Thermo Scientific LTQ XL Getting Started

LTQ XL ®

Getting Started

97355-97042 Revision A June 2006

For Research Use Only
Not for use in Diagnostic Procedures

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Printing History: Revision A printed June 2006.
Software Revision: LTQ 2.2, Xcalibur 2.2

Regulatory Compliance

Thermo Electron San Jose performs complete testing and evaluation of its products to ensure full compliance
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pertinent electromagnetic compatibility (EMC) and safety standards as described below.

EMC Directive 89/336/EEC

• EMC compliance has been evaluated by TUV Rheinland of North America, Inc.

EN 55011 1999 EN 61000-4-3 2002 EN 55011 1998

IEC 61000-4-3 A1-1998

EN 61000-3-2 1995, A1; 1998,

A2; 1998, A14;
2000

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IEC 61000-4-4

IEC 61000-4-5 2005

IEC 61000-4-6 2004

1995, A1; 2001, A2;
2001;
A2-1995

2002

EN 61000-3-2 1995, A1; 1998,

A2; 1998, A14;
2000

Low Voltage Safety Compliance

This device complies with Low Voltage Directive EN 61010-1:2001.

Changes that you make to your system may void compliance with one or more of these EMC and safety
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FCC Compliance Statement

THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES. OPERATION IS
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INTERFERENCE RECEIVED, INCLUDING INTERFERENCE THAT MAY CAUSE
UNDESIRED OPERATION.

CAUTION: Read and understand the various precautionary notes, signs, and symbols
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Contents

Preface .............................................................................................xi

About This Guide ...................................................................... xi

Related Documentation .............................................................xi

Safety and Special Notices..........................................................xi

Contacting Us...........................................................................xii

Assistance...............................................................................xii

Changes to the Manual and Online Help...............................xii

Chapter 1 Introduction..........................................................................................1

Why Use the LTQ XL MS Detector?..........................................2

Which MS Detector Technique—ESI or APCI—Is Better for

Analyzing My Samples?............................................................4

Using ESI/MS..........................................................................4

Using APCI/MS.......................................................................5

Should I Use Sheath, Auxiliary, and/or Sweep Gases?..................7

How Can I Introduce My Samples into the MS Detector?..........8

What Types of Buffers Should I Use? What Types Should I

Avoid?....................................................................................10

How Should I Set Up the MS Detector for Various LC Flow

Rates?.....................................................................................11

What is Tuning and Calibration of the MS Detector All About?...

13
What Types of Experiments Can I Perform with the LTQ XL MS

Detector?................................................................................16

General MS or MSn Experiments ..........................................16

Data-Dependent Experiments................................................17

Ion Mapping Experiments .....................................................20

Ion Tree Experiments.............................................................23

Chapter 2 Setting Up the Ion Source for Tuning and Calibrating the MS

Detector27

Placing the LC/MS System in Standby......................................28

Removing the APCI Probe........................................................29

Removing the Ion Max Ion Source Housing .............................33

Installing the Ion Sweep Cone...................................................34

Installing the Ion Max Ion Source Housing ..............................36

Installing the ESI Probe ............................................................39

Thermo Electron Corporation LTQ XL Getting Started vii

Contents

Chapter 3 Tuning and Calibrating Automatically in the ESI/MS Mode.... 43

Setting Up the Syringe Pump for Tuning and Calibration ........45

Setting Up the MS Detector in the Xcalibur Data System for

Tuning and Calibration .........................................................47

Testing the Operation of the MS Detector in the ESI/MS Mode..

52

Tuning the MS Detector Automatically in the ESI/MS Mode ..56

Saving Your ESI/MS Tune Method ..........................................60

Calibrating the MS Detector Automatically ..............................62

Cleaning the MS Detector after Tuning and Calibrating...........65

Chapter 4 Tuning with Your Analyte in LC/ESI/MS Mode........................... 69

Setting Up to Introduce Sample by Syringe Pump into Solvent

Flow from an LC ...................................................................71

Setting Up to Tune the MS Detector with Your Analyte...........74

Optimizing the MS Detector Tune Automatically with Your

Analyte...................................................................................77

Saving the ESI/MS Tune Method.............................................81

Chapter 5 Acquiring ESI Sample Data Using the Tune Plus Window ...... 83

Setting Up to Acquire MS/MS Data in the Full Scan Type.......84

Optimizing the Isolation Width and Setting Up to Optimize

the Collision Energy............................................................84

Optimizing the Collision Energy Automatically for an MS/MS

Experiment .........................................................................89

Setting Up to Introduce Sample by Loop Injection into Solvent

Flow from an LC ...................................................................92

Acquiring MS Data in the SIM Scan.........................................94

Chapter 6 Setting Up the Ion Source for Acquiring Data in APCI/MS/MS

Mode101

Removing the ESI Probe .........................................................102

Removing the Ion Max Ion Source Housing ...........................105

Removing the Ion Sweep Cone ...............................................106

Installing the Corona Needle ..................................................107

Installing the Ion Max Ion Source Housing ............................108

Installing the APCI Probe .......................................................111

Chapter 7 Optimizing the MS Detector with Your Analyte in APCI/MS

Mode115

Setting Up the Inlet for Tuning Using High-Flow Infusion....116

Setting Up the MS Detector for APCI/MS Operation ............119

viii LTQ XL Getting Started Thermo Electron Corporation

Contents

Optimizing the Tune of the MS Detector Automatically in

APCI/MS Mode...................................................................123

Saving the APCI/MS Tune Method........................................126

Cleaning the MS Detector after Tuning in APCI Mode..........128

Chapter 8 Acquiring APCI Sample Data Using the Tune Plus Window..131

Setting Up to Introduce Sample by Loop Injection into Solvent

Flow from an LC .................................................................132

Acquiring APCI Data in the SIM Scan Mode .........................134

Appendix A Sample Formulations......................................................................139

Caffeine, MRFA, and Ultramark 1621 Stock Solutions ..........140

Stock Solution: Caffeine.......................................................141

Stock Solution: MRFA.........................................................142

Stock Solution: Ultramark 1621 ..........................................142

ESI Calibration Solution: Caffeine, MRFA, Ultramark 1621..143

Reserpine ................................................................................144

Stock Solution: Reserpine ....................................................144

Reserpine Tuning Solution and Reserpine APCI Sample

Solution ............................................................................144

Appendix B LTQ XL High Mass Range Calibration .........................................147

High Mass Range Calibration Solution...................................148

Normal Mass Range Calibration .............................................149

Enter Normal Mass Range Data into Tune Plus .....................150

Tune on m/z 524.3 .................................................................152

High Mass Range Calibration Procedure.................................154

Notes ......................................................................................159

Index..................................................................................................161

Thermo Electron Corporation LTQ XL Getting Started ix

Preface

About This Guide Welcome to the Thermo Electron, LTQ XL™ system. The LTQ XL is a

member of the Thermo family of MS detectors.

This LTQ XL Getting Started manual provides information on how to set up,
calibrate, and tune the LTQ XL MS detector, and how to acquire LC/MS
data. All of these procedures can be performed from the Xcalibur® Tune Plus
window.

To perform analyses in ESI mode, see Chapters 2, 3, 4, and 5. To perform
analyses in APCI mode, see Chapters 2, 3, 6, 7, and 8.

Related

Documentation

Safety and Special

Notices

In addition to this guide, Thermo Electron provides the following
documents for the LTQ XL system:

• LTQ XL Preinstallation Guide

• LTQ XL Getting Connected

• LTQ XL Hardware Manual

Help is also available from within the software.

Make sure 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 laser-related hazards to human beings. It includes
information specific to the class of laser involved. Each DANGER notice
is accompanied by the international laser radiation symbol.

CAUTION Highlights hazards to humans, property, or the environment.
Each CAUTION notice is accompanied by an appropriate CAUTION
symbol.

Thermo Electron Corporation LTQ XL Getting Started xi

Preface

Contacting Us

IMPORTANT Highlights information necessary to avoid damage to
software, loss of data, invalid test results, or information critical for
optimal performance of the system.

Note Highlights information of general interest.

Note Helpful information that can make a task easier.

Contacting Us There are several ways to contact Thermo Electron.

Assistance For new product updates, technical support, and ordering information,

contact us in one of the following ways:

Visit Us on the Web

Changes to the Manual

and Online Help

www.thermo.com/finnigan

Contact Technical Support

Phone: [phone]
Fax: [phone]

[e-mail]

Contact Customer Service

In the US and Canada for ordering information:
Phone: [phone]
Fax: [phone]

International contacts for ordering information:
Visit www.thermo.com/finnigan for the current listing,

To suggest changes to this guide or to the Help, use either of the following
methods:

• Fill out a reader survey online at www.thermo.com/lcms-techpubs

• Send an e-mail message to the Technical Publications Editor at

[e-mail]

xii LTQ XL Getting Started Thermo Electron Corporation

Chapter 1 Introduction

The LTQ XL™ is a member of the Thermo family of MS detectors. The
LTQ XL MS detector is an advanced analytical instrument that includes a
syringe pump, a divert/inject valve, an atmospheric pressure ionization
(API) source, an MS detector, and the Xcalibur data system. In a typical
analysis, a sample can be introduced in any one of the following ways:

• Using the syringe pump (direct infusion)

• Using the inject valve fitted with a loop and an LC pump (flow injection
analysis)

• Using a valve and an LC system fitted with a column (LC/MS)

In analysis by LC/MS, a sample is injected onto an LC column. The sample
is then separated into its various components. The components elute from
the LC column and pass into the MS detector where they are analyzed.
Analysis by direct infusion or flow injection provides no chromatographic
separation of components in the sample before it passes into the MS
detector. The data from the MS detector is then stored and processed by the
Xcalibur data system.

This introduction answers the following questions:

• Why Use the LTQ XL MS Detector?

• Which MS Detector Technique—ESI or APCI—Is Better for Analyzing

My Samples?

• How Can I Introduce My Samples into the MS Detector?

• What Types of Buffers Should I Use? What Types Should I Avoid?

• How Should I Set Up the MS Detector for Various LC Flow Rates?

• What is Tuning and Calibration of the MS Detector All About?

• What Types of Experiments Can I Perform with the LTQ XL MS

Detector?

Thermo Electron Corporation LTQ XL Getting Started 1

1

Introduction

Why Use the LTQ XL MS Detector?

Why Use the LTQ XL

MS Detector?

The attribute that sets the LTQ XL MS detector apart from other LC
detectors is the high level of analytical specificity that it provides. The LTQ
XL MS detector can provide multiple levels of analysis. Each level of analysis
adds a new dimension of specificity for positive compound identification.
The various levels of analysis are as follows:

• Chromatographic separation and compound detection (non MS
technique utilizing chromatographic retention time)

• Mass analysis (molecular mass information)

• Two-stage mass analysis, MS/MS (structural information)

• Multiple MS-MS mass analysis, MSn (structural information)

• ZoomScan™ analysis (charge state information)

Chromatographic separation and compound detection can be obtained by
all LC/detector systems. 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, even if a
compound is identified correctly by retention time, quantitation results can
be in error because other compounds in the sample might co-elute with the
compound of interest.

Single stage mass analysis allows for the identification of analytes of interest.
Atmospheric pressure ionization typically produces mass spectra that
provide molecular mass information.

Two-stage mass analysis allows for even more positive compound
identification. MS/MS analysis monitors how a parent ion fragments when
exposed to an additional stage of ionization. There are two types of MS/MS
analysis: Full Scan MS/MS and Selective Reaction Monitoring (SRM). Full
Scan MS/MS monitors the production of all product ion from a specific
parent ion. SRM MS/MS analysis monitors a specific reaction path: the
production of a specific product ion from a specific parent ion. Using
MS/MS analysis, you can easily quantitate 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, quantitative target
compound analysis is easily accomplished using the LTQ XL MS detector.

Multiple MS/MS mass analysis provides a unique capability to obtain
structural information that can be useful in structure elucidation of

n

metabolites, natural products, and sugars. MS

techniques on the LTQ XL
MS detector allow for stepwise fragmentation pathways, making
interpretation of MSn spectra relatively straightforward. The LTQ XL MS

2 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

Why Use the LTQ XL MS Detector?

detector has several advanced features that make its MSn capabilities
extremely powerful for qualitative analysis. (See “What Types of

Experiments Can I Perform with the LTQ XL MS Detector?” on page 16.)

ZoomScan analysis provides information about the charge state of one or
more mass ions of interest. ZoomScan data is collected by using slower scans
at higher resolution. This allows for unambiguous determination of charge
state, which in turn allows for the correct determination of molecular mass.

In addition to the aforementioned levels of analysis, there is an additional
technique called Wideband Activation. The Wideband Activation option
allows the LTQ XL MS detector to apply collision energy to ions during
MS/MS fragmentation over a fixed mass range of 20 u. This option allows
the LTQ XL MS detector to apply collision energy to both the parent ion, as
well as to product ions created as a result of non-specific losses of water
(18 u) or ammonia (17 u), for example, or to product ions formed from the
loss of fragments less than 20 u. When you want enhanced structural
information and you do not want to perform MS3 analysis with the LTQ XL
MS detector, choose the Wideband Activation option for qualitative
MS/MS. Because the collision energy is applied to a broad mass range,
signal sensitivity is somewhat reduced when you choose this option.
Therefore, increase the value of the collision energy (Activation Amplitude)
to compensate somewhat for the reduction of sensitivity.

Thermo Electron Corporation LTQ XL Getting Started 3

1

Introduction

Which MS Detector Technique—ESI or APCI—Is Better for Analyzing My Samples?

Which MS Detector

Technique—ESI or

APCI—Is Better for

Analyzing My

Samples?

Using ESI/MS The ESI mode typically produces mass spectra consisting of multiply

The LTQ XL MS detector includes two standard atmospheric pressure
ionization source probes:

• Electrospray ionization (ESI) probe

• Atmospheric pressure chemical ionization (APCI) probe

Typically, more polar compounds such as amines, peptides, and proteins are
best analyzed by ESI, and nonpolar compounds such as steroids are best
analyzed by APCI.

Sample ions can carry a single charge or multiple charges. The number of
charges carried by the sample ions depends on the structure of the analyte of
interest, the mobile phase, and the ionization mode.

charged ions (for proteins and peptides) depending on the structure of the
analyte and the solvent. For example, the resulting mass spectrum of a
higher molecular mass protein or peptide typically consists of a distribution
of multiply charged analyte ions. The resulting mass spectrum can be
mathematically manipulated to determine the molecular mass of the sample.

1

The ESI mode transfers ions in solution into the gas phase. Many samples
that previously were not suitable for mass analysis (for example, heat-labile
compounds or high molecular mass compounds) can be analyzed by ESI.
ESI can be used to analyze any polar compound that makes a preformed ion
in solution. The term preformed ion can include adduct ions. For example,
polyethylene glycols can be analyzed from a solution containing ammonium
acetate, because of adduct formation between the NH
and oxygen atoms in the polymer. With ESI, the range of molecular masses
that can be analyzed by the LTQ XL MS detector is greater than 100,000 u,
due to multiple charging. ESI is especially useful for the mass analysis of
polar compounds, which include: biological polymers (for example,
proteins, peptides, glycoproteins, and nucleotides); pharmaceuticals and
their metabolites; and industrial polymers.

You can use the ESI mode in either positive or negative ion polarity mode.
The ion polarity mode is determined by the polarity of the preformed ions
in solution: Acidic molecules form negative ions in high pH solution, and
basic molecules form positive ions in low pH solution. A positively charged
ESI needle is used to generate positive ions and a negatively charged needle
is used to generate negative ions.

+

ions in the solution

4

1

Optional ionization sources [atmospheric photo ionization (APPI), atmospheric pressure matrix

assisted laser desorption ionization (AP MALDI), and nanospray] are also available.

4 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

Which MS Detector Technique—ESI or APCI—Is Better for Analyzing My Samples?

You can vary the flow rate from the LC into the MS detector over a range
from 1 μL/min to 1000 μL/min. See Ta bl e 3 . (In ESI, the buffer and the
buffer strength both have a noticeable effect on sensitivity. Therefore, it is
important to choose these variables correctly.) In the case of higher
molecular mass proteins or peptides, the resulting mass spectrum consists
typically of a series of peaks corresponding to a distribution of multiply
charged analyte ions.

The ESI process is affected by droplet size, surface charge, liquid surface
tension, solvent volatility, and ion solvation strength. Large droplets with
high surface tension, low volatility, strong ion solvation, low surface charge,
and high conductivity prevent good electrospray.

Mixed organic/aqueous solvent systems that include organic solvents such as
methanol, acetonitrile, and isopropyl alcohol are superior to water alone for
ESI. Volatile acids and bases are good, but salts above 10 mM are not
recommended. Strong mineral acids and bases are extremely detrimental to
the instrument.

The rules for a good electrospray are as follows:

• Keep non-volatile salts and buffers out of the solvent system. For
example, avoid the use of salts containing sodium or potassium and
avoid the use of phosphates. If necessary, use ammonium salts instead.

• Use organic/aqueous solvent systems and volatile acids and bases.

• If possible, optimize the pH of the solvent system for your analyte of
interest. For example, if your analyte of interest contains a primary or
secondary amine, your mobile phase should be slightly acidic
(pH 2 to 5). The acid pH tends to keep positive ions in solution.

Using APCI/MS Like ESI, APCI is a soft ionization technique. 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.

APCI is a 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 is a very robust ionization technique. It is not affected by minor
changes in most variables such as changes in buffer or buffer strength. The
rate of solvent flowing from the LC into the MS detector in APCI mode is
typically high (between 0.2 and 2 mL/min). See Ta bl e 3 .

Thermo Electron Corporation LTQ XL Getting Started 5

1

Introduction

Which MS Detector Technique—ESI or APCI—Is Better for Analyzing My Samples?

You can use APCI in 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 which generally produce strong negative ions, with acidic
sites such as carboxylic acids and acid alcohols, are an exception to this
general rule.

Although, in general, fewer negative ions are produced than positive ions,
negative ion polarity can be more specific. This is because the negative ion
polarity mode sometimes generates less chemical noise than does the
positive mode. Thus, the signal-to-noise ratio might be better in the
negative ion mode than in the positive ion mode.

6 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

Should I Use Sheath, Auxiliary, and/or Sweep Gases?

Should I Use Sheath,

Auxiliary, and/or

Sweep Gases?

Nitrogen gas can be applied to the system using any combination of the
three gas sources: Auxiliary gas, Sweep gas, and/or Sheath gas. When Sheath
gas is used, nitrogen is applied as an inner coaxial gas (when used in tandem
with Auxiliary gas), helping to nebulize the sample solution into a fine mist
as the sample solution exits the ESI or APCI nozzle. (Sheath gas is not used
with the NSI source.) When Auxiliary gas is being used, nitrogen flows
through the ion source nozzle, the vapor plume is affected; the spray is
focused and desolvation is improved. When Sweep gas is used, the nitrogen
flows out from behind the sweep cone and can result in solvent declustering
and adduct reduction.

When you are analyzing complex matrices such as plasma or nonvolatile salt
buffers, Sweep gas is required for ruggedness. In full-scan MS or data
dependent scan experiments, the signal-to-noise ratio can be improved by
application of Sweep gas. In some cases, signal intensity can be increased by
using Auxiliary gas, particularly for higher LC flow rates.

All analyses are analyte dependent and require separate optimization with
Sheath gas, Sweep gas, and Auxiliary gas to determine which combination
will yield optimum performance. It is especially important to optimize with
each gas independently before you perform experiments using MS

techniques and before you perform any quantitative analysis experiments

because optimum results could be achieved with any combination of
Sheath, Sweep, and/or Auxiliary gas. See Ta bl e 2 and Tab le 3 for additional
information on using supplemental gas flows.

n

Thermo Electron Corporation LTQ XL Getting Started 7

1

Introduction

How Can I Introduce My Samples into the MS Detector?

How Can I Introduce
My Samples into the

MS Detector?

You can introduce your samples into the MS detector in a variety of ways.
Refer to Ta bl e 1 .

The syringe pump is often used to introduce 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 example, for determining the structure of an unknown
compound.

You can also use a Tee union to direct samples from the syringe pump into
an LC flow (without a column), which then enters the MS detector. This
technique is used to introduce sample at a steady rate and at higher solvent
flow rates; it is used especially for tuning in ESI or APCI on an analyte of
interest. You can also use this technique to introduce a solution of pure
analyte at a steady rate in ESI or APCI.

You can introduce samples from a syringe into the loop of the injector valve.
You can then use the divert valve to introduce the sample into an LC flow,
which then enters the MS detector. This technique is used in ESI or APCI
to introduce pure analytes into the MS detector in a slug. It is useful when
you have a limited quantity of pure analyte.

You can also use an LC autosampler to introduce samples into an LC flow.
This technique is also used in ESI or APCI to introduce a slug of pure
analyte into the LC flow and then into the MS detector.

Finally, you can perform LC/MS experiments by using an LC autosampler
to introduce a mixture onto an LC column. This technique is used with ESI
or APCI to separate the analytes before they are introduced sequentially into
the MS detector.

You can refer to subsequent chapters in this manual and to LTQ XL Getti n g
Connected for plumbing diagrams and methods of sample introduction.

Table 1. Sample introduction techniques

Syringe Pump
Flow
(no LC Flow)

Sample Introduction
Technique

Syringe pump* ESI automatic tuning

Analytical
Technique

and calibrating

ESI analysis of a
pure analyte solution

Figure Reference

LTQ XL Getting
Started

Figure 2-5

8 LTQ XL Getting Started Thermo Electron Corporation

How Can I Introduce My Samples into the MS Detector?

Table 1. Sample introduction techniques

1

Introduction

LC Flow Without
Chromatographic
Separation
(no column)

LC Flow With
Chromatographic
Separation

Sample Introduction
Technique

Syringe pump into LC flow
(connected by Tee union)*

Loop injection into LC flow ESI or APCI analysis

Autosampler injection into
LC flow
(one or multiple injections)

Autosampler injections
into LC column via LC flow
(one or multiple injections

Analytical
Technique

ESI or APCI
automatic
optimization of
tuning on analyte of
interest

ESI or APCI analysis
of a pure analyte
solution

of a pure analyte
solution

ESI or APCI analysis
of a pure analyte
solution

ESI or APCI analysis
of mixtures

Figure Reference

LTQ XL Getting
Started

Figure 4-1 (ESI)
Figure 6-1 (APCI)

LTQ XL Getting
Started

Figure 5-6 (ESI)
Figure 8-1 (APCI)

LTQ XL Getting
Connected

Figure 11-5 (ESI)
Figure 11-8 (APCI)

*Provides steady state introduction of sample (direct infusion)

Thermo Electron Corporation LTQ XL Getting Started 9

1

Introduction

What Types of Buffers Should I Use? What Types Should I Avoid?

What Types of Buffers

Should I Use? What

Types Should I Avoid?

Many LC applications use nonvolatile buffers such as phosphate and borate
buffers. It is best to avoid the use of nonvolatile buffers with the MS
detector because they can cause the following problems:

• Blocking the capillary in the probe

• Causing salt buildup on the spray head and thus compromising the
integrity of the spray

Use volatile buffers when you use the MS detector. Many volatile buffer
solutions are available that can be used instead of nonvolatile ones. Volatile
buffer solutions can include the following:

• Acetic acid

• Ammonium acetate

• Ammonium formate

• Ammonium hydroxide

• Triethylamine (TEA)

• Trifluoroacetic acid

10 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

How Should I Set Up the MS Detector for Various LC Flow Rates?

How Should I Set Up

the MS Detector for

Various LC Flow

Rates?

The ESI probe can generate ions from liquid flows2 of 1 μL/min to

1.0 mL/min. This flow rate range allows you to use a wide range of
separation techniques: CE, CEC, capillary LC, microbore LC, and
analytical LC.

The APCI probe can generate ions from liquid flows3 of 200 μL/min to

2.0 mL/min. This flow range allows you to use microbore LC, analytical
LC, and semi-preparative LC.

As you change the rate of flow of solvents entering the MS detector, you
need to adjust several of the MS detector parameters, as follows:

For ESI, you need to adjust the capillary temperature and adjust the gas flow
rates for the Sheath, Auxiliary, and/or Sweep gas.

For APCI, you need to adjust the capillary temperature and vaporizer
temperature and adjust the gas flow rates for the Sheath, Auxiliary, and/or
Sweep gas.

In general, an increase in the rate of liquid flowing into the MS detector,
requires a higher temperature of the ion transfer capillary (and vaporizer)
and the higher gas flow rate.

Ta bl e 2 provides guidelines for ESI operation for ion transfer capillary

temperatures and gas flow rates for various LC solvent flow rates.

Ta bl e 3 provides guidelines for APCI operation for the ion transfer capillary

temperature, vaporizer temperature, and gas flow rate for a range of LC
solvent flow rates.

2

The ESI probe can generate ions from liquid flows of 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.

3

For the APCI probe, flows below 200 μL/min require more care to maintain a stable spray.

Thermo Electron Corporation LTQ XL Getting Started 11

1

Introduction

How Should I Set Up the MS Detector for Various LC Flow Rates?

Table 2. Guidelines for setting operating parameters for LC/ESI/MS

Ion
Transfer
Capillary
Temperatu
re

setting:
150 to 200

°C

setting:
200 to 275

°C

setting:
250 to 350

°C

Sheath
Gas

Not
required

Typica l
setting:
5 to 15 units

Required

Typica l
setting:
20 to 40
units

Required

Typica l
setting:
40 to 60
units

Auxiliary and/or Sweep
Gas

Not required

Typical setting:
0 units

Not required, but might
help depending on
conditions

Typical setting:
0 to 20 units

Not required, but usually
helps to reduce solvent
background ions

Typical setting:
0 to 20 units

LC Flow Rates

Infusion or LC at
flow rates of <
10 μL/min

LC at flow rates
from 50 to
200 μL/min

LC at flow rates
from 100 to
500 μL/min

Suggeste
d Column
Size

Capillary Typ ic al

1 mm ID Ty pi ca l

2 to 3 mm IDTypi ca l

*

LC at flow rates
from 0.4 to
1mL/min

*

Note: Be sure to choose either Auxiliary gas and/or Sweep gas according to the hints in

Should I Use Sheath, Auxiliary, and/or Sweep Gases?

Table 3. Guidelines for setting operating parameters for LC/APCI/MS

LC Flow
Rate

LC at flow
rates from

0.2 to
2mL/min

*

Note: Be sure to choose either Auxiliary gas and/or Sweep gas according to the hints in Should

I Use Sheath, Auxiliary, and/or Sweep Gases?

4.6 mm ID Typ ic al

Ion Transfer
Capillary
Temperatur
e

Typi ca l
setting:
150 to
225

°C

setting:
300 to 400

°C

Vaporizer
Temperatur
e

Typi cal
setting:
400 to
550

°C

Required

Typica l
setting:
60 to 100
units

Sheath Gas

Required

Typi ca l
setting:
40 to 100
units

Required

Typical setting:
10 to 40 units

*

Auxiliary and/or Sweep
Gas

Not required, but usually
helps to reduce solvent
background ions

Typical setting:
0 to 20 units

12 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

What is Tuning and Calibration of the MS Detector All About?

What is Tuning and

Calibration of the MS

Detector All About?

To optimize the performance of data acquisition on the LTQ XL MS
detector, tune and calibrate in four steps:

• In ESI mode, you infuse a calibration solution into the MS detector at a
steady rate of 5 μL/min for several minutes. In Tune Plus, you observe
the signal at m/z 195, the mass-to-charge ratio of caffeine in the
calibration solution. Then, while observing the signal at m/z 195, you
adjust probe positions and gas flows to achieve the greatest signal
strength while still maintaining a stable spray of ions into the MS
detector.

• Once you have established a stable spray of ions into the MS detector,
tune the MS detector. In this step, you use the automatic tuning
procedure in Tune Plus to ensure that the transmission of ions into the
MS detector is optimum. You observe the Tune Plus window as the
Xcalibur data system tunes your LTQ XL MS detector automatically.

• After your tune method is optimized, calibrate the MS detector. In this
step, you want to ensure that the calibration parameters complete
automatic calibration successfully. The Calibrate dialog box in Tune
Plus provides a readback of the status of the calibration parameters, both
during the automatic calibration and when calibration is complete.

• Lastly, if you want to maximize the detection of one or more particular
ions, you can optimize the tune of the MS detector with your analyte of
interest in the ionization mode that you are going to use to analyze your
samples. You choose a mass-to-charge ratio of your analyte of interest.
Alternatively, you can choose an ion in the calibration solution that is
closest to the mass-to-charge ratio for your ion of interest. (It is
sometimes possible to acquire qualitative data without optimizing the
parameters, but detection sensitivity might be compromised.)

Calibration parameters are instrument parameters whose values do not vary
with the type of experiment. It is recommended that you calibrate the MS
detector at least once every three months and that you check the calibration
about once a week.

Automatic and semi-automatic calibration (including checking the
calibration) require that you introduce calibration solution into the MS
detector at a steady flow rate while the procedure is running. You introduce
the solution directly from the syringe pump into the MS detector in the
ESI/MS mode.

Thermo Electron Corporation LTQ XL Getting Started 13

1

Introduction

What is Tuning and Calibration of the MS Detector All About?

Tune parameters are instrument parameters whose values can vary with the
type of experiment. For example, if your experiment requires quantitative
data on one or more particular ions, you need to tune the MS detector with
your analyte if you change any one of the parameters specific to the
experiment or analyte.

Automatic and semi-automatic tuning procedures (including optimizing the
collision energy) require that you introduce calibration solution, or a tuning
solution of your analyte of interest, into the MS detector at a steady rate in
either of two ways:

• Introduce the solution directly from the syringe pump. See Setting Up

• Introduce the sample from the syringe pump into the effluent of the LC

the Syringe Pump for Tuning and Calibration in Chapter 3: “Tuning
and Calibrating Automatically in the ESI/MS Mode”.

by using a Tee union. See Setting Up to Introduce Sample by Syringe

Pump into Solvent Flow from an LC in Chapter 4: “Tuning with Your
Analyte in LC/ESI/MS Mode”.

Use the first method for tuning if you intend to use an experiment type at a
low flow rate involving the syringe pump. The second method is useful if
you intend to use an experiment type at a higher flow rate involving the LC.
However, the second method of introduction puts a comparatively large
amount of analyte into the MS detector. Therefore, before you can perform
an analytical run to analyze for the analyte, you might need to clean the API
spray shield.

CAUTION Do not use the calibration solution at flow rates above 10
μL/min. Ultramark 1621 can contaminate your system at high
concentrations.

In most cases, you can use the tune you obtain from the automatic or
semi-automatic tuning procedures for your analytical experiments.
However, for some applications, you might need to tune several MS detector
parameters. In that case, you would tune manually. With the manual tuning
process, you introduce a tuning solution at a steady flow rate.

Note The most important parameters that affect the signal quality
during ESI/MS operation are the ion transfer capillary temperature, tube
lens voltage, gases, and solution flow rate. For optimum sensitivity, tune
with the instrument in the same operational mode as the mode you use
for the analytical experiment.

14 LTQ XL Getting Started Thermo Electron Corporation

What is Tuning and Calibration of the MS Detector All About?

Ta bl e 4 summarizes methods of sample introduction for each of the

calibration and tuning procedures.

Table 4. Summary of methods of sampleintroduction for calibration and tuning

Calibrating Tuni ng

1

Introduction

Sample/
Sample Intro

Calibration solution/
Syringe pump

Your tune solution/
Syringe pump

Your tune solution/
Syringe pump into LC flow
by using Tee union

Check Auto

Semi­auto

Auto

Semi­auto

Manual

9 9 9 9 9 9 9

9 9 9 9

9 9 9 9

Collision
Energy

Thermo Electron Corporation LTQ XL Getting Started 15

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

What Types of

Experiments Can I

Perform with the LTQ

XL MS Detector?

General MS or MSn

Experiments

This topic describes several types of experiments that you can perform with
the LTQ XL MS detector. The experiments can be grouped into the
following categories:

• General MS or MS

• Data-Dependent™

• Ion Mapping™

• Ion Tree

You can specify which type of experiment you want to perform in the
Instrument Setup window, and then save it in an Instrument Method
(.METH) file.

Note Procedures for these experiments are beyond the scope of this LTQ
XL Getting Started manual. If you need more information, refer to online

Help.

You can use a General experiment to collect qualitative data for structural
analysis. The Xcalibur data system includes an Instrument Method template
in Instrument Setup so you can get started with a General MS or MSn
experiment. For an example of a General MS or MSn experiment template,
see Figure 1

n

In a General MS experiment, you need to specify the mass range of your
analyte(s) of interest. In a General MS/MS experiment, you need to specify a
parent (precursor ion) that fragments into distinctive product ions. In a

n

General MS
the parent ions of interest. The LTQ XL MS detector can then collect data
on the ions in the range or on the product ions of the parent ion(s) that you
specify.

If you use a General experiment to collect data for qualitative (structural)
analysis, you specify the scan mode (MS through MS
data in the Scan Event Settings group box. If you specify MS/MS or MS
you then choose the parent ion(s) for which you want data in the MSn
Settings table. The LTQ XL MS detector can then collect distinct qualitative
information for structural analysis or for spectral reference.

experiment, you need to specify the mass-to-charge ratios of all

n

) for which you want

n

,

16 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

Figure 1. MS Detector Setup page in Instrument Setup, showing a template for a General MS experiment

The LTQ XL MS detector can generate reproducible, analyte-specific
spectra, even from laboratory to laboratory. Consequently, reference spectra
that are generated with the LTQ XL MS detector can be used to confirm
structures of compounds generated with other LTQ XL systems.

Data-Dependent

Experiments

A Data-Dependent experiment is best used for the qualitative analysis of
unknown compounds for structure elucidation or confirmation. The LTQ
XL MS detector uses the information in a data-dependent experiment to
make decisions about the next step of the experiment
automatically—without input from a user. Instrument Setup contains the
Instrument Method templates that you need to get started with
data-dependent experiments. For an example of a data-dependent Triple
Play experiment template, see Figure 2.

Thermo Electron Corporation LTQ XL Getting Started 17

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

A data-dependent experiment produces a great deal of data from a single
sample analysis. You can run a data-dependent experiment even if you know
very little about your sample, and even if you are unfamiliar with the
variables of mass spectroscopy. In a data-dependent experiment, you can
specify parent ions for fragmentation or you can let the LTQ XL MS
detector automatically select the ions for fragmentation. The LTQ XL MS
detector can collect the structural information for every parent ion in the
sample automatically, even if the sample is a mixture of compounds.

A data-dependent experiment requires minimal input from a user about
how the experiment should best proceed. The user specifies that one or
more scan events of an experiment segment are to be run as data-dependent.
Then, the LTQ XL MS detector collects MS/MS or MSn data and makes
decisions about what the next step in the experiment should be to collect
even more data. For example, in a data-dependent Triple Play experiment
for a mixture of compounds, the LTQ XL MS detector can decide which
parent ion to isolate, the charge state of the parent ion, and the molecular
mass of the compound.

Ion Mapping experiments can be data-dependent. (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.

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.

You can approach the setup of data-dependent experiments in either of two
ways:

• If you have some idea of the parent ion, or if you expect a certain kind of
parent, you can set up a list of possible parent ions. Then, when one of
the parent ions you specified is detected, you can acquire product
spectra and analyze the information. Conversely, you can also set up a
list of ions that you do not want to be selected for fragmentation.

18 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

Figure 2. MS Detector Setup page in Instrument Setup, showing a template for a Data-Dependent Triple Play

experiment. (To select a scan event that makes active the Dependent Scan check box, click either the Scan
Event 2 or Scan Event 3 button.

• If you have little information about your 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 LTQ XL MS detector
generates product spectra. Parameters that you might specify, for

n

example, include threshold values for the intensity of the MS or MS

ion
signal. Whatever threshold values you choose should accomplish the
isolation of your parent ions of interest.

You can find useful structural information about your compound
automatically with the simplest data-dependent experiment,
Data-Dependent MS/MS. You specify the MS scan range, and you do not

Thermo Electron Corporation LTQ XL Getting Started 19

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

even need to specify a parent ion. The LTQ XL MS detector can then collect
full scan MS data, pick the most intense parent ion in the spectrum, and
fragment 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 LTQ XL 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
mass. The parent ion is then fragmented into product ions (MS/MS). For
example, if the LTQ XL MS detector determines a charge state equal to 2,
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 m/z 1000 (or 2 ×

500).

Use a data-dependent experiment (from templates in Instrument Setup) to
do the following:

• Identify low-level impurities in high-purity compounds
(Data-Dependent MS/MS)

• Identify metabolites in a complex mixture (Chromatographic Separation
with Data-Dependent MS/MS)

• Build a custom library of composite MSn spectra (Ion Tree)

You can use a Data-Dependent MSn experiment to identify process
impurities. In the quality assurance process for aspirin, for example, the
LTQ XL MS detector can identify impurities of less than 0.1%.

A Data-Dependent MS/MS experiment of a complex mixture of drug
metabolites can provide highly specific structural information.
Characteristic masses along the metabolic pathways of a drug, for example,
can produce MS/MS spectra that are specific to the structure of the drug.
These spectra are essential in metabolite identification.

A data-dependent experiment can produce a composite spectrum of, for
example, MS2, MS3, and MS4 data. The LTQ XL MS detector 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.

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

20 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

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.

The LTQ XL MS detector includes the following Ion Mapping templates in
Instrument Setup so you can get started with an Ion Mapping experiment:

• Total (or full scan) Ion Map

• Neutral Loss Ion Map

• Parent Ion Map

These Ion Mapping experiments, in general, require that sample solution
enter the MS Detector at a composition that is constant throughout.
Therefore, you use infusion to introduce your sample for these Ion Mapping
experiments. See Figure 3 for an example of an Ion Mapping experiment
template.

Thermo Electron Corporation LTQ XL Getting Started 21

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

Figure 3. Total Ion Map page in Instrument Setup, showing a template that contains parameters for an Ion Mapping

experiment

In a Total (or full scan) Ion Mapping experiment, you get product ion scans
for each parent ion, so you can determine which parent ions lost a particular
fragment to yield a particular product ion. Furthermore, you can determine
which parent ions are related to specific product ions. For example, you can
map the spectral peaks in a mass range from m/z 400 to m/z 2000 and
specify to scan 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.

A Neutral Loss Ion Mapping Experiment collects scans for masses that have
lost neutral fragments. As with Full Scan Ion Mapping, you can get product
ion scans on every parent ion. However, a Neutral Loss Ion Map identifies
which parent ions lost a neutral fragment of a particular mass. For example,
you can specify a neutral loss of 80 u (as in the case of a phosphorylated

22 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

peptide in a tryptic digest). A Neutral Loss Ion Mapping experiment can
step through each product mass in the mixture. The experiment searches for
evidence of the loss of a neutral moiety of mass 80 u.

A Parent Ion Mapping experiment identifies all the ions that produce a
particular molecular ion that you specify. For example, if you specify a
product ion mass of m/z 50, a Parent Ion Map includes all the parent ions
that yielded the specified product ion, m/z 50.

A Data-Dependent Zoom Map is an Ion Mapping experiment that collects
ZoomScan data on every scan interval in a mass range that you specify, as
well as Data-Dependent MS/MS product spectra on every mass above an
intensity threshold.

The results of any of the Ion Mapping experiments can be viewed in the
Xcalibur Qual Browser window.

Ion Tree Experiments In an Ion Tree experiment, the LTQ XL MS detector can collect MS

automatically. You can specify a particular parent ion for fragmentation, or
you can let the LTQ XL MS detector find the parent ions automatically and
fragment them to any level between MS2 and MS10. The LTQ XL MS
detector automates the collection of data by deciding what actions need to
occur next for the experiment to progress. See Figure 4 for an example of an
Ion Tree experiment template.

n

data

Thermo Electron Corporation LTQ XL Getting Started 23

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

Figure 4. Data-Dependent Ion Tree page in Instrument Setup, showing a template for an Ion Tree experiment

In an Ion Tree experiment, you can specify either of two options that
prioritize how the LTQ XL MS detector gathers information: depth focus
and breadth focus.

• Depth focus characterizes an ion by performing a series of MSn-level
fragmentations (for example, MS/MS, MS3, MS4, etc.) before
characterizing the next most intense ion in the MSn series.

• Breadth focus characterizes all ions to the same MSn level before

n

advancing to the next MS

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.

24 LTQ XL Getting Started Thermo Electron Corporation

1

Introduction

What Types of Experiments Can I Perform with the LTQ XL MS Detector?

First, with either depth or breadth focus, the LTQ XL MS detector scans for
parent ions (MS) over the specified mass range. The most intense ion of the
MS spectrum is selected for fragmentation (MS/MS).

• Second, if you chose the depth focus, after the most intense ion of the
MS spectrum is fragmented—producing an MS/MS spectrum—the
LTQ XL MS detector 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 LTQ XL MS detector 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 is then repeated for the second most intense
ion in the MS spectrum.

• If you chose the breadth focus, after the most intense ion of the MS
spectrum is fragmented—producing an MS/MS spectrum—the LTQ
XL MS detector selects and fragments the second-most intense ion of
the same MS spectrum. The fragmentation of parent ions continues to
the Max Breadth level that you specified (2, for this example). After the
two most intense peaks on the MS level are fragmented, the LTQ XL
MS detector scans the first MS/MS spectrum to select and fragment the
two most intense ions. This results in product ions on the level of MS3,
the level specified as the maximum depth for this example. This process
is then repeated for the second most intense ion in the MS spectrum.

The results of a Data-Dependent Ion Tree experiment can be viewed in the
Xcalibur Qual Browser window. The results are displayed as a structure tree
that originates from a particular parent ion.

Thermo Electron Corporation LTQ XL Getting Started 25

Chapter 2 Setting Up the Ion Source for

Tuning and Calibrating the
MS Detector

This chapter provides information on setting up the hardware for tuning
and calibrating your LTQ XL MS detector. You tune and calibrate the MS
detector in the ESI mode before you acquire data in either the ESI or APCI
mode.

This chapter contains the following topics:

• Placing the LC/MS System in Standby

• Removing the APCI Probe

• Removing the Ion Max Ion Source Housing (optional)

• Installing the Ion Sweep Cone (optional)

• Installing the Ion Max Ion Source Housing

• Installing the ESI Probe

Thermo Electron Corporation LTQ XL Getting Started 27

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Placing the LC/MS System in Standby

Placing the LC/MS

System in Standby

The LC/MS system needs to be placed in Standby condition before you can
remove the ion source.

Place the LC/MS system in Standby

1. If necessary, stop the flow of solvent to the API source as follows:

a. If the Xcalibur data system is not already open, choose Start >

Programs > Xcalibur > Xcalibur from the Windows® taskbar to
open the Xcalibur window.

b. In the Xcalibur Home Page window – Roadmap view, choose

GoTo > Instrument Setup to open the Instrument Setup window.

c. Click the Surveyor® MS Pump button on the view bar in the

Instrument Setup window to display the Surveyor MS Pump view.

d. Choose Surveyor MS Pump > Direct Control to open the Surveyor

MS Pump Direct Control dialog box.

e. In the Direct Control dialog box, click the Pump Off button to stop

the MS pump.

On Off Standby

2. If Tune Plus is not already open, choose Start > Programs > Xcalibur >
LTQ XLTune from the taskbar to open Tune Plus.

You can determine the state of the MS detector by observing the state of
the On/Standby button on the Control / Scan Mode toolbar. (The three
different states of the On/Standby button are shown at the left.)

3. If the MS detector is On, click the On/Standby button to place the MS
detector in the Standby mode. When the MS detector is in Standby, the
LTQ XL MS detector turns off the ion source sheath gas, auxiliary gas,
and high voltage.

The LC/MS system is now in Standby and it is safe to remove the ion
source.

If the ESI probe is already installed in the Ion Max™ ion source housing,
leave the LC/MS system in Standby and go to Chapter 3: “Tuning and

Calibrating Automatically in the ESI/MS Mode”.

If the ESI probe is not already installed in the Ion Max ion source housing,
go to the next section, Removing the APCI Probe.

28 LTQ XL Getting Started Thermo Electron Corporation

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Removing the APCI Probe

Removing the APCI

Probe

This topic describes how to remove the APCI probe from the Ion Max ion
source housing.

Note The following procedures assume that you are familiar with your
instrument and software. If you need additional guidance, refer to LTQ
XL online Help, LTQ XL Getting Connected, Ion Max API Source
Hardware Manual, or the LTQ XL Hardware Manual.

CAUTION AVOID BURNS. At operating temperatures, the APCI
vaporizer can severely burn you! The APCI vaporizer typically operates
between 400 and 600 °C. Always allow the heated vaporizer to cool to

room temperature (for approximately 20 min) before you touch or
remove this component.

Remove the APCI probe

1. Unplug the vaporizer heater cable from the vaporizer heater cable socket
on the APCI probe. See Figure 5.

2. Disconnect the sample transfer line from the APCI probe. (See

Figure 5.)

3. Remove the auxiliary gas line (green-colored fitting) from the APCI
probe. (Figure 5)

4. Remove the sheath gas line (blue-colored fitting) from the APCI probe.

Thermo Electron Corporation LTQ XL Getting Started 29

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Removing the APCI Probe

Vaporizer
Heater
Cable

Sheath
Gas
Fitting

Sample
Transfer
Line

Auxiliary
Gas
Fitting

8 kV
Cable

Corona
Needle
High
Voltage
Receptacle

Figure 5. Ion Max ion source housing with APCI probe installed

CAUTION AVOID BURNS. At operating temperatures, the APCI

vaporizer can severely burn you! The APCI vaporizer typically operates
between 400 and 600 °C. Always allow the heated vaporizer to cool to

room temperature (for approximately 20 min) before you touch or
remove this component.

5. Remove the APCI probe as follows:

a. Connect the vaporizer heater cable to the ESI interlock socket on

the ion source housing. See Figure 6.

b. Release the probe locking lever to loosen the probe collar. You might

need to unscrew the lever a few turns to permit probe movement.

c. Carefully pull the probe straight back in the port in the housing

until it meets with the slot in the API interlock block. The guide pin
on the probe manifold will prevent you from rotating the probe
until the pin is aligned with the slot in the API interlock block.
Once the probe is all the way back and aligned with the slot, turn
the probe 45 degrees counter-clockwise to free the probe from the
alignment notch.

d. Pull the probe straight out to remove it from the ion source housing.

e. Store the APCI probe in its original shipping container.

30 LTQ XL Getting Started Thermo Electron Corporation

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Removing the APCI Probe

ESI
Interlock
Socket

API
Interlock
Block

Probe
Collar

Probe
Locking
Lever

Door
Locks

Ion Source
Housing
Door

Figure 6. Ion Max ion source housing, detail of components

6. Remove the 8 kV cable from the corona needle high voltage receptacle
as follows:

a. Unlock the cable by rotating the locking ring counter-clockwise.

b. Unplug the 8 kV cable from the corona needle high voltage

receptacle.

CAUTION AVOID INJURY. The corona discharge needle is very sharp
and can puncture your skin. Handle it with care.

7. Remove the corona needle as follows:

a. Unlock the ion source housing door by turning the locks 90 degrees

so that the knobs are horizontal.

b. Open the ion source housing door.

c. Using pliers, grasp the corona needle and pull it straight out of the

corona needle contact. See Figure 7.

Thermo Electron Corporation LTQ XL Getting Started 31

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Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Removing the APCI Probe

d. Close and lock the ion source housing door.

8. Store the corona needle in its original shipping container.

The APCI probe and the corona needle are now properly removed from the
Ion Max ion source housing.

If you want to install the optional ion sweep cone, go to the next section,

Removing the Ion Max Ion Source Housing.

If you do not want to install the ion sweep cone, go to “Installing the ESI

Probe” on page 39.

Figure 7. Corona needle, view from rear

Corona Needle Contact

Corona Needle (grasp this
with pliers to remove it)

32 LTQ XL Getting Started Thermo Electron Corporation

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Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Removing the Ion Max Ion Source Housing

Removing the Ion Max

Ion Source Housing

The Ion Max ion source housing is removed to access the ion sweep cone.

Note If an ion source probe is still installed in the ion source housing,
the external liquid lines should first be disconnected before removing the
ion source housing.

Remove the ion source housing

1. Remove the drain tube from the ion source housing drain. See Figure 8.

2. Rotate the ion source housing locking levers 90 degrees to release the ion
source housing from the ion source mount assembly.

3. Remove the ion source housing by pulling the housing straight off of the
ion source mount assembly

4. Place the ion source housing in a safe location for temporary storage.

The Ion Max ion source housing is now properly removed.

Ion Source
Housing
Locking
Levers

Ion Source
Housing
Drain

Figure 8. Ion Max ion source housing, detail of components

Thermo Electron Corporation LTQ XL Getting Started 33

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Installing the Ion Sweep Cone

Installing the Ion

Sweep Cone

The ion sweep cone is a metallic cone that is installed over the ion transfer
tube. The ion sweep cone channels the sweep gas towards the entrance of
the capillary. 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 that can be analyzed without a loss of signal intensity. In addition,
keeping the ion transfer tube entrance cleaner reduces the need for frequent
MS detector maintenance.

Install the ion sweep cone

1. Remove the ion sweep cone from its storage container. Inspect and clean
it if necessary.

2. Note the location of the sweep gas supply port in the API cone seal. The
gas inlet on the ion sweep cone is placed in this port. See Figure 9 and

Figure 10.

Sweep Gas
Supply Port

Figure 9. Sweep gas supply port in the API cone seal

34 LTQ XL Getting Started Thermo Electron Corporation

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Figure 10. Ion sweep cone, showing the gas inlet

Installing the Ion Sweep Cone

Gas Inlet

CAUTION AVOID BURNS. At operating temperatures, the ion
transfer tube can severely burn you! The ion transfer tube typically
operates between 200 and 400 °C. Always allow the ion transfer

capillary to cool to room temperature (for approximately 20 min)
before you install the ion sweep cone. Always be careful not to touch

the entrance end of the ion transfer tube when it is exposed.

3. After the ion transfer tube has cooled to room temperature, carefully
align the gas inlet on the ion sweep cone with the sweep gas supply port
in the API cone seal. Firmly press the ion sweep cone into position.

4. If necessary to achieve a proper ion sweep cone installation, you might
adjust the set screws around the perimeter of the ion sweep cone.

The ion sweep cone is now properly installed on the MS detector.

Thermo Electron Corporation LTQ XL Getting Started 35

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Installing the Ion Max Ion Source Housing

Installing the Ion Max

Ion Source Housing

Reinstall the Ion Max ion source housing

1. Carefully align the two guide pin holes on the rear of the ion source
housing with the ion source housing guide pins on the MS detector, and
carefully press the ion source housing onto the ion source mount. See

Figure 11 and Figure 12.

Ion Source Housing
Locking Levers

Guide
Pin Holes

Ion Source
Housing Drain

Figure 11. Rear view of the Ion Max ion source housing

36 LTQ XL Getting Started Thermo Electron Corporation

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Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Installing the Ion Max Ion Source Housing

Ion Source Housing Guide Pins

Figure 12. Ion source mount showing ion source housing guide pins

2. Rotate the ion source housing locking levers 90 degrees to lock the ion
source housing onto the ion source mount assembly.

CAUTION Prevent solvent waste from backing up into the ion source
and MS detector. Always ensure that liquid in the drain tube is able to
drain to a waste container.

3. Reinstall the ion source drain tube as follows:

Thermo Electron Corporation LTQ XL Getting Started 37

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Installing the Ion Max Ion Source Housing

CAUTION Do not vent the API source drain tube (or any vent tubing
connected to the waste container) to the same fume exhaust system to
which you have connected the forepumps. The analyzer optics can
become contaminated if the API source drain tube and the (blue)
forepump exhaust tubing are connected to the same fume exhaust
system.

CAUTION Your laboratory must be equipped with at least two fume
exhaust systems. Route the (blue) forepump exhaust tubing to a
dedicated fume exhaust system. Route the drain tube from the API
source to a waste container. Vent the waste container to a dedicated fume
exhaust system.

a. Connect the 1-in. ID Tygon® tubing to the ion source housing drain.

b. Attach the free end of the hose to a dedicated drain system. Ideally,

the drain system should be vented to a fume exhaust system.

The Ion Max ion source housing is now properly installed on the MS
detector.

38 LTQ XL Getting Started Thermo Electron Corporation

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Installing the ESI Probe

Installing the ESI

Probe

Install the ESI probe

1. Remove the ESI probe from its storage container. Inspect and clean it if
necessary.

Note If your ESI probe does not already have a sample tube
(fused-silica capillary or metal needle) and safety sleeve attached,
follow the procedure for installing a sample tube and PEEK safety
sleeve that is outlined in Installing a New Fused-Silica Sample

Tube and PEEK Safety Sleeve in the Ion Max API Source Hardware
Manual.

2. Ensure that the probe locking lever on the ion source housing is
unlocked (opened to its widest position). See Figure 13.

3. Insert the ESI probe into the port in the ion source housing, align the
guide pin on the probe body at a minus 45 degree angle from the API
interlock block. See Figure 14

ESI Interlock Plug

API Interlock Block

Probe Port

Grounding Bar

Probe Collar

APCI Vaporizer Heater
Cable

Aux Gas Fitting

8kV Cable

Sheath Gas Fitting

Probe Locking
Lever (open position)

Figure 13. Ion Max ion source housing probe locking lever open

Thermo Electron Corporation LTQ XL Getting Started 39

2

Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Installing the ESI Probe

ESI Needle High Voltage

Connector Receptacle

Sheath Gas

Inlet (S)

Guide

Pin

Auxiliary Gas

Inlet (A)

Sample

Inlet Fitting

Sheath Liquid /

Calibrant Inlet (C)

Figure 14. ESI probe, side view

4. Push the probe into the port until the guide pin meets with the probe
collar on the ion source housing.

5. Turn the probe 45 degrees clockwise and align the guide pin with the
slot in the API interlock block (you might need to pull the probe
towards you slightly to properly align the pin with the notch). Once you
have turned the probe far enough to align the pin with the alignment
notch at the rear of the port, push the probe straight in until the guide
pin stops at the bottom of the alignment notch.

6. Lock the probe in place by rotating the probe locking lever towards the
front of the housing; closing the probe locking lever towards the rear of
the ion source housing might make it difficult to unlock. You might first
need to tighten the locking lever threaded shaft by rotating it clockwise a
few turns if rotating the lever does not tighten the probe collar enough.

7. Insert the APCI vaporizer heater cable into the API interlock socket.

40 LTQ XL Getting Started Thermo Electron Corporation

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Setting Up the Ion Source for Tuning and Calibrating the MS Detector

Installing the ESI Probe

8. Insert the stainless steel ZDV fitting (grounding union) into the
grounding bar on the ion source housing. See Figure 15.

ESI Interlock
Socket

Sheath Gas Inlet (S)

Grounding Bar

Grounding Union

PEEK Safety
Sleeve

8 kV Cable

Auxiliary Gas Inlet (A)

Sample Inlet

Figure 15. Ion Max ion source housing with ESI probe installed

9. Connect the sheath gas fitting (blue) to the sheath gas inlet (S) on the
probe. (See Figure 15.)

10. Connect the auxiliary gas fitting (green) to the auxiliary gas inlet (A) on
the probe. (See Figure 15.)

11. Connect the 8 kV cable to the ESI needle high voltage receptacle on the
ESI probe. Tighten the locking ring on the 8 kV connector.

12. Connect the sample transfer tubing to the grounding union.

The ESI probe is now properly installed in the Ion Max ion source housing.

Leave the LC/MS system in Standby and go to Chapter 3: “Tuning and

Calibrating Automatically in the ESI/MS Mode”.

Thermo Electron Corporation LTQ XL Getting Started 41

Chapter 3 Tuning and Calibrating

Automatically in the ESI/MS
Mode

This chapter provides information on how to tune and calibrate the LTQ
XL MS detector in the ESI/MS mode. For most applications, you tune and
calibrate in the ESI mode through automatic procedures. The procedures
use a calibration solution that is introduced into the MS detector in low
flow mode. The procedures properly tune and calibrate the MS detector for
ESI operation (refer to Table 2 on page 2 for ESI operating parameter
guidelines). You need to calibrate the MS detector every one to three
months of operation for optimum performance over the entire mass range
of the detector.

To tune and calibrate your MS detector automatically in the ESI/MS mode,
you do the following:

• Infuse a low concentration calibration solution containing caffeine,
MRFA, and Ultramark 1621 into the ESI source by using the syringe
pump. (Refer to the section, Setting Up the Syringe Pump for Tuning

and Calibration.)

• Test the efficiency and stability of the spray of calibration solution into
the MS detector. You can observe the following singly-charged, positive
ions for caffeine, MRFA, and Ultramark 1621 in the Tune Plus window:
m/z 195, 524, 1222, 1522, and 1822.

• Tune the MS detector from the Tune Plus window to optimize
automatically the lenses.

• Calibrate the MS detector to adjust automatically the voltages of the
linear trap.

This chapter contains the following sections:

• Setting Up the Syringe Pump for Tuning and Calibration

• Setting Up the MS Detector in the Xcalibur Data System for Tuning

and Calibration

• Testing the Operation of the MS Detector in the ESI/MS Mode

Thermo Electron Corporation LTQ XL Getting Started 43

3

Tuning and Calibrating Automatically in the ESI/MS Mode

• Tuning the MS Detector Automatically in the ESI/MS Mode

• Saving Your ESI/MS Tune Method

• Calibrating the MS Detector Automatically

• Cleaning the MS Detector after Tuning and Calibrating

44 LTQ XL Getting Started Thermo Electron Corporation

3

Tuning and Calibrating Automatically in the ESI/MS Mode

Setting Up the Syringe Pump for Tuning and Calibration

Setting Up the Syringe

Pump for Tuning and

Calibration

You introduce tuning and calibration solution into the API source with a
syringe infusion pump. A syringe pump allows you to infuse a sample
solution into the API source for extended periods of time.

The syringe pump and syringe are located on the front panel of your LTQ
XL MS detector. To infuse solution for tuning and calibration, you install
on the pump a 500-mL Unimetrics® syringe containing the calibration
solution.

Note To minimize the possibility of cross-contamination, use a different
syringe and section of fused silica tubing for the calibration solution than
you do for your sample solution.

Set up the syringe pump for infusion

1. Connect a 4 cm (1.5 in.) segment of Teflon® tube with a (brown)
fingertight fitting and a (brown) ferrule to the (black) LC union. See

Figure 16.

LC Union

(

P/N

00101-18202)

Fingertight fitting

P/N

00101-18081)

(

Ferrule

(

P/N

00101-18196)

Teflon Tube

(

P/N

00301-22803)

Figure 16. Plumbing connections for the syringe

2. Load a clean, 500-μL Unimetrics syringe with 450 μL of the calibration
solution. (Refer to Appendix A: “Sample Formulations” on page 139 for
a procedure for making the calibration solution.)

3. Insert the syringe needle into the segment of Teflon tube.

4. Place the syringe into the syringe holder of the syringe pump.

5. While squeezing the blue release button on the syringe pump handle,
push the handle forward until it just contacts the syringe plunger.

6. Connect a fused-silica infusion line from the LC union to the (stainless
steel) grounding union as follows. See Figure 17.

Thermo Electron Corporation LTQ XL Getting Started 45

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Tuning and Calibrating Automatically in the ESI/MS Mode

Setting Up the Syringe Pump for Tuning and Calibration

a. Connect the infusion line with a (brown) fingertight fitting and a

b. Connect the other end of the infusion line with a (red) fingertight

The syringe pump is now properly set up for infusing solution into the MS
detector.

Go to the next section, Setting Up the MS Detector in the Xcalibur Data

System for Tuning and Calibration.

(brown) ferrule to the free end of the LC union.

fitting and a (brown) ferrule to the grounding union.

Figure 17. ESI/MS plumbing connections for the fused-silica infusion line

46 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Setting Up the MS Detector in the Xcalibur Data System for Tuning and Calibration

Setting Up the MS

Detector in the

Xcalibur Data System

for Tuning and

Calibration

You first tune manually with calibration solution to establish a stable spray
of solution and to ensure that enough ions are detected to calibrate the MS
detector. You then calibrate the MS detector automatically to optimize the
parameters that affect ion detection. With the optimized MS detector, the
Xcalibur data system can isolate and fragment ions and determine their
mass-to-charge ratios. Perform a calibration periodically, every one to three
months, for optimum performance of the MS detector.

Note The following procedures assume that you are familiar with your
LTQ XL instrument and the Tune Plus window. If you need additional
guidance, see LTQ XL online Help, LTQ XL Getting Connected, and/or
the LTQ XL Hardware Manual.

CAUTION Before you begin normal operation each day, ensure that you
have sufficient nitrogen for your API source. If you run out of nitrogen,
the LTQ XL MS detector automatically turns Off to prevent the
possibility of atmospheric oxygen from entering the ion source. The
presence of oxygen in the ion source when the MS detector is On could
be unsafe. (In addition, if the LTQ XL MS detector automatically turns
Off during an analytical run, you could lose data.)

On Off Standby

Set up the MS detector in the Xcalibur data system for tuning and
calibration in the ESI/MS mode

1. If you have not already done so, open the Tune Plus window from the
Start button on your Windows XP task bar, as follows:

a. Choose Start > Programs > Xcalibur > Xcalibur to display the

Xcalibur Home Page – Roadmap view.

b. Click the Instrument Setup button to display the Instrument Setup

window.

c. Click the Finnigan LTQ XL button to display the New Method

page.

d. Click the Tune Plus button to display the Tune Plus window. See

Figure 18.

2. In the Tune Plus window, on the Control/Scan Mode toolbar, click the
On/Off/Standby button to take the MS detector out of the Standby (or

Thermo Electron Corporation LTQ XL Getting Started 47

3

Tuning and Calibrating Automatically in the ESI/MS Mode

Setting Up the MS Detector in the Xcalibur Data System for Tuning and Calibration

Off) mode and turn it On. When you turn the MS detector to On, you
initiate the following events:

• The MS detector begins scanning.

• Nitrogen flows into the ESI probe.

• The LTQ XL MS detector applies high voltage to the ESI probe.

• The Xcalibur data system shows a real-time display in the Spectrum
view.

Figure 18. Tune Plus window, showing the MS detector in the Standby mode

48 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Setting Up the MS Detector in the Xcalibur Data System for Tuning and Calibration

Note The Xcalibur data system contains customized tune files for
different applications in the folder C:\Xcalibur\methods, including
one for low flow LC/ESI/MS operation.

3. Open the Tune Method file that stores the factory default tune settings
for low-flow ESI operation, as follows:

a. Choose File>Open to display the Open dialog box.

b. Browse for the folder C:\Xcalibur\methods. Select the file

AutoTune.LTQTune.

c. Click Open to open the file. Tune Plus downloads the Tune Method

parameters to the MS detector.

4. Examine the pre-tune ESI source settings as follows:

a. From the Instrument Setup toolbar, click the API Source button to

open the ESI Source dialog box. Verify that the settings in your
dialog box are the same as those shown in Figure 19.

b. Click OK to return to the Tune Plus window.

Figure 19. ESI Source dialog box, showing the settings to start a typical low

flow experiment

5. Set the scan parameters for tuning and calibration, as follows:

Thermo Electron Corporation LTQ XL Getting Started 49

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Tuning and Calibrating Automatically in the ESI/MS Mode

Setting Up the MS Detector in the Xcalibur Data System for Tuning and Calibration

a. On the Control/Scan Mode toolbar, click the Define Scan button

to open the Define Scan dialog box. See Figure 20. (If your dialog
box appears different from the one shown in the figure, it is
probably because the advanced settings are not displayed. You can
turn on the advanced settings as follows: In Tune Plus, choose
ScanMode, and then click Advanced Scan Features to select the
option.)

b. In the Scan Description group box, in the Mass Range list box,

select Normal to allow for a selection of mass ranges between m/z
150 to 2000.

c. In the Scan Rate list box, select Normal to specify a normal scan

rate.

d. In the Scan Type list box, select Full specify a full scan.

e. In the Scan Time group box, in the Microscans spin box, enter 1 to

set the total number of microscans to 1.

f. In the Max. Inject Time spin box, enter 200.000 to specify a 200 ms

maximum injection time.

g. In the Source Fragmentation group box, confirm that the On check

box is not selected ( ) to specify that the ion source fragmentation
option is turned off.

h. In the Scan Ranges group box, in the Input list box, select From/To

to make available the First Mass and Last Mass text boxes in the
Scan Ranges table.

50 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Setting Up the MS Detector in the Xcalibur Data System for Tuning and Calibration

Figure 20. Define Scan dialog box, showing the default settings for ESI/MS operation

i. In the Scan Ranges group box, in the Scan Ranges table, in the First

Mass text box, enter 150 to set the first mass for the scan range to
m/z 150.

j. In the Last Mass text box, enter 2000 to set the last mass for the scan

range to m/z 2000.

k. Ensure that the settings in your Define Scan dialog box are the same

as those shown in Figure 20.

l. Click OK to apply the MS detector scan parameters and to close the

Define Scan dialog box.

6. On the Control/Scan Mode toolbar, click the Centroid/Profile button
to toggle the data type to profile. (The picture on the button should be
the same as that shown here.)

7. Click the Positive/Negative button to toggle the ion polarity mode to
positive. (The picture on the button should be the same as that shown
here).

The MS detector is now properly set up in the Xcalibur data system for
tuning and calibration in the ESI/MS mode.

Thermo Electron Corporation LTQ XL Getting Started 51

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Tuning and Calibrating Automatically in the ESI/MS Mode

Testing the Operation of the MS Detector in the ESI/MS Mode

Testing the Operation

of the MS Detector in

the ESI/MS Mode

You are now ready to test whether your MS detector is operating properly.
To test for proper operation, you infuse the calibration solution into the ESI
source, and then you monitor the real-time display of the mass spectrum of
calibration solution. You want to ensure that a stable spray of solution enters
the MS detector.

Test the operation of the MS detector in the ESI/MS mode

1. Click the Syringe Pump button to display the Syringe Pump dialog
box. See Figure 21.

Figure 21. Syringe Pump dialog box

2. Turn on the syringe pump and set an infusion flow rate of 5 μL/min, as
follows:

a. In the Flow Control group box, click the On option button to make

active the Flow Rate spin box.

b. Type 5 in the Flow Rate spin box to specify a rate of 5 μL/min.

Note This procedure assumes that you are using the 500-μL
Unimetrics syringe that is provided with your LTQ XL system. If
you are using another type of syringe, select the option button
corresponding to your syringe.

c. If you are using a standard Unimetrics (or Hamilton) syringe, set up

the syringe parameters as follows:

52 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Testing the Operation of the MS Detector in the ESI/MS Mode

i. In the Type group box, click the Unimetrics (or Hamilton)

option button to specify the proper syringe type.

ii. Click the Vo lu m e list box arrow to display the list of available

volumes, and then select 500 (or your syringe size) from the list
to set the proper syringe volume. Note that, if you are using a
Unimetrics syringe, the LTQ XL MS detector automatically sets
the syringe ID to its proper value of 3.260 mm.

d. If you are not using a Unimetrics (or Hamilton) syringe, set up the

syringe parameters as follows:

i. In the Type group box, click the Other option button to make

active the syringe ID spin box.

ii. Type the inner diameter of your syringe in the Syringe ID spin

box.

e. Click OK to apply the syringe parameters, start the syringe pump,

and close the Syringe Pump dialog box.

3. On the File/Display toolbar, click the Display Spectrum View button
to ensure that the Spectrum view is displayed.

4. Monitor the data for the calibration solution, as follows:

a. In the Spectrum view of the Tune Plus window, observe the mass

spectra of the singly-charged ions of calibration solution. The ions
are as follows. See Figure 22.

• Caffeine: m/z 195

• MRFA: m/z 524

• Ultramark 1621: m/z 1022, 1122, 1222, 1322, 1422, 1522, 1622,

1722, 1822

Note Based on the LC flow rate of your experiment, you can
specify the value of each of the following tuning parameters on
the LTQ XL MS detector: sheath, Auxiliary, and Sweep gas
pressures, ESI needle (or “spray”) voltage, ion transfer capillary
temperature, and probe position. Automatic tuning sets the
values of the other parameters.

b. At the top of the Spectrum view, notice the values for the ionization

time (IT) and normalization level (NL). See Figure 22.

c. Click the API Source button to open the ESI Source dialog box.

(See the Spray Current readback shown in Figure 19.)

Thermo Electron Corporation LTQ XL Getting Started 53

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Tuning and Calibrating Automatically in the ESI/MS Mode

Testing the Operation of the MS Detector in the ESI/MS Mode

d. Observe the value for the Spray Current readback and the values for

If you answered “yes” to the questions in step 4.d, then your MS detector is
operating properly.

If you answered “no” to either of these questions, try the following
troubleshooting measures:

• Ensure that the fused-silica sample tube does not extend beyond the tip
of the ESI needle.

NL and IT in the Spectrum view. As calibration solution infuses,
and the readback values fluctuate, ask yourself 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?

• Ensure that the entrance to the ion transfer capillary is clean, and is not
covered with a piece of septum.

• Ensure that the solution entering the probe is free of air bubbles and
that the tubing and connectors are free of leaks.

Congratulations! You have demonstrated that your MS detector is operating
properly in the ESI mode. You are now ready to tune and calibrate the MS
detector. Leave your LTQ XL MS detector as it is, and go to the next
section, Tuning the MS Detector Automatically in the ESI/MS Mode.

54 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Testing the Operation of the MS Detector in the ESI/MS Mode

IT=ionization time

NL=normalization level

Figure 22. Spectrum view of the Tune Plus window, showing ionization time (IT) and normalization level (NL) of the

calibration solution

Thermo Electron Corporation LTQ XL Getting Started 55

3

Tuning and Calibrating Automatically in the ESI/MS Mode

Tuning the MS Detector Automatically in the ESI/MS Mode

Tuning the MS

Detector

Automatically in the

ESI/MS Mode

You tune the MS detector automatically in the ESI/MS mode to optimize
important parameters, including heated capillary voltage and tube lens
voltage.

Tune the MS detector automatically

1. On the Control/Scan Mode toolbar, click the Tu n e button to display
the Tune dialog box.

2. If necessary, click the Automatic tab to display the Automatic tuning
page. See Figure 23.

3. In the What to Optimize On group box, select the Mass option button
to make active the Mass spin box.

Figure 23. Tune dialog box, showing the Automatic tuning page

56 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Tuning the MS Detector Automatically in the ESI/MS Mode

4. In the Mass spin box, enter 195.1 to specify that the LTQ XL MS

detector optimize your Tune Method on the peak at m/z 195.1.

Note In this example, you use the mass peak at m/z 195.1 to
optimize the Tune Method. However, you can optimize the tune on
any mass peak of the calibration solution.

5. Start the automatic tuning procedure, as follows:

a. Click Start. A message box displays the following message:

Please ensure that the 500 microliter syringe is full.

Ensure that the syringe contains at least 450 μL calibration solution.

b. Click OK to close the message box, and return to the Tune dialog

box.

6. On the File/Display toolbar, click the Graph View button to display the
Graph view. See Figure 24.

7. Observe the Tune Plus window and the Tune dialog box. While
automatic tuning is in progress, the LTQ XL MS detector displays
various tests in the Spectrum and Graph views in Tune Plus and displays
various messages in the Status group box in the Tune dialog box. Your
Tune Plus window should now look similar to the one shown in

Figure 24.

8. Click the ESI Source dialog box to examine the ESI source parameters
after tuning. Compare the settings shown in Figure 25 with the pre-tune
settings shown in Figure 19 on page 3-49.

9. Click the Ion Optics toolbar button to display the Ion Optics dialog
box. The parameters in the Ion Optics dialog box are optimized
automatically by the LTQ XL MS detector. See Figure 26.

You have now successfully tuned the MS detector in ESI/MS mode using
the calibration solution. Go to the next section, Saving Your ESI/MS Tune

Method.

Thermo Electron Corporation LTQ XL Getting Started 57

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Tuning and Calibrating Automatically in the ESI/MS Mode

Tuning the MS Detector Automatically in the ESI/MS Mode

Figure 24. Tune Plus window, showing the results of a typical automatic tune procedure

58 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Tuning the MS Detector Automatically in the ESI/MS Mode

Figure 25. ESI Source dialog box, showing typical parameters after automatic

tuning

Figure 26. Ion Optics dialog box, showing examples of voltages of lenses and

Intermultipoles, which are optimized by the LTQ XL automatic tuning
procedure

Thermo Electron Corporation LTQ XL Getting Started 59

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Tuning and Calibrating Automatically in the ESI/MS Mode

Saving Your ESI/MS Tune Method

Saving Your ESI/MS

Tune Method

You can save the parameters you just set in a Tune Method specific to your
particular analyte and solvent flow rate. (In this case, you save settings
obtained using calibration solution.) You can recall the Tune Method and
use it as a starting point for optimizing the MS detector on a different
analyte of interest or at a different flow rate.

Note You must save the Tune Method while the MS detector is On.

Save your ESI/MS Tune Method (for low-flow operation) when automatic
tuning is complete

1. Choose File > Save As to display the Save As dialog box. See Figure 27.

Figure 27. Save As dialog box, showing files in the folder C:\Xcalibur\methods

2. Select the C:\Xcalibur\methods folder.

60 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Saving Your ESI/MS Tune Method

3. Click the File Name text box, and type ESImyTune to name the Tune
Method ESImyTune.LTQTune.

4. Click Save to save the Tune Method, and return to the Tune Plus
window. Note that the Tune Method is named ESImyTune.LTQTune.

Once you have tuned the MS detector, you are now ready to calibrate.

Thermo Electron Corporation LTQ XL Getting Started 61

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Tuning and Calibrating Automatically in the ESI/MS Mode

Calibrating the MS Detector Automatically

Calibrating the MS

Detector

Automatically

Calibrate the MS detector automatically from the Tune Plus window

1. Choose Control > Calibrate to display the Calibrate dialog box.

2. If necessary, click the Automatic tab to display the Automatic
calibration page. See Figure 28.

Figure 28. Calibrate dialog box, showing the Automatic calibration page

3. Start the automatic calibration procedure, as follows:

a. Click Start. A message box displays the following message:

Please ensure that the 500 microliter syringe is full.

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Tuning and Calibrating Automatically in the ESI/MS Mode

Calibrating the MS Detector Automatically

Ensure the syringe contains at least 450 μL calibration solution.

b. Click OK to close the message box, and return to the Calibrate

dialog box.

4. Observe the Tune Plus window and the Calibrate dialog box. While the
automatic calibration is in progress, the LTQ XL MS detector displays a
variety of test results in the Spectrum and Graph views and displays a
variety of messages in the Status box of the Calibrate dialog box.

The automatic calibration procedure typically takes about 40 min.

When the LTQ XL MS detector completes the calibration procedure it
restores the full scan ESI mass spectrum in the Spectrum view. The
Instrument Messages dialog box is displayed, which indicates whether or not
the calibration procedure for an item is successful.

• If a calibration item is successful, the LTQ XL MS detector saves the
new calibration parameter automatically to the hard disk.

• If a calibration item fails, you can try calibrating on that item again after
you ensure the following: 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. If the sensitivity of the ions is low,
increase the solution flow rate somewhat, and then use the
semi-automatic calibration procedure to calibrate the specific parameter
that failed. See Figure 29. Consider deselecting the ZoomScan Mode
option if repeated failures occur.

When all calibration items are successful, your MS detector is properly
tuned and calibrated for low-flow experiments. A successful calibration
exhibits adequate intensities of the following calibrant ions: m/z 195, 524,
1222, 1522, and 1822. In many cases, fine tuning on your particular analyte
is not necessary if the intensity of these ions is sufficient. You are ready to
analyze samples if you do not need to maximize the intensity of the ion
signals for your analyte.

The procedures for changing the solution flow rate and optimization of the
MS detector parameters for reserpine, or your particular analyte, are
explained in Chapter 4: “Tuning with Your Analyte in LC/ESI/MS Mode”.
Before you tune with your analyte, go to the next section, Cleaning the MS

Detector after Tuning and Calibrating.

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Tuning and Calibrating Automatically in the ESI/MS Mode

Calibrating the MS Detector Automatically

Figure 29. Tune Plus window with Calibrate dialog box, showing the results of a successful semi-automatic calibration

procedure

64 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Cleaning the MS Detector after Tuning and Calibrating

Cleaning the MS

Detector after Tuning

and Calibrating

On Standby

This topic describes how to clean your MS detector after using the
calibration solution, in preparation for acquiring data on your analyte of
interest.

Clean the MS detector after calibrating

1. Click On/Standby to put the MS detector in Standby mode. When the
MS detector is in Standby, the LTQ XL MS detector turns off the sheath
gas, Auxiliary gas, Sweep gas, ESI high voltage, and syringe pump. The
MS detector stops scanning, and the LTQ XL MS detector freezes the
displays for the Spectrum and Graph views.

CAUTION Always place the MS detector in Standby (or Off) before you
open the API source to atmospheric oxygen. The presence of oxygen in
the ion source when the MS detector is On could be unsafe. (The LTQ
XL MS detector automatically turns the MS detector Off when you
open the API source, however, it is best to take this added precaution.

2. Remove the syringe from the syringe pump holder, as follows:

a. Squeeze the blue buttons, and pull back on the syringe pump handle

to free the syringe.

b. Remove the syringe from the holder.

c. Disconnect the tip of the syringe needle from the Teflon tubing.

3. Clean the syringe thoroughly, as follows:

a. Clean the syringe with a solution of 5% formic acid in water.

b. Rinse the syringe with a solution of 50:50 methanol:water.

c. Use acetone to rinse the syringe. (Repeat this step several times.)

4. To gain access to the ion transfer capillary, the Ion Max ion source
housing and the ion sweep cone need to be removed. Refer to the topic

“Removing the Ion Max Ion Source Housing” on page 33 for

instructions for removing the Ion Max ion source housing.

5. Remove the ion sweep cone as follows:

a. Put on a pair of talc-free gloves.

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Tuning and Calibrating Automatically in the ESI/MS Mode

Cleaning the MS Detector after Tuning and Calibrating

CAUTION AVOID BURNS. At operating temperatures, the ion
transfer tube can severely burn you! The ion transfer tube typically
operates between 200 and 400 °C. Always allow the ion sweep cone to

cool to room temperature (for approximately 20 min) before you touch
or remove this component. Always be careful not to touch the entrance

end of the ion transfer tube when it is exposed.

b. Grasp the outer ridges of the ion sweep cone and pull the cone

6. Remove the ion transfer capillary by using the custom tool provided.

7. Clean the ion sweep cone and the ion transfer capillary as follows:

a. Place the ion sweep cone and the ion capillary tube in a beaker of

b. Sonicate the components for 15 min.

straight off of the API cone seal. Note, you might need to loosen the
set screws on the ion sweep cone in order to remove it.

50:50 methanol/water.

8. Reinstall the ion transfer capillary.

9. Reinstall the ion sweep cone as described in “Installing the Ion Sweep

Cone” on page 34.

10. Place a small Teflon-coated septum over the entrance end of the ion
transfer capillary to seal the vacuum chamber of the MS detector.

11. Flush the sample transfer line, sample tube, and ESI probe thoroughly
with a solution of 5% formic acid in water (or with another appropriate
solvent), as follows:

Note The solvent that you use to flush the sample transfer line,
sample tube, and ESI probe assembly depends on the solvent system
you use to dissolve your samples. For example, if you are using a
buffered solution of a high concentration, an acidic solution is
appropriate.

a. Fill a clean, 250 μL Unimetrics syringe with a solution an

appropriate solvent.

b. While holding the plunger of the syringe in place, carefully insert

the needle of the syringe into the free end of the Teflon tube.

c. Flush the sample transfer line, sample tube, and ESI probe with the

solution by slowly depressing the syringe plunger. Visually confirm
that the solution is exiting the tip of the ESI probe on the inside of

66 LTQ XL Getting Started Thermo Electron Corporation

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Tuning and Calibrating Automatically in the ESI/MS Mode

Cleaning the MS Detector after Tuning and Calibrating

the probe assembly. Use a lint-free tissue to gently remove the excess
solution as it exits the probe.

d. Remove the needle of the syringe from the Teflon tube.

12. Repeat step 11 with a solution of 50:50 methanol:water.

13. Repeat step 11 with acetone.

14. Clean the spray shield as follows:

a. Fill a spray bottle with solvent solution.

b. Temporarily place a large Kimwipe (or other lint-free tissue) beneath

of the spray shield. (The Kimwipe is required to absorb the solution
used to flush the ion transfer capillary and spray shield.)

c. Use the spray bottle to flush contaminants from the exterior surface

of the spray shield.

d. Remove the Kimwipe you used to absorb the solution. Swab the

surface of the spray shield with a dry Kimwipe.

e. Repeat step 14.a through step 14.d with acetone to remove the

(high molecular weight) Ultramark 1621.

15. Being careful not to touch the ion transfer capillary with your hand,
remove the septum from the entrance end of the ion transfer capillary.

16. Reinstall the Ion Max ion source housing as described in “Installing the

Ion Max Ion Source Housing” on page 36.

The MS detector is now clean and ready for acquiring data on your analyte
of interest.

If you plan to run analytical samples in high-flow ESI mode (using flow
rates between 50 and 1000 μL/min), the procedures in Chapter 4: “Tuning

with Your Analyte in LC/ESI/MS Mode” explain how to optimize the tune

for this situation.

Thermo Electron Corporation LTQ XL Getting Started 67

Chapter 4 Tuning with Your Analyte in

LC/ESI/MS Mode

This chapter provides information on tuning the MS detector in the
LC/ESI/MS mode using your analyte. You optimize the sensitivity of the
MS detector to your analyte through an automatic procedure.

The customized Tune Methods contained in your LTQ XL data system are
optimized for a wide range of applications, and they can be used without
further tuning of your MS detector. However, for certain applications you
might need to tune and optimize several MS detector parameters.

For instance, the most important parameters that interact with the ESI
interface and signal quality are as follows:

• Electrospray voltage

• Heated capillary temperature (voltage)

• Tube lens voltage

• Capillary voltage

• Sheath gas flow rate

• Auxiliary gas flow rate

• Sweep gas flow rate

The settings for these parameters depend on the solvent flow rate and target
analyte composition. In general, you should fine tune your MS detector
whenever you change the solvent flow rate conditions of your particular
application. In this procedure, you use the ESI low-flow Tune Method
ESImyTune.LTQTune as a starting point, then further optimize the MS
detector parameters using an automatic procedure. The automatic
procedure adjusts the tube lens voltage, capillary voltage, and voltages
applied to the ion optics until the ion transmission of your analyte is
maximized.

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Tuning with Your Analyte in LC/ESI/MS Mode

The capillary is heated to maximize the ion transmission to the MS detector.
For ESI only, you set the ion transfer capillary temperature proportional to
the flow rate of your solution. Refer to Table 1-2 for guidelines for setting
operating parameters for LC/ESI/MS. For this procedure, the ion transfer
capillary temperature is set to 350 °C, and the sheath gas is set to 30.

Note

1. If your experiment is performed at a flow rate below 10 μL/min, and
the results you want can be obtained without optimizing the MS
detector on your particular analyte, go to Chapter 5: “Acquiring ESI

Sample Data Using the Tune Plus Window” to acquire sample data.

2. Before you optimize the tune for your analyte of interest, ensure that
the LTQ XL MS detector has been calibrated within the previous
three months. If the system needs to be calibrated, refer to the
procedures in Chapter 3: “Tuning and Calibrating Automatically in

the ESI/MS Mode”.

To tune the MS detector in the ESI/MS (high-flow) mode using your
analyte, perform the following tasks:

• Set up the MS detector for your specific analyte from the Tune Plus
window.

• Infuse your analyte into the MS detector using a syringe pump
connected to the LC with a Tee union.

• Optimize the MS detector parameters for your analyte while the
solution flows into the MS detector.

This chapter contains the following topics:

• Setting Up to Introduce Sample by Syringe Pump into Solvent Flow

from an LC

• Setting Up to Tune the MS Detector with Your Analyte

• Optimizing the MS Detector Tune Automatically with Your Analyte

• Saving the ESI/MS Tune Method

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Tuning with Your Analyte in LC/ESI/MS Mode

Setting Up to Introduce Sample by Syringe Pump into Solvent Flow from an LC

Setting Up to

Introduce Sample by

Syringe Pump into

Solvent Flow from an

LC

This topic describes setting up the MS detector to introduce your analyte by
syringe pump into solvent flow from an LC.

Plumbing connections for ESI/MS sample introduction into LC solvent
flow from the syringe pump

1. Connect a 4 cm (1.5 in.) segment of Teflon tubing with a (brown)
fingertight fitting and a (brown) ferrule to the (black) LC union. (See

Figure 17 on page 46.)

2. Fill a clean, 500-μL Unimetrics syringe with the 125 fg/μL solution of
reserpine or your analyte of interest. (See Appendix A: “Sample

Formulations” for a procedure for making the reserpine tuning

solution.)

3. Insert the needle of the syringe into the segment of Teflon tube. Check
that the needle tip of the syringe fits readily into the opening in the free
end of the Teflon tubing. If necessary, you can enlarge the opening in
the end of the tubing slightly.

4. Place the syringe into the syringe holder of the syringe pump.

5. While squeezing the blue release buttons on the syringe pump handle,
push the handle forward until it just contacts the syringe plunger.

6. Connect the fused-silica infusion line from the (black) LC union to the
(black) LC Tee union, as follows. See Figure 30.

a. Connect the infusion line with a (brown) fingertight fitting and a

(brown) ferrule to the free end of the LC union.

b. Connect the other end of the infusion line with a (red) fingertight

fitting and a (brown) ferrule to the side arm of the LC Tee union.

7. Connect an appropriate length of (red) PEEK tubing from the (stainless
steel) grounding union to the (black) LC Tee union, as follows:

a. Use a PEEK tubing cutter to cut a 4 cm (1.5 in.) length of the PEEK

tubing.

b. Connect the PEEK tubing with a (brown) fingertight fitting and a

(brown) ferrule to the grounding union.

c. Connect the other end of the PEEK tubing with a (brown)

fingertight fitting and a (brown) ferrule to the LC Tee union.

Thermo Electron Corporation LTQ XL Getting Started 71

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Tuning with Your Analyte in LC/ESI/MS Mode

Setting Up to Introduce Sample by Syringe Pump into Solvent Flow from an LC

From Divert /

Inject Valve

Stainless Steel

Ferrule

Ferrules

(

P/N

00101-18120)

Fingertight

Fitting

P/N

00101-18195)

(

PEEK Tubing

(

P/N

00301-22912)

Stainless
Steel Nut

LC Tee Union

(

P/N

00101-18204)

From LC Union

Figure 30. ESI/MS plumbing connections for the LC Tee union

PEEK Tubing

(

P/N

00301-22912)

Ferrules

P/N

00101-18196)

(

Infusion Line

Fused-Silica Capillary

(

P/N

00106-10504)

Grounding Union

(

P/N

00101-18182)

Fingertight

Fittings

P/N

00101-18081)

(

8. If you have not already done so, connect the PEEK safety sleeve and
fused-silica sample tube from the grounding union to the ESI probe
sample inlet as described in the section, Installing a New Fused-Silica

Sample Tube and PEEK Safety Sleeve in the Ion Max API Source
Hardware Manual.

If you have installed the stainless steel needle in the ESI probe, connect
the PEEK safety sleeve and fused-silica capillary tube from the
grounding union to the ESI probe sample inlet as described in the
section, Installing a New Stainless Steel Needle in the ESI Probe and

Installing a New Fused-Silica Sample Tube and PEEK Safety Sleeve in the
Ion Max API Source Hardware Manual.

9. Connect an appropriate length of PEEK tubing (transfer line from the
divert/inject valve) from the divert/inject valve to the free end of the
(black) LC Tee union, as follows.

a. Connect a length of PEEK tubing with a (stainless steel) nut and a

(stainless steel) ferrule to port 3 of the divert/inject valve.
See Figure 31.

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Tuning with Your Analyte in LC/ESI/MS Mode

Setting Up to Introduce Sample by Syringe Pump into Solvent Flow from an LC

b. Connect the other end of the PEEK tubing with a (brown)

fingertight fitting and a (brown) ferrule to the free end of the LC
Tee union. (See Figure 30.)

To LC Tee

Union

Plug

(optional)

3

4

5

6

Detector Position

2

1

To Wast e

From

LC

To LC Tee

Union

Plug

(optional)

3

4

5

6

To Wast e

Waste Position

From

LC

2

1

Figure 31. Divert/Inject valve, showing the correct setup for tuning by syringe

infusion and showing the flow of liquid through the valve in the
Detector and Waste positions

10. Connect an appropriate length of PEEK tubing (transfer line from the
LC) from the divert/inject valve to the LC, as follows:

a. Connect a length of PEEK tubing with a (stainless steel) nut and a

(stainless steel) ferrule to port 2 of the divert/inject valve.

b. Connect the other end of the PEEK tubing with a proper fitting and

a ferrule to the outlet of the LC.

11. Connect an appropriate length of PEEK tubing (waste line) from the
divert/inject valve to a waste container, as follows:

a. Connect a length of PEEK tubing with a (stainless steel) nut and a

(stainless steel) ferrule to port 1 of the divert/inject valve.

b. Insert the other end of the PEEK tubing in a suitable waste

container.

The MS detector is now properly set up to introduce your analyte by syringe
pump into solvent flow from an LC.

Thermo Electron Corporation LTQ XL Getting Started 73

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Tuning with Your Analyte in LC/ESI/MS Mode

Setting Up to Tune the MS Detector with Your Analyte

Setting Up to Tune the

MS Detector with

Your Analyte

Set up the MS detector to tune automatically on your analyte in ESI/MS
mode

In this procedure, you can use the reserpine solution described in Appendix

A: “Sample Formulations”, or you can use a solution of an analyte of interest

to you.

CAUTION Do not infuse calibration solution at flow rates above 10
μL/min. Ultramark 1621 can contaminate your system at high
concentrations.

Note The following procedures assume that you are familiar with your
LTQ XL instrument and the Tune Plus window. If you need additional
guidance, see LTQ XL online Help and/or the LTQ XL Hardware

Manual.

1. Open the Tune Plus window from the Start button on your
Windows XP Desktop, as follows:

a. Choose Start > Programs > Xcalibur > Xcalibur to display the

Xcalibur Home Page – Roadmap view.

On Standby

b. Click the Instrument Setup button to display the Instrument Setup

window.

c. Click the LTQ XL button to display the New Method page.

d. Click the Tune Plus button to display the Tune Plus window.

2. In Tune Plus, click the On/Standby button to take the MS detector out
of Standby mode and turn it On. The MS detector begins scanning, the
LTQ XL MS detector applies high voltage to the ESI probe, and the
LTQ XL MS detector shows a real-time display in the Spectrum view.

3. Open the ESImyTune.LTQTune Tune Method, the Tune Method you
saved in Chapter 3, as follows:

a. On the File/Display toolbar, click the Open File icon to display the

Open dialog box.

b. Browse to the folder C:\Xcalibur\methods. Then, select the file

ESImyTune.LTQTune.

c. Click Open to open the file. Tune Plus downloads the Tune Method

parameters to the MS detector, and the title bar in the Tune Plus
window should read as follows:
C:\Xcalibur\methods\ESImyTune.LTQTune – Tune Plus

74 LTQ XL Getting Started Thermo Electron Corporation

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Tuning with Your Analyte in LC/ESI/MS Mode

Setting Up to Tune the MS Detector with Your Analyte

4. Define the scan parameters for tuning with your analyte in the ESI/MS
mode, as follows:

a. In the Instrument Control toolbar, click Define Scan to open the

Define Scan dialog box. See Figure 32.

b. In the Scan Description group box, in the Mass Range list box,

select Normal to allow for a selection of mass ranges between m/z
150 to 2000.

c. In the Scan Rate list box, select Normal to specify a normal scan rate.

d. In the Scan Type list box, select SIM specify a Selected Ion

Monitoring experiment.

e. In the Scan Time group box, in the Number of Microscans spin box,

type 1 to set the total number of microscans to 1.

f. In the Max. Inject Time spin box, type 200.000 to specify a 200 ms

maximum injection time.

g. In the Scan Ranges group box, in the Input list box, select

Center/Width to make available the Center Mass and Width text
boxes in the Scan Ranges table.

h. In the Source Fragmentation group box, confirm that the On check

box is not selected ( ) to specify that the ion source fragmentation
option is turned off.

i. In the Scan Ranges group box (Scan Ranges table), in the Center

Mass text box, type the mass of your analyte to set the center of mass
for the scan range. If reserpine is the analyte you would enter 609.20
to set the center mass for the scan range to m/z 609.20. If you are
using another analyte enter its mass value into the Center Mass text
box (Figure 32)

j. In the Width text box, type 2.0 to set the width of the scan range to

2.0 daltons.

k. Verify that the settings in your Define Scan dialog box are the same

as those shown in Figure 32.

l. Click OK to apply the MS detector scan parameters and to close the

Define Scan dialog box.

Thermo Electron Corporation LTQ XL Getting Started 75

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Tuning with Your Analyte in LC/ESI/MS Mode

Setting Up to Tune the MS Detector with Your Analyte

Figure 32. Define Scan dialog box, showing typical settings for acquiring reserpine data of the SIM type

5. On the Control/Scan Mode toolbar, click Centroid/Profile to toggle
the data type to profile. (The picture on the button should be the same
as that shown here.)

6. Click Positive/Negative to toggle the ion polarity mode to positive.
(The picture on the button should be the same as that shown here).

You have completed setting up to tune your MS detector with your analyte
in ESI/MS mode.

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Tuning with Your Analyte in LC/ESI/MS Mode

Optimizing the MS Detector Tune Automatically with Your Analyte

Optimizing the MS

Detector Tune

Automatically with

Your Analyte

Optimize the MS detector tune automatically to maximize the ion
transmission of reserpine (or your analyte of interest) for a high-flow
experiment. Thermo Electron recommends that you begin optimizing after
you have successfully passed an automatic tuning procedure and an
automatic calibration procedure with the calibration solution infused at
5 μL/min.

The following procedure describes how to optimize the MS detector Tune
Method with the reserpine m/z 609.2 peak at an LC flow rate of
400 μL/min. You can also carry out this procedure with your analyte of
interest and at your particular LC flow rate. (Refer to Tab le 2 for guidelines
about setting flow rates and temperatures.)

Optimize the MS detector Tune Method

1. On the Control/Scan Mode toolbar, click the Tu n e button to display
the Tune dialog box.

2. If necessary, click the Automatic tab to display the Automatic tuning
page. See Figure 33.

3. In the What to Optimize On group box, select the Mass option button
to make active the Mass spin box.

4. In the Mass spin box, enter 609.2 (or the appropriate mass of your
analyte of interest) to specify that the LTQ XL MS detector is to use the
peak at m/z 609.2 (or the appropriate mass of your analyte of interest) to
optimize your tune.

5. Ensure that the Divert/Inject valve is in the Detector position, as
follows:

a. Click the Divert/Inject button to open the Divert/Inject Valve

dialog box. See Figure 34.

b. Click the Detector option button.

c. Click Close.

6. Start the automatic tuning procedure from the Tune dialog box, as
follows:

a. Click Start. A message box displays the following message:

Please ensure that the 500 microliter syringe is full.

Thermo Electron Corporation LTQ XL Getting Started 77

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Tuning with Your Analyte in LC/ESI/MS Mode

Optimizing the MS Detector Tune Automatically with Your Analyte

b. Click OK to close the message box, and return to the Tune Plus

7. In the File/Display toolbar, click the Graph View button to display the
Graph view.

Ensure the syringe pump contains at least 450 μL of the 125 fg/μL
reserpine tuning solution.

window.

Figure 33. Tune dialog box, showing the Automatic tuning page

78 LTQ XL Getting Started Thermo Electron Corporation

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Tuning with Your Analyte in LC/ESI/MS Mode

Optimizing the MS Detector Tune Automatically with Your Analyte

Figure 34. Divert/Inject Valve dialog box

8. Observe the Tune Plus window and the Tune dialog box. While
automatic tuning is in progress, the LTQ XL MS detector displays
various tests in the Spectrum and Graph views in the Tune Plus window
and displays various messages in the Status group box in the Tune dialog
box. Your Tune Plus window should now look similar to the one shown
in Figure 35.

Note The most important parameters that affect the signal quality
during ESI/MS operation are electrospray voltage, ion transfer
capillary temperature, heated capillary voltage, tube lens voltage,
gases, and solution flow rate. If any one of these parameters is
changed, you need to reoptimize MS detector parameters. You can
use the Semi-Automatic tune procedure to tune the MS detector on
individual parameters.

You have now successfully tuned the MS detector in ESI/MS mode for the
compound reserpine (or your analyte of interest).

Thermo Electron Corporation LTQ XL Getting Started 79

4

Tuning with Your Analyte in LC/ESI/MS Mode

Optimizing the MS Detector Tune Automatically with Your Analyte

Figure 35. Tune Plus window with the Tune dialog box, showing the Automatic tuning page

80 LTQ XL Getting Started Thermo Electron Corporation

4

Tuning with Your Analyte in LC/ESI/MS Mode

Saving the ESI/MS Tune Method

Saving the ESI/MS

Tune Method

Save your ESI/MS Tune Method (for a high-flow experiment using your
analyte) when automatic tuning is complete

Note Save the Tune Method while the MS detector is On, if any of the

ion source parameters are different from those with which you started.

1. Choose File > Save As to display the Save As dialog box. See Figure 36.

Figure 36. Save As dialog box, showing files in the folder C:\Xcalibur\methods

2. Select the C:\Xcalibur\methods folder.

3. Click the File Name text box, and enter reserpine (or the name of your
analyte of interest).

4. Click Save to save the Tune Method, and return to the Tune Plus
window. Note that the Tune Method is named reserpine.LTQTune.

Thermo Electron Corporation LTQ XL Getting Started 81

The Tune Method is now properly saved and you are ready to acquire data
on your analyte of interest.

Chapter 5 Acquiring ESI Sample Data

Using the Tune Plus Window

This chapter describes how to acquire LC/ESI/MS sample data using the
Tune Plus window. This experiment uses reserpine but you can use the same
procedure with your analyte of interest.

Note The following procedures assume that you are familiar with your
LTQ XL instrument and the Tune Plus window. If you need
information, refer to the LTQ XL online Help, LTQ XL Getting
Connected, and/or the LTQ XL Hardware Manual.

Ensure that you have completed the procedures in Chapter 3: “Tuning

and Calibrating Automatically in the ESI/MS Mode” and Chapter 4:
“Tuning with Your Analyte in LC/ESI/MS Mode”.

This chapter contains the following sections:

• Setting Up to Acquire MS/MS Data in the Full Scan Type

• Setting Up to Introduce Sample by Loop Injection into Solvent Flow

from an LC

• Acquiring MS Data in the SIM Scan

Thermo Electron Corporation LTQ XL Getting Started 83

5

Acquiring ESI Sample Data Using the Tune Plus Window

Setting Up to Acquire MS/MS Data in the Full Scan Type

Setting Up to Acquire

MS/MS Data in the

Full Scan Type

Optimizing the Isolation
Width and Setting Up to

Prepare to acquire MS/MS data in the Full scan type on reserpine (or on
your analyte of interest). You need to optimize the isolation width and the
relative collision energy parameters before you acquire MS/MS data.

You first optimize the isolation width to ensure that the ion of interest is
isolated effectively, and then you optimize the collision energy to ensure that
fragmentation of the parent ion is efficient. The relative collision energy for
a particular analysis depends on the type of sample you are analyzing.

The information in this topic applies to operation of the LTQ XL MS
detector in either the ESI or the APCI mode.

This topic contains the following subtopics:

• Optimizing the Isolation Width and Setting Up to Optimize the

Collision Energy

• Optimizing the Collision Energy Automatically for an MS/MS

Experiment

Optimize the isolation width and set up to optimize the collision energy
for an MS/MS experiment

Optimize the Collision

Energy

On Standby

Note The collision energy is optimized on the LTQ XL MS detector by
changing the values for the parameter Normalized Collision Energy in
the MSn Settings group box of the Define Scan dialog box. For this
experiment, and for most applications, leave the parameters Activation
Q and Activation Time set to their default values. For more information
about these parameters, refer to the online Help.

1. If you have not already done so, from the Tune Plus window, click the
On/Standby button to take the MS detector out of Standby mode and
turn it On.

2. Ensure that the Centroid data type is selected. (The picture on the
button should be the same as that shown here.)

3. Ensure that the scan parameters are defined to acquire MS/MS Full scan
data for reserpine (or your analyte of interest), as follows:

a. Click the Define Scan button to open the Define Scan dialog box.

See Figure 37.

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Acquiring ESI Sample Data Using the Tune Plus Window

Setting Up to Acquire MS/MS Data in the Full Scan Type

Figure 37. Define Scan dialog box, showing initial settings to optimize the isolation width of an MS/MS experiment for

reserpine

b. Verify that the values in your dialog box are the same as those shown

in Figure 37. Start with a relatively wide Isolation Width. Leave the
Define Scan dialog box open.

4. At this time you might want to turn on your LC pump and specify a
flow rate of 0.4 mL/min, for example, to ensure that your system does
not leak.

5. Click the Syringe Pump button to display the Syringe Pump dialog
box. See Figure 38.

6. Turn on the syringe pump and set an infusion flow rate of 5 μL/min, as
follows:

a. In the Flow Control group box, click the On option button to make

active the Flow Rate spin box.

b. Type 5 in the Flow Rate spin box to specify a rate of 5 μL/min.

Note This procedure assumes that you are using the 500-μL
Unimetrics syringe that is provided with your LTQ XL system. If
you are using another type of syringe, select the option button
corresponding to your syringe.

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Acquiring ESI Sample Data Using the Tune Plus Window

Setting Up to Acquire MS/MS Data in the Full Scan Type

Figure 38. Syringe Pump dialog box

c. If you are using a standard Unimetrics or Hamilton syringe, set up

the syringe parameters as follows:

i. In the Type group box, click the Unimetrics or Hamilton

option button to specify the proper syringe type.

ii. Click the Vo lu m e list box arrow to display the list of available

volumes, and then select 500 (or your syringe size) from the list
to set the proper syringe volume. Note that, if you are using a
Unimetrics syringe, the LTQ XL MS detector automatically sets
the syringe ID to its proper value of 3.260 mm.

d. If you are not using a Unimetrics or Hamilton syringe, set up the

syringe parameters as follows:

i. In the Type group box, click the Other option button to make

active the syringe ID spin box.

ii. Enter the inner diameter of your syringe in the Syringe ID spin

box.

e. Click Apply to apply the syringe parameters and start the syringe

pump.

f. Finally, move the Syringe Pump dialog box out of the way, to the top

of the monitor screen.

7. In the Tune Plus window, observe the mass spectrum of reserpine (or

your analyte of interest). Also observe the values for NL and IT

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Acquiring ESI Sample Data Using the Tune Plus Window

Setting Up to Acquire MS/MS Data in the Full Scan Type

(Normalization Level and Ion Time), while you optimize the value of the
Isolation Width in the Define Scan dialog box, as follows:

a. In the Define Scan dialog box, in the MSn Settings group box, in the

Isolation Width box, type 3 to specify an isolation width of m/z 3.
Then, click Apply.

b. In the Tune Plus window, observe the mass spectrum for the parent

ion of reserpine, m/z 609.2. Ensure that the readback values for NL
and IT are relatively stable.

c. Again, in the Define Scan dialog box, in the MSn Settings group

box, in the Isolation Width box, type 2.8 to specify an isolation
width of m/z 2.8. Then, click Apply.

Note The optimum value for the Isolation Width is the smallest
m/z width (instrument minimum width =m/z 0.4) that gives a
mass spectrum of maximum intensity for only the ions of
interest. 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 substaitial drop in the NL reading. A significant drop in
sensitivity indicates that the ions of interest are not effectively
isolated.

d. Repeat steps b and c above, entering successively smaller values for

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 you make to the Isolation Width.

Note After the Isolation Width is optimized, you can
compensate for minor changes in tune stability by increasing the
Isolation Width value a small amount. This adjustment should
be no larger than m/z=1.

8. In the Define Scan dialog box, in the MSn Settings group box, in the
Normalized Collision Energy box, type 20 to specify an initial value of
20 for the collision energy. Click Apply.

9. In the Tune Plus window, observe the mass spectrum of the product ions
of reserpine (or your analyte of interest). If necessary, increase the value
for the Normalized Collision Energy in increments of 5% to cause the
clear display of product ion mass spectrum. (After each change in value,
click Apply to implement the change.) See Figure 39.

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Acquiring ESI Sample Data Using the Tune Plus Window

Setting Up to Acquire MS/MS Data in the Full Scan Type

10. When you have clearly identified a product ion mass-to-charge ratio for

11. In the Tune dialog box, click the Collision Energy tab to display the

reserpine (or your analyte of interest), click the Tu n e button to display
the Tune dialog box.

page. See Figure 40.

Figure 39. Define Scan dialog box, showing typical settings for acquiring MS/MS data in the Full scan type on reserpine

12. Click the Product Ion Mass option button to make active the spin box.

Ty pe 397.2 to specify the product ion at m/z 397.2 for reserpine. The
LTQ XL MS detector can optimize collision energy automatically by
using this product ion of reserpine.

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