lancaster Synthesis, Inc.
P.O. Box 1000
Windham, NH U.S. 03087-9977
(603) 889-3306, (800) 238-2324 (in the U.S. and Canada)
+44 (0)1524 36101 (U.K. and International)
www.lancastersynthesis.com
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Sample Formulations for the LXQ and LTQ XL Mass Spectrometers
Preparing the Normal Mass Range Calibration Solution for ESI Mode
CAUTION Always wear protective gloves and safety glasses when you use solvents or
corrosives.
Caffeine Stock Solution
The LXQ and LTQ XL mass spectrometers ships with a 1 mg/mL stock solution of caffeine in
100% methanol. You can also order this solution through Sigma Chemical (P/N C6035).
Preparing the MRFA Stock Solution
 To prepare the MRFA stock solution
1. Weigh out 6 mg of the MRFA compound provided in the accessory kit, and then transfer
the sample to a clean, minimum 20 mL glass vial.
The MRFA provided in the accessory kit has an average molecular weight of 523.7 Da.
2. Add 2.0 mL of 50:50 methanol/water to the vial.
3. Mix the solution thoroughly.
4. Label the vial MRFA Stock Solution 3 mg/mL.
 To prepare the MRFA diluted stock solution
1. Transfer 100 μL of the 3 mg/mL MRFA stock solution into a clean, minimum 20 mL
glass vial.
2. Add 2.9 mL of 50:50 methanol/water to the vial.
3. Mix the solution thoroughly.
4. Label the vial Diluted MRFA Stock Solution 0.1 mg/mL.
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Preparing the Normal Mass Range Calibration Solution for ESI Mode
Preparing the Ultramark 1621 Stock Solution
 To prepare the Ultramark 1621 stock solution
1. Use a syringe to transfer 25 μL of Ultramark 1621 to a clean 25 mL volumetric glass flask.
2. Fill the flask to volume with 100% acetonitrile.
3. Mix the solution thoroughly.
4. Transfer the solution to a vial and label the vial
Ultramark 1621 stock solution (1/1000 dilution).
Preparing the Normal Mass Range Calibration Solution for ESI Mode
The following ready-to-use normal mass range calibration solution is available from Thermo
Fisher Scientific at www.thermo.com/pierce:
P/N 88322, Pierce LTQ ESI Positive Ion Calibration Solution, 10 mL
IMPORTANT Use only glass pipets or stainless steel syringes when measuring glacial
acetic acid. Using plastic pipet tips causes contamination of acid stock solutions that can
introduce contaminants in the calibration solution.
 To prepare the normal mass range calibration solution
1. Pipet 200 μL of the caffeine stock solution into a clean 10 mL volumetric glass flask.
2. Pipet 100 μL of the diluted MRFA stock solution (0.1 mg/mL) into the flask.
3. Pipet 100 μL of the Ultramark 1621 stock solution into the flask.
4. Pipet 100 μL of the LCMS-grade glacial acetic acid into the flask.
5. Pipet 5 mL of LCMS-grade acetonitrile into the flask.
6. Bring the flask to volume with 50:50 methanol/water.
7. Mix the solution thoroughly.
8. Transfer the solution to a clean, dry vial.
9. Label the vial LXQ/LTQ XL ESI Calibration Solution and store it in a refrigerator at
2–8 °C (36– 46 °F) until needed.
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Sample Formulations for the LXQ and LTQ XL Mass Spectrometers
Preparing the Reserpine Tuning and Sample Solutions for the ESI and APCI Modes
Preparing the Reserpine Tuning and Sample Solutions for the ESI and
APCI Modes
Ideally, you should prepare the reserpine solutions just before using them. If you must store
the solutions, keep them in a light-resistant container in the refrigerator until needed.
 To prepare the reserpine stock solution
1. Weigh out 10 mg of reserpine, and then transfer the sample into a clean 10 mL
volumetric glass flask.
The average molecular weight of reserpine is 608.7 Da.
2. Fill the flask to volume with a solution of 1% acetic acid in methanol.
3. Mix the solution thoroughly.
4. Transfer the solution to a clean, dry, light-resistant vial.
5. Label the vial Reserpine Stock Solution (1 μg/μL).
 To prepare the reserpine tuning solution
1. Pipet 100 μL of the reserpine stock solution (1 μg/μL) into a clean, minimum 1.5 mL
polypropylene microcentrifuge tube.
2. Add 900 μL of 1% acetic acid in 50:50 methanol/water to the tube.
3. Mix the solution (100 ng/μL) thoroughly.
4. Transfer 10 μL of the 100 ng/μL solution into a clean polypropylene tube.
5. Add 990 μL of 1% acetic acid in 50:50 methanol/water to the tube.
6. Mix the solution (1 ng/μL) thoroughly.
7. Transfer 100 μL of the 1 ng/μL solution into a clean, minimum 1.5 mL polypropylene
tube.
8. Add 900 μL of 1% acetic acid in 50:50 methanol/water to the tube.
9. Mix the solution thoroughly.
10. Label the tube Reserpine Tuning Solution (100 pg/μL).
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Sample Formulations for the LXQ and LTQ XL Mass Spectrometers
Preparing the High Mass Range Calibration Solution
 To prepare the reserpine sample solution
1. Transfer 100 μL of the 100 pg/μL reserpine tuning solution into a clean, minimum
1.5 mL polypropylene tube.
2. Add 900 μL of 1% acetic acid in 50:50 methanol/water to the tube.
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 100 μL of the 1 pg/μL solution into a clean, minimum 1.5 mL polypropylene
tube.
8. Add 700 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 (125 fg/μL).
Preparing the High Mass Range Calibration Solution
The high mass range calibrant is a solution of 3.5 mg/μL polypropylene glycol (PPG) in a
solvent of 70:30 methanol/23 mM sodium acetate.
The high mass range calibration procedure is designed to work with a PPG that has an average
molecular weight of approximately 2700 (Mn~2700), which is Aldrich product number
202347. PPG 2700 is a viscous liquid. To order this compound from Sigma-Aldrich, write
or call:
Sigma Chemical Company
P. O. Box 14508
St. Louis, Missouri U.S. 63178-9916
(800) 325-3010 (U.S.)
(905) 829-9500 (Canada)
(314) 771-3750 (outside the U.S. or Canada)
www.sigmaaldrich.com
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Sample Formulations for the LXQ and LTQ XL Mass Spectrometers
Preparing the High Mass Range Calibration Solution
 To prepare the sodium acetate stock solution
Dissolve 0.082 gm of sodium acetate in 10 mL of water in a clean 20 mL glass vial and
label the container Sodium Acetate Stock Solution.
 To prepare the PPG stock solution
1. Dissolve 0.7 gm of PPG 2700 in 7 mL of methanol in a clean 20 mL glass vial.
Tip Because PPG 2700 is a viscous liquid, use a glass pipette to transfer 0.7 gm of the
liquid into a weigh boat, or weigh the liquid directly into a minimum 20 mL glass
vial.
2. Add 2.3 mL of water to the vial.
3. Add 0.7 mL of the sodium acetate stock solution and label the container
PPG 2700 Stock Solution (70 mg/mL).
 To prepare the calibration solution
1. Pipette 7 mL of methanol into a clean 20 mL glass vial.
2. Add 2.3 mL of water to the vial.
3. Add 700 μL of the sodium acetate stock solution to the vial.
4. Add 10 μL of the PPG 2700 stock solution and label the container
LXQ/LTQ XL PPG 2700 Calibration Solution (70 ng/μL).
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Sample Formulations for the Velos Pro Mass
Spectrometer
This appendix describes how to prepare the tuning and calibration solutions for the Velos Pro
mass spectrometer.
CAUTION NEVER use the solutions described in this appendix to calibrate the LXQ and
LTQ XL mass spectrometers. Instead, use the solutions described in Appendix A, “Sample
Formulations for the LXQ and LTQ XL Mass Spectrometers.”
Contents
• Preparing the Normal Mass Range Calibration Solution for ESI Mode
• Preparing the Reserpine Tuning and Sample Solutions for ESI and
APCI Modes
• Preparing the High Mass Range Calibration Solution
The Chemical Accessory Kit provides the caffeine, MRFA, Ultramark 1621, and reserpine
needed to make the solutions. You can order replacement Chemical Accessory Kits
(P/N 97455-62045) from Thermo Fisher Scientific.
You can also order specific chemicals from Thermo Fisher Scientific, which are sold under its
Fisher Chemical brand. As specified in Table 9, use only LCMS-grade chemicals for
calibrating the mass spectrometers.
Table 9. Recommended chemicals
Thermo Scientific
Solvent/reagent
Specifications
Acetic acid (modifier)
LCMS-grade
Acetonitrile
LCMS-grade
Formic acid (modifier)
99–100%
(Must be supplied in a glass bottle.)
Isopropyl alcohol
LCMS-grade
Methanol
LCMS-grade
Water
LCMS-grade
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Sample Formulations for the Velos Pro Mass Spectrometer
Preparing the Normal Mass Range Calibration Solution for ESI Mode
For a complete selection of LCMS-grade consumables from Thermo Fisher Scientific, visit
www.FisherLCMS.com.
.
Note Do not filter solvents. Filtering solvents can introduce contamination.
Potentially hazardous chemicals used in the procedures throughout this appendix include the
following:
•
•
•
•
•
Acetonitrile
Formic acid
Glacial acetic acid
Methanol
Reserpine
IMPORTANT Do not use plastic pipettes to prepare the tuning and calibration standards.
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). The MSDSs describe the chemicals and must be freely
available to lab personnel to examine at any time. MSDSs provide summarized
information on the hazard and toxicity of specific chemical compounds. MSDSs also
provide information on the proper handling of compounds, first aid for accidental
exposure, and procedures for the remedy of spills or leaks.
Read the MSDS 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.
Preparing the Normal Mass Range Calibration Solution for ESI Mode
For tuning and calibrating the Velos Pro in ESI mode, use a calibration solution of caffeine,
MRFA, Ultramark 1621, and N-butylamine in an acetonitrile/methanol/water solution
containing 1% acetic acid. To prepare the ESI calibration solution, follow these procedures:
• “Caffeine Stock Solution” on page 150
• “Preparing the MRFA Stock Solution” on page 150
• “Preparing the Ultramark 1621 Stock Solution” on page 151
• “Preparing the N-butylamine Stock Solution” on page 151
• “Preparing the Normal Mass Range Calibration Solution for ESI Mode” on page 151
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Sample Formulations for the Velos Pro Mass Spectrometer
Preparing the Normal Mass Range Calibration Solution for ESI Mode
Note The Chemical Accessory Kit contains vials of caffeine and MRFA. The following
compound part numbers are available from Sigma Chemical Co.:
• P/N C6035 is for caffeine at a concentration of 1 mg/mL in methanol.
• P/N M1170 is for MRFA.
To order these compounds from Sigma, write or call:
Sigma Chemical Company
P. O. Box 14508
St. Louis, Missouri U.S. 63178-9916
(800) 325-3010 (U.S.)
(905) 829-9500 (Canada)
(314) 771-3750 (outside the U.S. or Canada)
www.sigmaaldrich.com
Note The Chemical Accessory Kit contains a vial of Ultramark 1621 (neat liquid). This
compound is available from Lancaster Synthesis.
The structure of Ultramark 1621 follows (x is 1, 2, or 3):
H(CF2CF2)x H2C O
H(CF2CF2)x H2C O
P
N
N
P
O CH2(CF2CF2)xH
O CH2(CF2CF2)xH
N
P
H(CF2CF2)x H2C O
O CH2(CF2CF2)xH
The Lancaster product number for Ultramark 1621 is L16698 (Ultramark 1621,
Mass Spec Std.). To order this compound from Lancaster Synthesis, write or call:
Lancaster Synthesis, Inc.
P.O. Box 1000
Windham, NH U.S. 03087-9977
(603) 889-3306, (800) 238-2324 (in the U.S. and Canada)
+44 (0)1524 36101 (U.K. and International)
www.lancastersynthesis.com
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Sample Formulations for the Velos Pro Mass Spectrometer
Preparing the Normal Mass Range Calibration Solution for ESI Mode
CAUTION Always wear protective gloves and safety glasses when you use solvents or
corrosives.
Caffeine Stock Solution
The Velos Pro mass spectrometer ships with a 1 mg/mL stock solution of caffeine in 100%
methanol. You can also order this solution through Sigma Chemical (P/N C6035).
Preparing the MRFA Stock Solution
 To prepare the MRFA stock solution
1. Weigh out 6 mg of the MRFA compound provided in the accessory kit, and then transfer
the sample to a clean, minimum 20 mL glass vial.
The MRFA provided in the accessory kit has an average molecular weight of 523.7 Da.
2. Add 2.0 mL of 50:50 methanol/water to the vial.
3. Mix the solution thoroughly.
4. Label the vial MRFA Stock Solution 3 mg/mL.
 To prepare the MRFA diluted stock solution
1. Transfer 100 μL of the 3 mg/mL MRFA stock solution into a clean, minimum 20 mL
glass vial.
2. Add 2.9 mL of 50:50 methanol/water to the vial.
3. Mix the solution thoroughly.
4. Label the vial Diluted MRFA Stock Solution 0.1 mg/mL.
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Preparing the Normal Mass Range Calibration Solution for ESI Mode
Preparing the Ultramark 1621 Stock Solution
 To prepare the Ultramark 1621 stock solution
1. Use a syringe to transfer 25 μL of Ultramark 1621 to a clean 25 mL volumetric glass flask.
2. Fill the flask to volume with 100% acetonitrile.
3. Mix the solution thoroughly.
4. Transfer the solution to a vial and label the vial
Ultramark 1621 stock solution (1/1000 dilution).
Preparing the N-butylamine Stock Solution
 To prepare the N-butylamine stock solution
1. Use a syringe to transfer 5 μL of N-butylamine to a clean, 25 mL volumetric glass flask.
2. Add 9.995 mL of 50:50 methanol/water to the vial.
3. Mix the solution thoroughly.
4. Transfer the solution to a vial and label the vial
N-butylamine (5/10 000 dilution) stock solution.
Preparing the Normal Mass Range Calibration Solution for ESI Mode
The following ready-to-use normal mass range calibration solution is available from Thermo
Fisher Scientific at www.thermo.com/pierce:
P/N 88323, Pierce LTQ Velos Positive Ion Calibration Solution, 10 mL
IMPORTANT Use only glass pipets or stainless steel syringes when measuring glacial
acetic acid. Using plastic pipet tips causes contamination of acid stock solutions that can
introduce contaminants in the calibration solution.
 To prepare the normal mass range calibration solution
1. Pipet 20 μL of the caffeine stock solution into a clean 10 mL volumetric glass flask.
2. Pipet 100 μL of the diluted MRFA stock solution (0.1 mg/mL) into the flask.
3. Pipet 100 μL of the Ultramark 1621 stock solution into the flask.
4. Pipet 100 μL of the N-butylamine stock solution into the flask.
5. Pipet 100 μL of LCMS-grade glacial acetic acid into the flask.
6. Pipet 5 mL of LCMS-grade acetonitrile into the flask.
7. Bring the flask to volume with 50:50 methanol/water.
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Preparing the Reserpine Tuning and Sample Solutions for ESI and APCI Modes
8. Mix the solution thoroughly.
9. Transfer the solution to a clean, dry vial.
10. Label the vial Velos Pro ESI Calibration Solution and store it in a refrigerator at 2–8 °C
(36–46 °F) until needed.
Preparing the Reserpine Tuning and Sample Solutions for ESI and
APCI Modes
Ideally, you should prepare the reserpine solutions just before using them. If you must store
the solutions, keep them in a light-resistant container in the refrigerator until needed.
 To prepare the reserpine stock solution
1. Weigh out 10 mg of reserpine, and then transfer the sample into a clean 10 mL
volumetric glass flask.
The average molecular weight of reserpine is 608.7 Da.
2. Fill the flask to volume with a solution of 1% acetic acid in methanol.
3. Mix the solution thoroughly.
4. Transfer the solution to a clean, dry, light-resistant vial.
5. Label the vial Reserpine Stock Solution (1 μg/μL).
 To prepare the reserpine tuning solution
1. Pipet 100 μL of the reserpine stock solution (1 μg/μL) into a clean, minimum 1.5 mL
polypropylene microcentrifuge tube.
2. Add 900 μL of 1% acetic acid in 50:50 methanol/water to the tube.
3. Mix the solution (100 ng/μL) thoroughly.
4. Transfer 10 μL of the 100 ng/μL solution into a clean polypropylene tube.
5. Add 990 μL of 1% acetic acid in 50:50 methanol/water to the tube.
6. Mix the solution (1 ng/μL) thoroughly.
7. Transfer 100 μL of the 1 ng/μL solution into a clean, minimum 1.5 mL polypropylene
tube.
8. Add 900 μL of 1% acetic acid in 50:50 methanol/water to the tube.
9. Mix the solution thoroughly.
10. Label the tube Reserpine Tuning Solution (100 pg/μL).
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Preparing the High Mass Range Calibration Solution
 To prepare the reserpine sample solution
1. Transfer 100 μL of the 100 pg/μL reserpine tuning solution into a clean, minimum
1.5 mL polypropylene tube.
2. Add 900 μL of 1% acetic acid in 50:50 methanol/water to the tube.
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).
Preparing the High Mass Range Calibration Solution
The high mass range calibrant is a solution of 3.5 mg/μL polypropylene glycol (PPG) in a
solvent of 65:35 methanol/10 mM sodium acetate.
The high mass range calibration procedure is designed to work with a PPG that has an average
molecular weight of approximately 2700 (Mn~2700), which is Aldrich product number
202347. PPG 2700 is a viscous liquid. To order this compound from Sigma-Aldrich, write
or call:
Sigma Chemical Company
P. O. Box 14508
St. Louis, Missouri U.S. 63178–9916
(800) 325-3010 (U.S.)
(905) 829-9500 (Canada)
(314) 771-3750 (outside the U.S. or Canada)
www.sigmaaldrich.com
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Sample Formulations for the Velos Pro Mass Spectrometer
Preparing the High Mass Range Calibration Solution
 To prepare the sodium acetate stock solution
Dissolve 0.082 gm of sodium acetate in 10 mL of water in a clean 20 mL glass vial and
label the container Sodium Acetate Stock Solution.
 To prepare the PPG stock solution
1. Dissolve 0.7 gm of PPG 2700 in 7 mL of methanol in a clean 20 mL glass vial.
Tip Because PPG 2700 is a viscous liquid, use a glass pipette to transfer 0.7 gm of the
liquid into a weigh boat, or weigh the liquid directly into a minimum 20 mL glass
vial.
2. Add 2.3 mL of water to the vial.
3. Add 0.7 mL of the sodium acetate stock solution and label the container
PPG 2700 Stock Solution (70 μg/μL).
 To prepare the calibration solution
1. Pipette 6.65 mL of methanol into a clean 20 mL glass vial.
2. Add 2.15 mL of water to the vial.
3. Add 700 μL of the sodium acetate stock solution to the vial.
4. Add 500 μL of the PPG 2700 stock solution and label the container
Velos Pro PPG 2700 Calibration Solution (3.5 μg/μL).
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This appendix applies to all LTQ Series mass spectrometers, unless otherwise noted.
Before calibrating an LTQ Series mass spectrometer in the high mass range, you must
calibrate it in the normal mass range as described in Chapter 3, “Automatic Tuning and
Calibration in ESI Mode.”
Note
Prepare the appropriate Normal mass range calibration solution as follows:
• For the LXQ and LTQ XL, see “Preparing the Normal Mass Range Calibration
Solution for ESI Mode” on page 140 in Appendix A.
• For the Velos Pro, see “Preparing the Normal Mass Range Calibration Solution for
ESI Mode” on page 148 in Appendix B.
Prepare the appropriate High mass range calibration solution as follows:
• For the LXQ and LTQ XL, see “Preparing the High Mass Range Calibration
Solution” on page 145 in Appendix A.
• For the Velos Pro, see “Preparing the High Mass Range Calibration Solution” on
page 153 in Appendix B.
Follow these procedures:
1. “Verifying Coarse Calibration for the High Mass Range by Using the Normal Mass Range
Calibration Solution” on page 156
2. “Calibrating the High Mass Range by Using the Normal Mass Range
Calibration Solution” on page 162
3. “Performing Two-Point Manual Coarse Calibration—High Mass Range Mode by Using
Normal Mass Range Calibration Solution” on page 164
4. “Calibrating the High Mass Range by Using the High Mass Range Calibration Solution”
on page 168
5. “Cleaning the System After Calibration” on page 171
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Verifying Coarse Calibration for the High Mass Range by Using the Normal Mass
Range Calibration Solution
To verify that the high mass range is coarsely calibrated, use the normal mass range calibration
solution to view the peaks in the normal and high mass ranges. When you are sure that the
system performs properly in the normal and high mass ranges with the normal mass range
calibration solution, proceed to “Calibrating the High Mass Range by Using the Normal Mass
Range Calibration Solution” on page 162.
To verify coarse calibration, follow these procedures:
1. Checking the Mass Calibration in the Normal Mass Range
2. Checking the Mass Calibration in the High Mass Range on page 160
Checking the Mass Calibration in the Normal Mass Range
View the peaks in the spectrum in the normal mass range to determine if the system is
calibrated correctly in this mass range.
 To check the mass calibration in the normal mass range
1. Load the syringe with the normal mass range calibration solution that is appropriate for
your mass spectrometer and turn on the syringe pump.
2. Open the Tune Plus window (see page 32).
3. Click the On/Standby button to select the On mode.
On Standby
The mass spectrometer begins scanning, nitrogen flows into the ESI probe, and high
voltage is applied to the ESI nozzle.
4. Click the Display Spectrum View button.
A real-time mass spectrum view appears in the Spectrum view of the Tune window.
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5. Click the Define Scan button to open the Define Scan dialog box (Figure 89).
Figure 89. Define Scan dialog box
6. Under Scan Description, do the following:
• In the Mass Range list, select Normal.
• In the Scan Rate list, select Normal.
• In the Scan Type list, select Full.
7. Under Scan Time, do the following:
• In the Microscans box, enter 1.
• In the Max. Inject Time (ms) box, enter 10.000.
8. Under Source Fragmentation, clear the On check box.
Note Selecting the Source Fragmentation check box allows collision-induced
fragmentation to occur in the ion source. Because collision-induced fragmentation
performed in the ion source is not very specific, this feature is rarely used.
9. Under Scan Ranges, do the following:
a. In the Input list, select From/To.
b. In the First Mass (m/z) column, type 150.00.
c. In the Last Mass (m/z) column, type 2000.00.
10. Click Apply.
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11. Observe the mass spectra of the singly-charged ions in the calibration solution (Figure 90
for the LXQ and LTQ XL, or Figure 91 for the Velos Pro). The integer m/z values of the
calibration ions are as follows:
• Caffeine: m/z 195 (only for the LXQ and LTQ XL)
• MRFA: m/z 524
• Ultramark 1621: m/z 1022, 1122, 1222, 1322, 1422, 1522, 1622, 1722, and 1822
Figure 90. Calibration solution in the normal mass range spectrum (LTQ XL, coarse calibration)
Caffeine peak
Ultramark peaks
MRFA peak
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Figure 91. Calibration solution in the normal mass range spectrum (Velos Pro, coarse calibration)
12. Do one of the following:
• If the m/z of the observed ions are ±1 Da for the LXQ and LTQ XL or ±3 Da for the
Velos Pro of the integer mass-to-charge ratios stated in step 11 (see the example in
Figure 90), proceed to section “Checking the Mass Calibration in the High Mass
Range.”
• If the ions are not ±1 Da for the LXQ and LTQ XL or ±3 Da for the Velos Pro of the
integer mass-to-charge ratios stated in step 11, perform the normal mass range
calibration before proceeding (see “Calibrating Automatically in the Normal Mass
Range” on page 79).
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Checking the Mass Calibration in the High Mass Range
The next step in the process is to view the peaks of the normal mass range calibration solution
when the scan is set to the high mass range.
 To check the mass calibration in the high mass range
1. In the Tune Plus window, click the Define Scan button to open the Define Scan
dialog box.
2. Under Scan Description, do the following:
• In the Mass Range list, select High.
• In the Scan Rate list, select Normal.
• In the Scan Type list, select Full.
3. Under Scan Time, do the following:
• In the Microscans box, enter 1.
• In the Max. Inject Time (ms) box, enter 10.000.
4. Under Source Fragmentation, clear the On check box.
Note Selecting the Source Fragmentation check box allows collision-induced
fragmentation to occur in the ion source. Because collision-induced fragmentation
performed in the ion source is not very specific, this feature is rarely used.
5. Under Scan Ranges, do the following:
a. In the Input list, select From/To.
b. In the First Mass (m/z) column, do one of the following:
–
For the LXQ and LTQ XL, type 150.00.
–
For the Velos Pro, type 400.00.
c. In the Last Mass (m/z) column, type 2000.00.
6. Click Apply.
7. Observe the mass spectra of the singly-charged ions in the calibration solution (Figure 90
on page 158 for the LXQ and LTQ XL, or Figure 91 on page 159 for the Velos Pro). The
ions are as follows:
• Caffeine: m/z 195 (only for the LXQ and LTQ XL)
• MRFA: m/z 524
• Ultramark 1621: m/z 1022, 1122, 1222, 1322, 1422, 1522, 1622, 1722, and 1822
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8. Do one of the following:
• If the high mass range spectrum shows that the observed ions are no more than
±10 Da for the LXQ and LTQ XL or ±5 Da for the Velos Pro at m/z 1822, calibrate
in the high mass range with the normal mass range calibration solution (see
“Calibrating the High Mass Range by Using the Normal Mass Range
Calibration Solution” on page 162).
• If the ions are more than ±10 Da for the LXQ and LTQ XL or ±5 Da for the
Velos Pro at m/z 1822, note the observed low mass (195) and observed high mass
(1822), and then perform the two-point calibration (see “Performing Two-Point
Manual Coarse Calibration—High Mass Range Mode by Using Normal Mass Range
Calibration Solution” on page 164).
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Calibrating the High Mass Range by Using the Normal Mass Range
Calibration Solution
 To calibrate in the high mass range by using the normal mass calibration solution
1. Load the syringe pump with the normal mass range calibration solution that is
appropriate for your mass spectrometer and turn on the syringe pump.
2. Click the On/Standby button to select the On mode.
On Standby
3. In the Tune Plus window, click the Calibrate button to open the Calibrate dialog box.
4. For Mass Range, select the High option (Figure 92).
Figure 92. Calibrate dialog box showing the High Mass Range Calibration page
5. Under What To Do, select the Calibrate option.
6. Under What to Cal/Check, select all four check boxes: Mass for Turbo,
Mass for Normal, Mass for Zoom, and Isolation Waveforms.
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7. (For the LXQ and LTQ XL only) Under Calibration Mass List, in the Name list, select
Calmix (factory) (Figure 93).
Figure 93. Calibrate dialog box showing the Calmix calibration mix (LXQ and LTQ XL only)
8. Specify the instrument state after completing the calibration:
• To switch to Standby mode after the calibration, select the Set Instrument to
Standby when Finished check box.
• To remain in the On mode after the calibration, clear the Set Instrument to Standby
when Finished check box.
9. Click Start.
A message appears:
Please ensure that the 500 microliter syringe is full.
10. Ensure that the syringe contains at least 450 μL of the normal mass range calibration
solution and that the syringe pump is turned on.
11. Click OK.
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12. Observe the Tune Plus window and the Calibrate dialog box.
While the automatic calibration is in progress, test results appear in the Spectrum and
Graph views and messages appear in the Status box in the Calibrate dialog box. The
system automatically uses the average mass values for the Turbo Scan calibration. It
automatically uses monoisotopic masses for the normal, Zoom Scan, and isolation
waveform calibrations. The results appear when the calibration process finishes.
13. Check the calibration results.
14. Do one of the following:
• If the calibration passes and the masses are correct, proceed to “Calibrating the High
Mass Range by Using the High Mass Range Calibration Solution” on page 168.
• If the calibration does not pass or if the masses are off by more than ±10 Da, note the
observed low mass (195) and observed high mass (1822) before “Performing
Two-Point Manual Coarse Calibration—High Mass Range Mode by Using Normal
Mass Range Calibration Solution.”
Performing Two-Point Manual Coarse Calibration—High Mass Range Mode by
Using Normal Mass Range Calibration Solution
 To perform a two-point calibration in the high mass range mode by using the normal
mass range calibration solution
1. In the Tune Plus window, click the Define Scan button to open the Define Scan dialog
box (Figure 94).
Figure 94. Define Scan dialog box (default ESI settings)
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2. Under Scan Description, do the following:
• In the Mass Range list, select High.
• In the Scan Rate list, select Normal.
• In the Scan Type list, select Full.
3. Under Scan Time, do the following:
• In the Microscans box, enter 1.
• In the Max. Inject Time (ms) box, enter 10.000.
4. Under Source Fragmentation, clear the On check box.
Note Selecting the Source Fragmentation check box allows collision-induced
fragmentation to occur in the ion source. Because collision-induced fragmentation
performed in the ion source is not very specific, this feature is rarely used.
5. Under Scan Ranges, do the following:
a. In the Input list, select From/To.
b. In the First Mass (m/z) column, do one of the following:
–
For the LXQ and LTQ XL, type 150.00.
–
For the Velos Pro, type 400.00.
c. In the Last Mass (m/z) column, type 2000.00.
6. Click Apply.
7. Observe the mass spectra of the singly-charged ions in the calibration solution.
The m/z of the expected ions are as follows:
• Caffeine: m/z 195 (only for the LXQ and LTQ XL)
• MRFA: m/z 524
• Ultramark 1621: m/z 1022, 1122, 1222, 1322, 1422, 1522, 1622, 1722, and 1822
See Figure 90 for the LXQ and LTQ XL, or Figure 91 for the Velos Pro.
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8. In the Tune Plus window, choose Diagnostics > Diagnostics to open the Diagnostics
dialog box (Figure 95).
Figure 95. Diagnostics dialog box
9. In the Tools list, click Mass Calibration to open the Manual Coarse Calibration page
(Figure 96).
Figure 96. Diagnostics dialog box showing the mass calibration page (LXQ and LTQ XL)
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Figure 97. Diagnostics dialog box showing the mass calibration page (Velos Pro)
10. Under Calibrate Current Scan Type, do the following:
• In the Expected Low Mass box, do one of the following:
–
For LXQ and LTQ XL, type 195.10.
–
For Velos Pro, type 524.3.
• In the Expected High Mass box, type 1822.00.
• In the Observed Low Mass box, do one of the following:
–
For LXQ and LTQ XL, type the mass closest to caffeine (195.10).
–
For Velos Pro, type the observed mass closest to MRFA (524.3).
• In the Observed High Mass box, type whatever mass was the closest to 1822,
observed in “Checking the Mass Calibration in the High Mass Range” on page 160.
11. Under Calibrate Current Scan Type, click Execute.
12. Click OK.
13. Again, observe the masses in the spectrum, which should be very close to the ones shown
in Figure 90 on page 158.
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High Mass Range Calibration
14. Do one of the following:
• If any of the midrange Ultramark peaks (1122, 1222, 1322, 1422, 1522, or 1622) are
more than ±10 Da off, pick a different peak for 1822. If the Ultramark peaks are less
than 100 Da apart, pick the next lower mass peak (~1725), or if they are greater than
100 Da apart, pick the next higher peak (~1925). Enter this peak as the observed
mass and repeat step 11 through step 13 before proceeding.
• If the masses are now ±3 Da of those specified above, click Execute under Estimate
Other Calibration Modes Using Normal Scan Rate Calibration, and then click Save.
IMPORTANT Ensure that the mass range in the Define Scan dialog box is set to
High so that the normal mass range calibration values are not affected.
15. Do one of the following:
• If the calibration passed, proceed to the next procedure, “Calibrating the High Mass
Range by Using the High Mass Range Calibration Solution.”
• If the calibration failed, repeat this entire procedure.
If the calibrations become severely off, or if error messages are reported in the process
of doing this procedure, you can restore the factory defaults to return to the valid
calibration default values.
Calibrating the High Mass Range by Using the High Mass Range Calibration
Solution
The final step is to calibrate in the high mass range with the high mass range calibration
solution (PPG 2700). Follow these procedures:
1. To prepare the mass spectrometer
2. To calibrate in the high mass range by using the high mass range calibration solution
IMPORTANT To minimize the possibility of cross-contamination, use a clean syringe and
a new length of PEEK tubing for the high mass range calibration solution.
 To prepare the mass spectrometer
1. Install new red PEEK tubing.
2. Load a clean, 500 μL Unimetrics syringe with 450 μL of the PPG 2700 calibration
solution.
3. Turn on the mass spectrometer.
4. Turn the syringe pump on at 5 μL/min and purge the system until high mass peaks
appear.
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High Mass Range Calibration
 To calibrate in the high mass range by using the high mass range calibration solution
1. In the Tune Plus window, click the Calibrate button to open the Calibrate dialog box.
2. For the Mass Range, select the High option.
Figure 98. Calibrate dialog box showing the high mass range calibration page with
PPG 2700 (factory) (LXQ and LTQ XL)
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High Mass Range Calibration
Figure 99. Calibrate dialog box showing the high mass range calibration page (Velos Pro)
3. Under What To Do, select the Calibrate option.
4. Under What to Cal/Check, select all four check boxes: Mass for Turbo,
Mass for Normal, Mass for Zoom, and Isolation Waveforms.
5. (For the LXQ and LTQ XL only) Under Calibration Mass List, in the Name list, select
PPG 2700 (factory) (Figure 98).
6. Specify the instrument state after completing the calibration:
• To switch to Standby mode after the calibration, select the Set Instrument to
Standby when Finished check box.
• To remain in the On mode after the calibration, clear the Set Instrument to Standby
when Finished check box.
7. Click Start.
A message appears:
Please ensure that the 500 microliter syringe is full.
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High Mass Range Calibration
8. Ensure that the syringe contains at least 450 μL of the high mass range calibration
solution, that the syringe pump is on, and that it is set to 5 μL/min.
9. Click OK.
10. Observe the Tune Plus window and the Calibrate dialog box.
While the automatic calibration is in progress, test results appear in the Spectrum and
Graph views and messages appear in the Status box in the Calibrate dialog box. The
system automatically uses the average mass values for the Turbo Scan calibration. It
automatically uses monoisotopic masses for the normal, Zoom Scan, and isolation
waveform calibrations. The results appear when the calibration process finishes.
Cleaning the System After Calibration
After infusing the high mass calibration solution, you must clean the system. For instructions,
see Chapter 8, “Cleaning the Mass Spectrometer After Tuning and Calibrating.”
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G
Glossary
A
B
C
D
E
F
G
H
I
J
K
L M N O
A
Activation Q Directly relates to the rf frequency used
to fragment ions in an ion trap. If the Advanced
Features option is turned off, the default value for
Activation Q is 0.25. Activation Q can be set from
the Instrument Setup window or the Tune Plus
window by using the Advanced Features option.
activation time The time in milliseconds that the rf
used for fragmentation is applied in an ion trap. The
activation time default value is 10 ms for the
Velos Pro mass spectrometer and 30 ms for the LXQ
and LTQ XL mass spectrometers. In general, shorter
activation time results in less fragmentation and a
longer activation time results in more fragmentation.
API ion transfer tube A tube assembly that assists in
desolvating ions that are produced by the ESI, NSI,
or APCI probe.
API ion transfer tube offset voltage A dc voltage
applied to the ion transfer tube. The voltage is
positive for positive ions and negative for negative
ions.
API source The sample interface between the LC and
the mass spectrometer. It consists of the API probe
(ESI, HESI-II, or APCI) and API stack.
API tube lens A lens in the API source that separates
ions from neutral particles as they leave the ion
transfer tube. A potential applied to the tube lens
focuses the ions toward the opening of the skimmer
and helps to dissociate adduct ions.
Thermo Scientific
P
Q
R
S
T
U
V W X
Y
Z
API tube lens offset voltage A DC voltage applied to
the tube lens. The value is normally tuned for a
specific compound.
API tube-skimmer region The area between the tube
and the skimmer, which is surrounded by the tube
lens. It is also the area of first-stage evacuation in the
API source.
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. A reagent gas forms,
which efficiently produces positive and negative ions
of the analyte through a complex series of chemical
reactions.
atmospheric pressure ionization (API) Ionization
performed at atmospheric pressure by using
atmospheric pressure chemical ionization (APCI),
electrospray ionization (ESI), or nanospray
ionization (NSI).
atmospheric pressure photoionization (APPI) A
soft ionization technique that shows an ion generated
from a molecule when it interacts with a photon
from a light source.
Automatic Gain Control™ (AGC) Sets the ion
injection time to maintain the optimum quantity of
ions for each scan. With AGC on, the scan function
consists of a prescan and an analytical scan.
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Glossary
autosampler The device used to inject samples
automatically into the inlet of a chromatograph.
C
collision energy The energy used when ions collide
with the collision gas.
F
full-scan type Provides a full mass spectrum of each
analyte or parent ion. With the full-scan type, the
mass analyzer is scanned from the first mass to the
last mass without interruption. Also known as
singlestage full-scan type.
collision-induced dissociation (CID) A method of
fragmentation where molecular ions are accelerated
to high-kinetic energy and then allowed to collide
with neutral gas molecules such as helium for the
LTQ Series mass spectrometer. The collisions break
the bonds and fragment the ions into smaller pieces.
H
consecutive reaction monitoring (CRM) scan type
high performance liquid chromatography (HPLC)
heated-electrospray ionization (H-ESI) Converts
ions in solution into ions in the gas phase by using
electrospray ionization (ESI) in combination with
heated auxiliary gas.
A scan type with three or more stages of mass analysis
and where a particular multi-step reaction path is
monitored.
conversion dynode A highly polished metal surface
that converts ions from the mass analyzer into
secondary particles, which enter the electron
multiplier.
D
divert/inject valve A valve on the mass spectrometer
that can be plumbed as a divert valve or as a loop
injector.
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
an HCD collision cell. A voltage offset between the
mass analyzer and HCD collision cell accelerates
parent ions into the collision gas inside the HCD
cell, which causes the ions to fragment into product
ions. The product ions are then returned to the mass
analyzer for mass analysis. HCD produces triple
quadrupole-like product ion mass spectra.
I
E
electron multiplier A device used for current
amplification through the secondary emission of
electrons. Electron multipliers can have a discrete
dynode or a continuous dynode.
electrospray ionization (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.
ion detection system The ion detection system is a
high sensitivity, off-axis system for detecting ions. It
produces a high signal-to-noise ratio and allows for
switching of the voltage polarity between positive ion
and negative ion modes of operation. The ion
detection system includes a ±15 kV conversion
dynode and a channel electron multiplier.
ion optics Focuses and transmits ions from the API
source to the mass analyzer.
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.
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Glossary
L
LC pump A high pressure solvent pump in the liquid
chromatograph (LC) that provides the pressure on
the input side of a column to drive the eluent and
sample through the column.
lens A metal disk with a circular hole in the center that
allows the ion beam to pass.
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-to-charge ratio (m/z) An abbreviation used to
denote the quantity formed by dividing the mass of
an ion (in u) by the number of charges carried by the
ion. For example, for the ion C7H72+, m/z = 45.5.
mass analyzer A device that determines the mass-tocharge ratios of ions by one of a variety of techniques.
product ion An electrically charged product of
reaction of a selected parent ion. In general, product
ions have a direct relationship to a particular parent
ion and can correlate to a unique state of the parent
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.
pulsed Q collision-induced dissociation (PQD) A
method of fragmentation where precursor ions are
activated at high Q, a time delay occurs to allow the
precursor to fragment, and then a rapid pulse is
applied to low Q where all fragment ions are trapped.
The product ions can then be scanned out of the ion
trap and detected. PQD fragmentation produces
precise, reproducible fragmentation and has been
used for iTRAQ peptide quantitation on the LTQ by
using both electrospray and MALDI source
ionization. PQD eliminates the “1/3 Rule” low mass
cut-off for MS/MS data.
Q
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.
qualitative analysis Chemical analysis designed to
determine the identity of the components of a
substance.
N
quantitative analysis Chemical analysis designed to
determine the quantity or concentration of a specific
substance in a sample.
nanospray ionization (NSI) A type of electrospray
ionization (ESI) that accommodates very low flow
rates of sample and solvent on the order of 1 to
20 nL/min (for static nanospray) or 100 to
1000 nL/min (for dynamic nanospray).
P
parent ion An electrically charged molecular species
that can dissociate to form fragments. The fragments
can be electrically charged or neutral species. A
parent ion can be a molecular ion or an electrically
charged fragment of a molecular ion. Also called a
precursor ion.
Thermo Scientific
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 quadrupole rods in the mass
analyzer to transmit ions from the lowest mass to the
highest mass of a specified scan range.
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Glossary
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.
ZoomScan scan type, multi-stage A scan type with
three or more stages of mass analysis and where a
particular multi-step reaction path is monitored to
determine the charge-state of one final product ion.
ZoomScan scan type, single-stage A ZoomScan scan
type with one stage of mass analysis and where ions
in one to ten 10-u scan-range windows are
monitored to determine the charge-state of up to ten
ions.
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.
selected reaction monitoring (SRM) scan type A
scan type with two stages of mass analysis and where
a particular reaction or set of reactions, such as the
fragmentation of an ion or the loss of a neutral
moiety, is monitored. In SRM a limited number of
product ions is monitored.
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.
syringe pump A device that delivers a solution from a
syringe at a specified rate.
W
WideBand Activation A type of resonance excitation
in ion traps with a wide range of excitation
frequencies, used during mass analyzer, collisioninduced dissociation. Wideband activation causes
multiple fragmentations of the parent ion, providing
more structural information but less sensitivity.
Z
ZoomScan scan type A scan type that provides
information about the charge state of one or more
ions of interest. ZoomScans are slower scans with
higher resolution than normal scans.
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Index
Numerics
C
8 kV cable locking ring 49
caffeine
calibration solution 143, 151
spectral ion from
high mass range, LXQ and LTQ XL 160, 165
normal mass range, LXQ and LTQ XL 158
testing in ESI mode 69
stock solution 142, 150
calibration
automatic 79
description 13
high mass 155
setup 65
solution, preparing 143, 151
calibration parameters 13
calibration solution
infusing 68
Pierce, ready-to-use 143, 151
preparing for LXQ and LTQ XL 146
preparing for Velos Pro 154
centroid data 15
cleaning. See mass spectrometer
collision energy
optimizing automatically 103
optimizing manually 102
compliance
FCC vii
regulatory iii
WEEE ix
consecutive reaction monitoring (CRM) 19
corona needle
installing 54
removing 59–60
A
acetonitrile 139, 147
APCI mode
description 6
scan parameters 66, 119
setting up parameters for 117
tune method, saving 124
tune, optimizing automatically 120
APCI probe
depth markers 57
installing 54
installing corona needle 53
removing 59
APCI source parameters 118
API modes 4
API probe
cleaning 133
flushing 133
API source housing
drain assembly 38
installing 34
removing 36
auxiliary gas, description 5
B
Breadth Focus in Ion Tree experiment 28
buffers, description 11
D
data
acquiring, in SIM scan type 108, 127
acquiring, prerequisites for 99
reviewing, in Qual Browser window 111, 130
Data Dependent experiment, description 23
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LTQ Series Getting Started Guide
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Index: E
data types
description 15
setting 67, 93, 120
data types, description 15
Depth Focus in Ion Tree experiment 28
depth markers on probe body 44, 57
direct infusion, description 7
divert/inject valve
connecting to the LC tee union 88
connecting to waste container 89
documentation, related xx
drain, API source housing 38
H
HCD. See Trap-HCD license
HESI-II probe
depth markers 44
installing 40
removing 47
high mass range calibrant
LXQ and LTQ XL 145
Velos Pro 153
high mass range calibration 155
high-flow infusion
description 8
setting up 115
E
electromagnetic compatibility vii
electrospray ionization. See ESI mode
EMC compliance iii
ESI mode
description 5
setting up parameters for 92
setting up the API source 40
ESI or HESI-II probe, removing 47
ESI probe
depth markers 43
installing 40
removing 47
ESI source parameters 66
experiment types
Data Dependent 23
General MS 21
Ion Mapping 26
Ion Tree 28
MSn 21
F
FCC compliance vii
flow rates, setting up 11
flow-injection analysis
description 9
setting up 126
forepumps 39
full scan type
single-stage 17
two-stage 17
G
gas flow rates, adjusting for LC flow rate 12
Graph view, displaying 96, 122
grounding union, connecting to ESI probe sample inlet 91
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inlet plumbing, setting up 85, 114
instrument setup 20
Ion Max and Ion Max-S ion source housings, description 34
ion polarity mode, specifying 93, 120
ion polarity modes 14
ion source
APCI probe, installing and removing 53
ESI or HESI-II probe, installing and removing 40
ion sweep cone
cleaning 136–137
description 4
removing 136
ion transfer tube
and signal quality 14
cleaning 137
installing 137
removal tool 137
removing 136
Ion Tree experiment
Breadth Focus, description 28
Depth Focus, description 28
Ion Tree experiments
Breadth Focus, description 28
Depth Focus, description 28
isolation width, optimizing 100
L
LC tee union
connecting to the divert/inject valve 88
connecting to the ion source 90
LC/APCI/MS operational guidelines 13
LC/ESI/MS operational guidelines 12
locking ring, 8 kV cable 49
Thermo Scientific
Index: M
loop injection
and liquid chromatography description 9
setting up 106, 126
LTQ mass spectrometer, note xix, 1
LTQ Velos mass spectrometer, note xix, 1
M
mass analysis
single-stage (qualitative) 17
two-stage 17
mass spectrometer
cleaning after tuning and calibration 131
features 2
flow rates, setting up 11
introducing sample 7
modes
polarity 14
power 33
scan 15
scan types 16
setting up for APCI mode operation 117
setting up for ESI mode operation 92
mass spectrum, viewing
high mass range 160, 165
normal mass range 158
testing in ESI mode 69
methanol 139, 147
micrometer, Ion Max 61
MRFA
stock solution 142, 150
stock solution, diluted 150
MRFA, mass-to-charge ratio
high mass range 160, 165
normal mass range 158
testing in ESI mode 69
MS scan mode 16
MS/MS and MSn scan mode 16
MS/MS and MSn, description 3
MSn scan, consecutive reaction monitoring 19
N
N-butylamine
stock solution 151
Neutral Loss Ion Mapping experiment, description 26
P
polarity mode, setting 67
product ions 15
profile data 15
Thermo Scientific
Q
Qual Browser window 111, 130
qualitative experiments 21
quantitation experiments 21
R
reagents 139, 147
red alignment markers on probe body 51–52
regulatory compliance iii
reserpine
sample solution 145
sample solution, preparing 153
stock solution 144
stock solution, preparing 152
tuning solution 144
tuning solution, preparing 152
S
safety standards iii
sample formulations 139, 147
sample introduction, for calibration and tuning 14
sample transfer line, flushing 133
sample tube, flushing 133
samples, introduction techniques 7
scan parameters
APCI mode 66, 119
LC/ESI/MS mode 92
scan power 15
scan types
full scan 17
selected ion monitoring (SIM) 18
selected reaction monitoring (SRM) 18
single-stage full scan 17
two-stage full scan 17
ZoomScan 19
selected ion monitoring (SIM), description 18
selected reaction monitoring (SRM), description 18
sheath gas, description 4
signal quality
APCI 113, 120
effect of ion transfer tube 14
ESI 14, 83
SIM scan type, acquiring data in 108, 127
single-stage mass analysis, description 2
solvent waste system 38
solvents
acetonitrile 139, 147
contamination of 143, 151
methanol 139, 147
recommendations 139, 147
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Index: T
Spectrum view, displaying 69, 156
spray cone, cleaning 137
Standby mode 33
stock solutions
caffeine, for LXQ and LTQ XL 150
N-butylamine, for Velos Pro 151
PPG, for LXQ and LTQ XL 146
PPG, for Velos Pro 154
sodium acetate, for LXQ and LTQ XL 146
sodium acetate, for Velos Pro 154
Ultramark 1621, for LXQ and LTQ XL 151
sweep cone. See ion sweep cone
sweep gas, description 4
syringe pump
plumbing 64, 86
setting up for tuning and calibrating 86
W
waste container 36, 38, 89
water, purity requirements 139, 147
WEEE compliance ix
Wideband Activation, description 3
Z
ZoomScan, description 3, 19
T
Total (or full scan) Ion Mapping experiment, description 26
Trap-HCD license xxi, 16, 21
tube temperature, adjusting for LC flow rate 12
tune method
description 13
for APCI mode 118
for ESI mode 66
opening saved 92, 117
saving 78, 98, 124
tune parameters, description 13
tuning
automatic 13
automatically in ESI/MS mode 74
description 13
optimizing for target analyte 95
semi-automatic 13
setup 65
U
Ultramark 1621
calibration solution, for LXQ and LTQ XL 143
calibration solution, for Velos Pro 151
spectral ions from
high mass range 160, 165
normal mass range 158
testing in ESI mode 69
stock solution 143, 151
V
vaporizer temperature, adjusting for LC flow rate 12
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