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
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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: 1-800-685-9535
Fax: 1-561-688-8736
techsupport.finnigan@thermo.com
Contact Customer Service
In the US and Canada for ordering information:
Phone: 1-800-532-4752
Fax: 1-561-688-8731
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
techpubs.finnigan-lcms@thermo.com
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
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