LXQ Start Man9705597042 B EN

Finnigan™
LXQ™
Getting Started
97055-97042 Revision B June 2006
For Research Use Only

Not for use in Diagnostic Procedures
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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 Finnigan LXQ MS Detector?.................................2
Which Ion Source—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 Source? .............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 Finnigan
LXQ MS Detector? ................................................................16
General MS or MSn Experiments ..........................................16
Data-Dependent Experiments................................................17
Ion Mapping Experiments .....................................................20
Ion Tree Experiments.............................................................22
Chapter 2 Setting Up the Ion Source for Tuning and Calibrating the MS
Detector25
Placing the LC/MS System in Standby......................................26
Removing the APCI Probe........................................................27
Removing the Ion Max Ion Source Housing .............................31
Installing the Ion Sweep Cone...................................................32
Installing the Ion Max Ion Source Housing ..............................34
Installing the ESI Probe ............................................................37
Thermo Electron Corporation Finnigan LXQ Getting Started vii

Contents
Chapter 3 Tuning and Calibrating Automatically in the ESI/MS Mode .... 41

Setting Up the Syringe Pump for Tuning and Calibration ........43
Setting Up the MS Detector in the Xcalibur Data System for
Tuning and Calibration .........................................................45
Testing the Operation of the MS Detector in the ESI/MS Mode ..
50
Tuning the MS Detector Automatically in the ESI/MS Mode ..54
Saving Your ESI/MS Tune Method ..........................................58
Calibrating the MS Detector Automatically ..............................60
Cleaning the MS Detector after Tuning and Calibrating...........63
Chapter 4 Tuning with Your Analyte in LC/ESI/MS Mode........................... 67

Setting Up to Introduce Sample by Syringe Pump into Solvent
Flow from an LC ...................................................................69
Setting Up to Tune the MS Detector with Your Analyte...........72
Optimizing the MS Detector Tune Automatically ....................75
Saving the ESI/MS Tune Method .............................................79
Chapter 5 Acquiring ESI Sample Data Using the Tune Plus Window ...... 81
Setting Up to Acquire MS/MS Data in the Full Scan Type.......82
Optimizing the Isolation Width and Setting Up to Optimize
the Collision Energy............................................................82
Optimizing the Collision Energy Automatically for an MS/MS
Experiment .........................................................................87
Setting Up to Introduce Sample by Loop Injection into Solvent
Flow from an LC ...................................................................90
Acquiring MS Data in the SIM Scan Type................................92
Chapter 6 Setting Up the Ion Source for Acquiring Data in APCI/MS/MS

Mode97
Removing the ESI Probe ...........................................................98
Removing the Ion Max Ion Source Housing ...........................101
Removing the Ion Sweep Cone ...............................................103
Installing the Corona Needle ..................................................104
Installing the Ion Max Ion Source Housing ............................105
Installing the APCI Probe .......................................................108
Chapter 7 Optimizing the MS Detector with Your Analyte in APCI/MS

Mode111
Setting Up the Inlet for Tuning Using High-Flow Infusion ....112
Setting Up the MS Detector for APCI/MS Operation ............115
viii Finnigan LXQ Getting Started Thermo Electron Corporation

Contents
Optimizing the Tune of the MS Detector Automatically in

APCI/MS Mode...................................................................118
Saving the APCI/MS Tune Method........................................121
Cleaning the MS Detector after Tuning in APCI Mode..........123
Chapter 8 Acquiring APCI Sample Data Using the Tune Plus Window ..125
Setting Up to Introduce Sample by Loop Injection into Solvent
Flow from an LC .................................................................126
Acquiring APCI Data in the SIM Scan Mode .........................128
Appendix A Sample Formulations......................................................................133

Caffeine, MRFA, and Ultramark 1621 Stock Solutions ..........134
Stock Solution: Caffeine.......................................................135
Stock Solution: MRFA.........................................................136
Stock Solution: Ultramark 1621 ..........................................136
ESI Calibration Solution: Caffeine, MRFA, Ultramark 1621..137
Reserpine ................................................................................138
Stock Solution: Reserpine ....................................................138
Reserpine Tuning Solution and Reserpine APCI Sample
Solution ............................................................................138
Appendix B LXQ High Mass Range Calibration...............................................141

High Mass Range Calibration Solution ...................................142
Normal Mass Range Calibration .............................................143
Enter Normal Mass Range Data into Tune Plus .....................144
Tune on m/z 524.3 .................................................................146
High Mass Range Calibration Procedure.................................148
Notes ......................................................................................153
Index..................................................................................................155
Thermo Electron Corporation Finnigan LXQ Getting Started ix

Preface
About This Guide Welcome to the Thermo Electron, Finnigan™ LXQ™ system. The LXQ is a
member of the Finnigan family of MS detectors.
This Finnigan LXQ Getting Started manual provides you with information
on how to set up, calibrate, and tune the Finnigan LXQ 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 In addition to this guide, Thermo Electron provides the following

Documentation documents for the LXQ system as .PDF files:
• LXQ Preinstallation Guide
• LXQ Getting Connected
• LXQ Hardware Manual
Help is also available from within the software
Safety and Special Make sure you follow the precautionary statements presented in this guide.
Notices The safety and other special notices appear in boxes.
Safety and special notices include the following:
CAUTION Highlights hazards to humans, property, or the environment.

Each CAUTION notice is accompanied by an appropriate CAUTION
symbol.
IMPORTANT Highlights information necessary to 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.
Thermo Electron Corporation Finnigan LXQ Getting Started xi

Preface
Contacting Us
Tip 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
www.thermo.com/finnigan
Contact Technical Support
Phone: 1-800-685-9535
Fax: 1-561-688-8736
E-mail: techsupport.finnigan@thermo.com
Find software updates and utilities to download at

http://mssupport.thermo.com.
Contact Customer Service
In the US and Canada for ordering information:

Phone: 1-800-532-4752
Fax: 1-561-688-8731
Web site: www.thermo.com/finnigan
Changes to the Manual To suggest changes to this guide or to the online Help, use either of the
and Online Help 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 Finnigan LXQ Getting Started Thermo Electron Corporation

Chapter 1 Introduction
The LXQ™ is a member of the Finnigan™ family of MS detectors. The

Finnigan LXQ 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) or loop injection
• Using a valve and an LC system fitted with a column (LC/MS)
Analysis by direct infusion or flow injection provides no chromatographic

separation of components in the sample before it passes into the MS
detector. 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. The data from the MS detector are then stored and processed by
the Xcalibur data system.
This introduction answers the following questions:
• Why Use the Finnigan LXQ MS Detector?
• Which Ion Source—ESI or APCI—Is Better for Analyzing My Samples?
• How Can I Introduce My Samples into the MS Source?
• 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 Finnigan LXQ MS

Detector?
Thermo Electron Corporation Finnigan LXQ Getting Started 1

1Introduction
Why Use the Finnigan LXQ MS Detector?
Why Use the Finnigan The attribute that sets the Finnigan LXQ MS detector apart from other LC
LXQ MS Detector? detectors is the high level of analytical specificity that it provides. The
Finnigan LXQ MS detector can provide multiple levels of analysis. Each
level of analysis adds a new dimension of specificity for unequivocal
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)
• Multi-stage 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
based on molecular mass information. Atmospheric pressure ionization
typically produces mass spectra that provide molecular mass information.
Two-stage mass analysis allows for even more certainty in compound

identification. MS/MS analysis monitors how a parent ion fragments when
it is exposed to an additional stage of excitation. 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 ions 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 either type of MS/MS analysis, you can easily quantify target analytes
in complex matrices such as plant or animal tissue, plasma, urine,
groundwater, or soil. Because of the specificity of MS/MS measurements
and the ability to eliminate interferences by an initial mass selection stage,
quantitative target compound analysis is easily accomplished using the
Finnigan LXQ MS detector.
Multi-stage mass analysis provides a unique capability to obtain structural

information that can be useful in structure elucidation of metabolites,
natural products, and sugars. MSn techniques on the Finnigan LXQ MS
2 Finnigan LXQ Getting Started Thermo Electron Corporation

1 Introduction
Why Use the Finnigan LXQ MS Detector?
detector allow for stepwise fragmentation pathways, making interpretation

of MSn spectra relatively straightforward. The Finnigan LXQ MS detector
has several advanced features that make its MSn capabilities extremely
powerful for qualitative analysis. (Refer to the topic “What Types of
Experiments Can I Perform with the Finnigan LXQ MS Detector?” on
page 16.)
ZoomScan analysis provides information about the charge state of one or

more mass ions of interest. ZoomScan data are collected by using slower
scan rates that give higher resolution. This level of resolution allows the
unambiguous determination of charge state. This in turn allows for the
correct determination of molecular mass for compounds that have multiple
ionization sites.
In addition to the aforementioned levels of analysis, there is an additional

technique called Wideband Activation. The Wideband Activation option
allows the Finnigan LXQ MS detector to apply collision energy to ions
during MS/MS fragmentation over a fixed mass range of 20 u. This option
allows collision energy to be applied 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), or to product ions formed from the loss of fragments less
than 20 u. When you want enhanced structural information without
resorting to MS3 analysis, choose the Wideband Activation option for
qualitative MS/MS. Fragmentation efficienty is somewhat reduced and
therefore you will need to increase the value of the collision energy to
compensate for this.

1Introduction
Which Ion Source—ESI or APCI—Is Better for Analyzing My Samples?
Which Ion The Finnigan LXQ MS detector has two atmospheric pressure ionization
Source—ESI or sources:1
APCI—Is Better for • Electrospray ionization (ESI)
Analyzing My
• Atmospheric pressure chemical ionization (APCI)
Samples?
Typically, more polar compounds such as amines, peptides, and proteins are
best analyzed by ESI, and non-polar compounds such as steroids are best
analyzed by APCI.
Using ESI/MS ESI is considered to be a soft ionization technique. The ESI source transfers
ions in solution to 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 is an ion in solution. This can include
adduct ions. For example, polyethylene glycols can be analyzed from a
solution containing ammonium acetate, because of adduct formation
between NH4+ ions in the solution and oxygen atoms in the polymer. With
ESI, the range of molecular masses that can be analyzed by the Finnigan
LXQ MS detector can be greater than 50,000 u if there is multiple charging.
ESI is especially useful for the mass analysis of polar compounds. Included
in this class of compounds are: biological polymers (such as, proteins,
peptides, glycoproteins, and nucleotides), pharmaceuticals and their
metabolites, and industrial polymers.
The ESI source can produce multiply charged ions depending on the
structure of the analyte and the solvent. For example, the mass spectrum of a
protein or peptide typically consists of a distribution of multiply charged
analyte ions. This mass spectrum can be mathematically manipulated to
determine the molecular mass of the sample.
You can use the ESI source in either positive or negative ion polarity mode.
The ion polarity mode is determined by the polarity of the ions in solution:
acidic molecules form negative ions in high pH solution and basic molecules
form positive ions in low pH solution. The ESI needle may be either
positively or negatively charged. When it is positively charged it generates
positive ions. When it is negatively charged it generates negative ions.
You can vary the flow rate into the mass spectromrter over a range of
0.1 μL/min to 1000 μL/min. Refer to Table 2.
1Optional ionization sources [atmospheric pressure photo ionization (APPI), atmospheric pressure
matrix assisted laser desorption ionization (AP MALDI), and nanospray] are also available.

1 Introduction
In ESI, the buffer type and buffer concentration both have a noticeable
effect on sensitivity. Therefore, it is important to choose these variables
correctly.
The ESI process is affected by aerosol droplet size, liquid surface tension,
solvent volatility, surface charge, ion solvation strength and solution
conductivity. Specifically, ESI is not favored by large droplets with high
surface tension, low volatility, low surface charge, strong ion solvation and
high conductivity. Conversely, ESI is favored by small droplets with low
surface tension, high volatility, high surface charge, weak ion solvation and
low conductivity.
Mixed organic/aqueous solvent systems that include organic solvents such

as methanol, acetonitrile, and isopropyl alcohol are recommended for ESI.
Using only water as a solvent is not recommended. Volatile acids and bases
give good results. Salt solutions with concentrations above 10 mM are not
recommended. Strong mineral acids and bases can damage the instrument
and should not be used.
To obtain good ESI follow these rules:
• Keep non-volatile salts and buffers out of the solvent system. For
example, avoid the use of salts containing sodium, potassium or
phosphate. Use ammonium or acetate 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).
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 type 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). Refer to Table 3,
Guidelines for setting operating parameters for LC/APCI/MS.

1Introduction
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 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.

1 Introduction
Should I Use Sheath, Auxiliary, and/or Sweep Gases?
Should I Use Sheath, Nitrogen gas can be applied to the system using any combination of the
Auxiliary, and/or three gas sources: Auxiliary gas, Sweep gas, and/or Sheath gas. When
Auxiliary gas is being used, nitrogen flows through the ion source nozzle and
Sweep Gases? 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 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 solution exits the ESI or APCI nozzle. Note that
Sheath gas is not used with the nano spray ionization source.
When you are analyzing complex matrices such as plasma or non-volatile

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 must be individually optimized with Sheath gas, Sweep gas, and
Auxiliary gas to determine the combination that will provide optimum
performance. It is especially important to optimize with each gas
independently before you perform experiments using MSn techniques and
before you perform any quantitative analysis experiments. Refer to Table 2
and Table 3 for additional information on using supplemental gas flows.

1 Introduction
How Can I Introduce My Samples into the MS Source?
How Can I Introduce You can introduce your samples into the MS source in a variety of ways.
My Samples into the Refer to Table 1.
MS Source? 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 tuning purposes. This would be done to determine the structure
of an unknown compound or the fragmentation pattern of a standard.
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 source. This
technique is used to introduce a sample at a steady rate and at higher solvent
flow rates. This is done for tuning on an analyte of interest 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 pure samples into an LC

flow. This technique is also used in ESI or APCI to introduce analytes
automatically into the LC flow and then into the MS detector.
Finally, you can perform LC/MS experiments by using an 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 Finnigan LXQ
Getting Connected for plumbing diagrams for methods of sample
introduction.

1 Introduction
How Can I Introduce My Samples into the MS Source?
Table 1. Sample introduction techniques

Sample Introduction
Analytical Technique Figure Reference
Technique
Syringe Pump Flow Syringe pump* ESI automatic tuning and Finnigan LXQ Getting
(no LC Flow) calibrating Started
ESI analysis of a pure analyte Figure 2-5
solution
LC Flow Without Syringe pump into LC flow ESI or APCI automatic Finnigan LXQ Getting
Chromatographic (connected by Tee union)* optimization of tuning on analyte Started
Separation of interest Figure 4-1 (ESI)
(no column) ESI or APCI analysis of a pure Figure 6-1 (APCI)
analyte solution
Loop injection into LC flow ESI or APCI analysis of a pure Finnigan LXQ Getting
analyte solution Started
Figure 5-6 (ESI)
Figure 8-1 (APCI)
Autosampler injection into ESI or APCI analysis of a pure Finnigan LXQ Getting
LC flow analyte solution Connected
(one or multiple injections) Figure 11-5 (ESI)
Figure 11-8 (APCI)
LC Flow With Autosampler injections into ESI or APCI analysis of mixtures
Chromatographic LC column via LC flow
Separation (one or multiple injections)
*Provides steady state introduction of sample (direct infusion)

1Introduction
What Types of Buffers Should I Use? What Types Should I Avoid?
What Types of Buffers Many LC applications use nonvolatile buffers such as phosphate and borate
Should I Use? What buffers. It is best to avoid the use of nonvolatile buffers with the MS
detector because they can cause the following problems:
Types Should I Avoid?
• 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)
CAUTION Store and handle all chemicals in accordance with standard

safety procedures. The Material Safety Data Sheets (MSDS) describing
the chemicals being used are to be freely available to lab personnel for
them to examine at any time.

1 Introduction
How Should I Set Up the MS Detector for Various LC Flow Rates?
How Should I Set Up The ESI probe can volatilize ions from liquid flows2 of 1 μL/min to
the MS Detector for 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
Various LC Flow analytical LC.
Rates?
The APCI probe can volatilize 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.
Table 2 provides guidelines for ESI operation for ion transfer capillary
temperatures and gas flow rates for various LC solvent flow rates.
Table 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.

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
Suggested Column
LC Flow Rates Capillary Sheath Gas Auxiliary and/or Sweep Gas
Size
Temperature
Infusion or LC at flow Capillary Typical setting: Not required Not required

rates of < 10 μL/min 150 to 200 °C Typical setting: Typical setting:
5 to 15 units 0 units
LC at flow rates from 1 mm ID Typical setting: Required Not required, but might help
50 to 200 μL/min (microbore column) 200 to 275 °C Typical setting: depending on conditions
20 to 40 units Typical setting:
0 to 20 units
LC at flow rates from 2 to 3 mm ID Typical setting: Required Not required, but usually helps to
100 to 500 μL/min (narrow bore column) 250 to 350 °C Typical setting: reduce solvent background ions
0 to 20 units
LC at flow rates from 4.6 mm ID Typical setting: Required Required

0.4 to 1 mL/min (standard column) 300 to 400 °C Typical setting: Typical setting:
60 to 100 units 10 to 40 units
*
Note: Be sure to choose either Auxiliary gas and/or Sweep gas according to the hints in the topic Should I Use Sheath, Auxiliary, and/or
Sweep Gases?
Table 3. Guidelines for setting operating parameters for LC/APCI/MS*

Ion Transfer
Vaporizer
LC Flow Rate Capillary Sheath Gas Auxiliary and/or Sweep Gas
Temperature
Temperature
LC at flow rates from Typical setting: Typical setting: Required Not required, but usually helps to reduce
0.2 to 2 mL/min 150 to 350 °C 350 to 500 °C Typical setting: solvent background ions
0 to 20 units
*
Note: Be sure to choose either Auxiliary gas and/or Sweep gas according to the hints in the topic “Should I Use Sheath, Auxiliary, and/or Sweep
Gases?” on page 7.

1 Introduction
What is Tuning and Calibration of the MS Detector All About?
What is Tuning and To optimize the performance of data acquisition on the Finnigan LXQ MS
Calibration of the MS detector, you tune and calibrate in four steps:
Detector All About? • 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,
you 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 Finnigan LXQ MS detector
automatically.
• After your tune method is optimized, you 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.

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. You also have to tune the MS detector 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. Refer to the
topic: Setting Up the Syringe Pump for Tuning and Calibration in
Chapter 3.
• Introduce the sample from the syringe pump into the effluent of the LC
by using a Tee union. Refer to the topic: Setting Up to Introduce
Sample by Syringe Pump into Solvent Flow from an LC in Chapter 4.
The first method is good 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.
Table 4 summarizes methods of sample introduction for each of the

calibration and tuning procedures.
1 Introduction
What is Tuning and Calibration of the MS Detector All About?
Table 4. Summary of methods of sample introduction for calibration and tuning

Calibrating Tuning
Sample/ Semi- Semi- Collision
Check Auto Auto Manual
Sample Intro auto auto Energy
Calibration solution/
Syringe pump
9 9 9 9 9 9 9
Your tune solution/
Syringe pump
9 9 9 9
Your tune solution/
Syringe pump into LC flow by
9 9 9 9
using Tee union

1Introduction
What Types of Experiments Can I Perform with the Finnigan LXQ MS Detector?
What Types of This topic describes several types of experiments that you can perform with
Experiments Can I the Finnigan LXQ MS detector. The experiments can be grouped into the
following categories:
Perform with the
Finnigan LXQ MS • General MS or MSn
Detector? • Data-Dependent™
• Ion Mapping™
• Ion Tree
You can specify which type of experiment you want to perform in the
Xcalibur Instrument Setup window, and then save it in an Xcalibur
Instrument Method (.meth) file.
Note Procedures for these experiments are beyond the scope of this
Finnigan LXQ Getting Started manual. For more information you
may contact: a)Thermo North American Customer Support Center in
West Palm Beach, Florida, USA, at 1-800-765-4532 , b) Thermo
European Customer Institute, UK, at 44-1442-233-555, c)Thermo
Japan, 81-66387-6681, d) Thermo China, 86-10-6621-0839.
Customers in other regions please ask your Thermo representative
about the Thermo Support Center that serves you. Online Help may
also be useful.
General MS or MSn A General MS or MSn experiment is best used to collect qualitative data for
Experiments structural analysis . However, you can also use a General experiment for the
quantitative analysis of known compounds. The Xcalibur data system
includes an Instrument Method template in 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
In a General MS quantitation experiment, you need to specify: a) the mass

range of your analyte(s) of interest, b) a parent (precursor ion) that
fragments into distinctive product ions, and c) the mass-to-charge ratios of
all the parent ions of interest. The Finnigan LXQ 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 MSn) for which you want
data in the Scan Event Settings group box. If you specify MS/MS or MSn,

1 Introduction
you then choose the parent ion(s) for which you want data in the MSn
Settings table. The Finnigan LXQ MS detector can then collect distinct
qualitative information for structural analysis or for spectral reference.
Figure 1. MS Detector Setup page in Instrument Setup, showing a template for a General MS experiment
The Finnigan LXQ MS detector can generate reproducible, analyte-specific

spectra, even from laboratory to laboratory. Consequently, reference spectra
that are generated with the Finnigan LXQ MS detector can be used to
confirm structures of compounds generated with other Finnigan LXQ
systems.
Data-Dependent A Data-Dependent experiment is best used for the qualitative analysis of

Experiments unknown compounds for structure elucidation or confirmation. These
experiments are designed to maximize the data obtained while requiring
minimal user input. The Finnigan LXQ MS detector uses the information

1Introduction
in a Data-Dependent experiment to make decisions about subsequent

experiments automatically. Xcalibur Instrument Setup contains the
Instrument Method templates that you need to get started with
Data-Dependent experiments.
A Data-Dependent experiment produces a great deal of data from a single

sample analysis. In a Data-Dependent experiment, you can specify parent
ions for fragmentation or you can let the Finnigan LXQ MS detector
automatically select the ions for fragmentation. The Finnigan LXQ 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 that the user specify that one or

more scan events of an experiment segment are to be run as
Data-Dependent. For example, in a Data-Dependent Triple Play
experiment for a mixture of compounds, the Finnigan LXQ MS detector
decides which parent ion to isolate, estimates the charge state of the parent
ion (zoom scan) and performs MS/MS for the appropriate product ion mass
range. For an example of a Data-Dependent Triple Play experiment
template, see Figure 2.
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 specified parent ions is detected, you can acquire product spectra
and analyze the information.

1 Introduction
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, you click on 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 Finnigan LXQ MS
detector generates product spectra. Parameters that you might specify,
for example, include threshold values for the intensity of the MS or MSn
ion signal.

1Introduction
You can find useful structural information about your compound

automatically with the simplest Data-Dependent experiment,
Data-Dependent MS/MS. In this experiment you specify only the MS scan
range, you do not need to specify a parent ion. The Finnigan LXQ MS
detector can then collect full scan MS data, pick the most intense parent ion
in the spectrum, and then fragment the ion to generate product ions.
You can 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
Finnigan LXQ 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 Finnigan LXQ MS detector can
store the MSn fingerprint data in a custom MSn library spectrum. The data
are 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
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 Finnigan LXQ 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

1 Introduction
• 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 1-3 for an example of an Ion Mapping experiment
template.
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

1Introduction
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
peptide). 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 Finnigan LXQ MS detector can collect MSn
data automatically. You can specify a particular parent ion for
fragmentation, or you can let the Finnigan LXQ MS detector find the
parent ions automatically and fragment them to any level between MS2 and
MS10. The Finnigan LXQ 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.

1 Introduction
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 Finnigan LXQ 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
advancing to the next MSn level.
For example, if you specify a Maximum Depth of 3 and a Maximum Breadth

of 2 in an Ion Tree experiment, the following occurs.
With either Depth or Breadth Focus, the Finnigan LXQ 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).

1Introduction
• Then, if you chose the Depth Focus, after the most intense ion of the
MS spectrum is fragmented—producing an MS/MS spectrum—the
Finnigan LXQ 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 Finnigan LXQ
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
Finnigan LXQ 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 Finnigan LXQ 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.

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 Finnigan LXQ 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 sections:
• 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

2 Setting Up the Ion Source for Tuning and Calibrating the MS Detector
Placing the LC/MS System in Standby
Placing the LC/MS The LC/MS system needs to be placed in Standby condition before you can
System in Standby remove the ion source.
To place the LC/MS system in Standby:
1. If necessary, stop the flow of solvent to the API source:
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 on 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 on the Pump Off button to
stop the MS pump.
2. If Tune Plus is not already open, choose Start > Programs > Xcalibur >
LTQTune 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
OnOffStandby different states of the On/Standby button are shown at the left.)
3. If the MS detector is On, click on the On/Standby button to place the

MS detector in the Standby mode. When the MS detector is in Standby,
the Finnigan LXQ MS detector turns off the ion source sheath gas,
auxiliary gas, high voltage, and syringe pump.
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 topic: Removing the APCI Probe.

Removing the APCI Probe
Removing the APCI This topic describes how to remove the APCI probe from the Ion Max ion
Probe source housing.
Note The following procedures assume that you are familiar with your
instrument and software. If you need additional guidance, refer to
Finnigan LXQ online Help, Finnigan LTQ Getting Connected, Finnigan
Ion Max API Source Hardware Manual, or the Finnigan LTQ 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.
To 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.
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.

Vaporizer Heater Cable 8 kV Cable (white)

(black cable with silver plugged into Corona
cable plug) Needle High Voltage
Recepticle (on right side
of the Ion Max-S ion
Sheath Gas Fitting source housing)
(S on APCI probe body,
blue fitting)
Sample inlet fitting
Auxiliary Gas Fitting

(A on APCI probe
body, green fitting)
Figure 5. Ion Max ion source housing with APCI probe installed
ESI Interlock Socket

API Interlock Block
Ion Source Housing

Locking Levers
Probe Collar
Probe Locking Nut
Ion Source Housing Drain
Figure 6. Ion Max-S 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 by turning the ion source locking
levers 90 degrees (see Figure 6). Also see the next Section, Removing
the Ion Max Ion Source Housing and Figure 8.
b. Remove the ion source housing by pulling the housing straight off of
the ion source assembly.
c. The corona needle is in the corona assembly which is inside of the
ion source housing. Using pliers, grasp the corona needle and pull it
straight out of the corona needle contact (see Figure 7).
d. Remount the ion source housing per the Section “Installing the Ion
Max Ion Source Housing” on page 34 and Figure 11. Or, place the
ion source housing in a safe location for temporary storage.
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 topic:
Removing the Ion Max Ion Source Housing.
If you do not want to install the ion sweep cone, go to the topic: Installing
the ESI Probe.

Corona
Needle
Contact
Corona
Needle
(grasp this with pliers to
remove it)
Figure 7. Corona needle, view from inside of the Ion Max housing

Removing the Ion Max Ion Source Housing
Removing the Ion Max The Ion Max ion source housing is removed to access the ion sweep cone.
Ion Source Housing
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.
To remove the ion source housing:
1. Detach 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-S ion source housing, detail of components

Installing the Ion Sweep Cone
Installing the Ion The ion sweep cone is a metallic cone that is installed over the ion transfer
Sweep Cone 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 as follows:
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

Installing the Ion Sweep Cone
Gas
Inlet
Figure 10. Ion sweep cone, showing the gas inlet
CAUTION AVOID BURNS. At operating temperatures, the ion

transfer tube and spray shield 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.

Installing the Ion Max Ion Source Housing
Installing the Ion Max Reinstall the Ion Max ion source housing as follows:
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
Figure 11. Rear view of the Ion Max-S ion source housing

Ion Source
Housing
Guide
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. Reattach the ion source drain tube as follows:

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 with pump oil vapor if the API source drain tube
and the (blue) forepump exhaust tubing are connected to the same fume
exhaust system.
Your laboratory must be equipped with at least two independent fume
exhaust systems. Route the (blue) forepump exhaust tubing to one of
these fume exhaust systems. Route the drain tube from the API source to
a waste container. Vent the waste container to the other 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.

Installing the ESI Probe
Installing the ESI To install the ESI probe:

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, you need to follow
the procedure for installing a sample tube and PEEK safety sleeve that is
outlined in the topic Installing a New Fused-Silica Sample Tube and
PEEK Safety Sleeve in the Finnigan Ion Max API Source Hardware
Manual.
2. Ensure that the probe locking nut on the ion source housing (Ion Max
or Ion Max-S) is loosened. The probe locking nut is shown in 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

(behind API Interlock APCI Vaporizer
Block) Heater Cable (black)
API Interlock Block
Aux Gas outlet (A)
Sheath Gas outlet (S)
Aux Gas (green 8 kV Cable

fitting) (white)
Sheath Gas (blue

fitting)
Probe Locking Nut
Grounding Bar
Probe Collar
Probe Port
Figure 13. Ion Max-S ion source housing with some components labelled.

.
ESI Needle High Voltage
Connector Receptacle Aux Gas
Inlet (A)
Sheath Gas
Inlet (S) Sample
Inlet Fitting
Guide
Pin
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.
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 8 kV Cable
(black cable) (white)
Sheath Gas Inlet

(S on ESI Probe
body, blue
Grounding
Bar
Auxiliary Gas
Inlet (A on ESI
Probe body,
Grounding
green fitting)
Union
Probe locking
nut
Sample Inlet
Figure 15. Ion Max-S 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. (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.” .

Tuning and Calibrating
Chapter 3
Automatically in the ESI/MS Mode
This chapter provides information on how to tune and calibrate the

Finnigan LXQ 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 (refer to Table 2 on page 1-12.). 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 topic: 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 topics:
• 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
• Tuning the MS Detector Automatically in the ESI/MS Mode

3 Tuning and Calibrating 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

Setting Up the Syringe Pump for Tuning and Calibration
Setting Up the Syringe You introduce tuning and calibration solution into the ESI source with a
Pump for Tuning and syringe infusion pump. A syringe pump allows you to infuse a sample
solution into the ESI source for extended periods of time.
Calibration
The syringe pump and syringe are located on the front panel of your
Finnigan LXQ MS detector. To infuse solution for tuning and calibration,
you install 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.
To 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 Fingertight fitting

(P/N 00101-18202) (P/N 00101-18081)
Ferrule Teflon Tube

(P/N 00101-18196) (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.” for the
calibration solution formulation procedure.)
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.

Setting Up the Syringe Pump for Tuning and Calibration
7. Connect the infusion line with a (brown) fingertight fitting and a

(brown) ferrule to the free end of the LC union.
8. Connect the other end of the infusion line with a (red) fingertight
fitting and a (brown) ferrule to the grounding union.
The syringe pump is now properly set up for infusing solution into the MS
detector.
Figure 17. ESI/MS plumbing connections for the fused-silica infusion line

Setting Up the MS Detector in the Xcalibur Data System for Tuning and Calibration
Setting Up the MS You first tune manually with calibration solution to establish a stable spray
Detector in the 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
Xcalibur Data System parameters that affect ion detection.
for Tuning and
Calibration Note The following procedures assume that you are familiar with your
Finnigan LXQ instrument and the Tune Plus window. If you need
additional guidance, refer to: Finnigan LXQ online Help, Finnigan LXQ
Getting Connected, and/or the Finnigan LXQ Hardware Manual.
CAUTION Before you begin normal operation each day, ensure that you
have sufficient nitrogen for your ESI source. If you run out of nitrogen,
the Finnigan LXQ MS detector automatically turns Off to prevent
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 Finnigan LXQ MS detector automatically turns Off
during an analytical run, you could lose data.)
Use the following procedure to 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 on the Instrument Setup button to display the Instrument
Setup window.
c. Click the Finnigan LXQ 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 on

the On/Standby button to take the MS detector out of the Standby (or
OnOffStandby 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 ion source.
• The Finnigan LXQ MS system applies high voltage to the ESI ion
source.
• The Xcalibur data system shows a real-time mass spectrum display

in the Spectrum view.
Figure 18. Tune Plus window, showing the MS detector in the Standby mode
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. Then, 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 on 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:
a. On the Control/Scan Mode toolbar, click on 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 on 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.
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 on 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 on 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.

Testing the Operation of the MS Detector in the ESI/MS Mode
Testing the Operation You are now ready to test if your MS detector is operating properly. To test
of the MS Detector in 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 the
the ESI/MS Mode calibration solution.
The procedure to test the operation of the MS detector in the ESI/MS mode
is:
1. Click on 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. This
will: i) make active the Flow Rate spin box and, ii) the pump will
start when the dialog box is completed and OK is clicked.
b. Enter 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 Finnigan LXQ system. If you are using
another type of syringe, follow the procedures in (c) or (d) below.
c. If you are using the Unimetrics syringe supplied with the LXQ, or a
Hamilton syringe, set up the syringe parameters as follows:

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

option button to specify the syringe that you are using.
ii. Click on the Volume 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 Finnigan LXQ MS detector
automatically sets the syringe ID to its proper value of
3.260 mm.
d. If you are using a syringe that is not a Unimetrics or a Hamilton
syringe, set up the syringe parameters as follows:
i. In the Type group box, click on 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.
f. Click OK. This will start the syringe pump and close the Syringe
Pump dialog box.
3. On the File/Display toolbar, click on 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
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 on the API Source button to open the ESI Source dialog box.
(See the Spray Current readback shown in Figure 19.)
d. Observe the value for the Spray Current readback and the values for
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?
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.
• 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.
• Ensure that the sheath gas flow rate is optimal.
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 Finnigan LXQ MS detector as it is, and go to the next
topic: Tuning the MS Detector Automatically in the ESI/MS Mode.

NL=8.74E4
IT=1.778
Figure 22. Spectrum view of the Tune Plus window, showing ionization time (IT) and normalization level (NL) of the
calibration solution

Tuning the MS You tune the MS detector automatically in the ESI/MS mode to optimize
Detector important parameters, including heated capillary voltage and tube lens
voltage.
Automatically in the
ESI/MS Mode To tune the MS detector automatically:
1. On the Control/Scan Mode toolbar, click on the Tune button to display

the Tune dialog box.
2. If necessary, click on 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
4. In the Mass spin box, enter 195.1 to specify that the Finnigan LXQ MS
detector optimize your Tune Method on the peak at m/z 195.1.
Tuning the MS Detector Automatically in the ESI/MS Mode
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 of calibration solution (this
solution is specified in “ESI Calibration Solution: Caffeine, MRFA,
Ultramark 1621” on page 137).
b. Click OK to close the message box, and return to the Tune dialog
box.
6. On the File/Display toolbar, click on 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 Finnigan LXQ 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 on 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 3-47.
9. Click on 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 Finnigan LXQ MS detector. See Figure 26.
You have now successfully tuned the MS detector in ESI/MS mode using
the calibration solution.

Figure 24. Tune Plus window, showing the windows obtained from a typical automatic tune procedure

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 Finnigan LXQ automatic
tuning procedure

Saving Your ESI/MS Tune Method
Saving Your ESI/MS You can save the parameters you just set in a Tune Method specific to your
Tune Method particular analyte and solvent flow rate. (In this case, you save the settings
obtained using the 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, as follows:
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.

Saving Your ESI/MS Tune Method
3. Click on the File Name text box, and then enter the name under which
you want to save the tune method. As an example, you could enter
ESImyTune to name the Tune Method ESImyTune.LTQTune.
4. Click on Save to save the Tune Method and return to the Tune Plus
window. If you entered the name given in the example above, the Tune
Method is named ESImyTune.LTQTune.
Once you have tuned the MS detector, you are ready to calibrate.

Calibrating the MS Detector Automatically
Calibrating the MS Use the following procedure to calibrate the MS detector automatically
Detector from the Tune Plus window:
Automatically 1. Choose Control > Calibrate to display the Calibrate dialog box.
2. If necessary, click on 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 on Start. A message box displays the following message:

Ensure that the syringe contains at least 450 μL of calibration solution (this
solution is specified in “ESI Calibration Solution: Caffeine, MRFA,
Ultramark 1621” on page 137).
b. Click on 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 Finnigan LXQ 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 Finnigan LXQ 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 Finnigan LXQ 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.
Later you can change the solution flow rate and optimize the MS detector
parameters on reserpine or on your particular analyte. See “Optimizing the
MS Detector Tune Automatically” on page 4-75.

Before you tune with your analyte, go to the next topic: Cleaning the MS
Detector after Tuning and Calibrating.
Figure 29. Tune Plus window with Calibrate dialog box, showing the results of a successful semi-automatic calibration
procedure

Cleaning the MS Detector after Tuning and Calibrating
Cleaning the MS This topic describes how to clean your MS detector after using the
Detector after Tuning calibration solution, in preparation for acquiring data on your analyte of
interest.
and Calibrating
Use the following procedure to clean the MS detector after calibrating:
1. Click the On/Standby button to put the MS detector in Standby mode.

When the MS detector is in Standby, the Finnigan LXQ MS detector
OnStandby turns off the sheath gas, Auxiliary gas, Sweep gas, ESI high voltage, and
syringe pump. The MS detector stops scanning, and the Finnigan LXQ
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
Finnigan LXQ 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 31 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.

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
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.
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
50:50 methanol/water.
b. Sonicate the components for 15 min.
8. Reinstall the ion transfer capillary.
9. Reinstall the ion sweep cone as described in topic “Installing the Ion
Sweep Cone” on page 32.
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 check
that the solution is exiting the tip of the ESI probe on the inside of

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 34.
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), optimize the tune further by following
the procedures in the next chapter: Tuning with Your Analyte in
LC/ESI/MS Mode.

Tuning with Your Analyte in
Chapter 4
LC/ESI/MS Mode
This chapter provides information on tuning the MS detector in the

LC/ESI/MS mode using your analyte. You will optimize the sensitivity of
the MS detector to your analyte through an automatic procedure. The
customized Tune Methods contained in your Finnigan LXQ data system are
optimized for a wide range of applications, and they can often be used
without further tuning of your MS detector.
The most important parameters that affect signal quality with the ESI
interface 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, and 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.

4 Tuning with Your Analyte in LC/ESI/MS Mode
Note Based on the LC flow rate of your experiment, you can specify the
value of each of the following tuning parameters on the Finnigan LXQ MS
detector: sheath, Auxiliary, and Sweep gas pressures, ESI needle (or
“spray”) voltage, ion transfer capillary temperature, and probe position.
With the exception of ESI needle voltage and probe position,
recommended tuning parameter values can be obtained from Table 2. page
1-12. Automatic tuning sets the values of the parameters not mentioned
here.
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.
Note 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, you can go to Chapter 5, “Acquiring ESI
Sample Data Using the Tune Plus Window.” to acquire sample data.
Before you optimize the tune for your analyte of interest, ensure that the
Finnigan LXQ MS detector has been calibrated within the previous three
months. If the system needs to be calibrated, refer to the procedures in the
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, you perform the following tasks:
• Set up the MS detector for your specific analyte.
• 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.
• 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
• Saving the ESI/MS Tune Method

Setting Up to Introduce Sample by Syringe Pump into Solvent Flow from an LC
Setting Up to This topic describes setting up the MS detector to introduce your analyte by
Introduce Sample by syringe pump into solvent flow from an LC.
Syringe Pump into Make the plumbing connections for ESI/MS sample introduction from the
Solvent Flow from an syringe pump into solvent flow from an LC as following:
LC
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 3-44.)
2. Fill a clean, 500-μL Unimetrics syringe with the 10 pg/μL solution of

reserpine or your analyte of interest. If you are using the reserpine
tuning solution refer to Appendix A, “Sample Formulations.” for a
procedure to make this solution. Use the dilution made in step 9 of the
Reserpine Tuning Solution and Reserpine APCI Sample Solution
procedure on page 138.
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 (natural color)
fingertight fitting and a (natural color) ferrule to the LC Tee union.

PEEK Tubing LC Tee Union PEEK Tubing Grounding Union

(P/N 00301-22912) (P/N 00101-18204) (P/N 00301-22912) (P/N 00101-18182)
From Divert /
Inject Valve
Stainless Steel
Ferrule
Stainless
Steel Nut Ferrules
(P/N 00101-18196)
Infusion Line
Ferrules Fused-Silica Capillary
(P/N 00101-18120) (P/N 00106-10504)
Fingertight Fingertight
Fitting Fittings
(P/N 00101-18195) (P/N 00101-18081)
From LC Union
Figure 30. ESI/MS plumbing connections for the LC Tee union
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 topic: Installing a New Fused-Silica
Sample Tube and PEEK Safety Sleeve in the Finnigan 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 topic:
Installing a New Stainless Steel Needle in the ESI Probe and Installing a
New Fused-Silica Sample Tube and PEEK Safety Sleeve in the Finnigan
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.

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 From To LC Tee From

Union LC Union LC
3 3
4 2 4 2
5 1 5 1
Plug Plug
(optional) 6 (optional) 6
To Waste To Waste
Detector Position Waste Position
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
LC) from the divert/inject valve to the LC, as follows:

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:

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.

Setting Up to Tune the MS Detector with Your Analyte
Setting Up to Tune the Use the following procedure to set up the MS detector to tune automatically
MS Detector with on your analyte in ESI/MS mode. (In this example, you can use the
reserpine solution described in Appendix A: Sample Formulations, or you
Your Analyte can use a solution of your analyte of interest.)
CAUTION Do not infuse calibration solution at syringe pump flow rates

above 10 μL/min. Your system could be contaminated by high
concentrations of the Ultramark 1621 component of the calibration
solution.
additional guidance, refer to: Finnigan LXQ online Help and/or
Finnigan LXQ 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.
b. Click on the Instrument Setup button to display the Instrument
Setup window.
c. Click on the Finnigan LXQ button to display the New Method

page.
d. Click on the Tune Plus button to display the Tune Plus window.
2. In Tune Plus, click on the On/Standby button to take the MS detector

out of Standby mode and turn it On. The MS detector begins scanning,
the Finnigan LXQ MS detector applies high voltage to the ESI probe,
On Standby
and the Finnigan LXQ 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 on 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
4. Define the scan parameters for tuning with your analyte in the ESI/MS
mode, as follows:
a. In the Instrument Control toolbar, click on the Define Scan button

to open the Define Scan dialog box. See Figure 32.
150 to 2000.
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,
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
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, enter 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. See Figure 32.
j. In the Width text box, enter 2.0 to set the width of the scan range to
2.0 daltons. See Figure 32.
k. Ensure that the settings in your Define Scan dialog box are the same
as those shown in Figure 32 if reserpine is the analyte being used. If
you are using another analyte the Define Scan dialog box settings
will be appropriate for the 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 on the Centroid/Profile

button to toggle the data type to Profile (the picture on the button
should be as shown here).
6. Click on 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).
You have completed setting up to tune your MS detector with your analyte
in ESI/MS mode.

Optimizing the MS Detector Tune Automatically
Optimizing the MS Optimize the MS detector tune automatically to maximize the ion
Detector Tune transmission of reserpine (or your analyte of interest) for a high-flow
experiment. It is recommended that you begin optimizing after you have
Automatically 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 on the reserpine m/z 609.2 at an LC flow rate of 400 μL/min. You
can follow the same procedure with your analyte of interest and at your
particular LC flow rate. (Refer to Table 2 for guidelines about setting flow
rates and temperatures.)
1. On the Control/Scan Mode toolbar, click on the Tune button to display

the Tune dialog box.
2. If necessary, click on the Automatic tab to display the Automatic tuning

4. In the Mass spin box, enter 609.2 (or the appropriate mass of your
analyte of interest) to specify that the Finnigan LXQ MS detector is to
use the peak at m/z 609.2 to optimize your tune.
5. Ensure that the Divert/Inject valve is in the Detector position, as

follows:
a. Click on the Divert/Inject button to open the Divert/Inject Valve

dialog box. See Figure 34.
b. Select the Detector option button.
c. Click Close.
6. Start the automatic tuning procedure from the Tune dialog box, as
follows:

Ensure that the syringe pump contains at least 450 μL of the

10 pg/μL reserpine tuning solution. See Appendix A, “Sample
Formulations.” for a procedure for making the reserpine tuning

solution. Use the dilution made in step 9 of the Reserpine Tuning

Solution and Reserpine APCI Sample Solution procedure on
page 138
b. Click OK to close the message box, and return to the Tune Plus
window.
7. In the File/Display toolbar, click on the Graph View button to display

the Graph view.

Figure 34. Divert/Inject Valve dialog box
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 the 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).

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

Saving the ESI/MS Tune Method
Saving the ESI/MS To save your ESI/MS Tune Method (for a high-flow experiment using
Tune Method your analyte) when automatic tuning is complete:
Note Save the Tune Method while the MS detector is On.
1. Choose File > Save As to display the Save As dialog box. See Figure 36.
3. Click on 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.
The Tune Method is now properly saved and you are ready to acquire data
on your analyte of interest.

Acquiring ESI Sample Data
Chapter 5
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 follow the
same procedure with your analyte of interest.
information, refer to the Finnigan LXQ online Help, Finnigan LXQ
Getting Connected, and/or Finnigan LXQ Hardware Manual.
Ensure that you have completed the procedures in the topics Tuning and
Calibrating Automatically in the ESI/MS Mode and Tuning with Your
Analyte in LC/ESI/MS Mode.
• 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 Type

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 Prepare to acquire MS/MS data in the Full scan type on reserpine (or on
MS/MS Data in the your analyte of interest). You need to optimize the isolation width and the
relative collision energy parameters before you acquire MS/MS data.
Full Scan Type
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 Finnigan LXQ 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
Optimizing the Isolation Optimize the isolation width and set up to optimize the collision energy for
Width and Setting Up to an MS/MS experiment, as follows:
Optimize the Collision
Note The collision energy is optimized on the Finnigan LXQ MS
Energy 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.
The following procedure is based on the use of a reserpine solution. A

procedure for making a suitable reserpine solution is given in Appendix
A, “Sample Formulations.” . Use the dilution made in step 9 of the
Reserpine Tuning Solution and Reserpine APCI Sample Solution
procedure on page 138 (10 pg/mL). A different analyte may be used
with appropriate changes made to the procedure (Define Scan dialog box
settings, m/z values, etc).
1. From the Tune Plus window, click On/Standby to take the MS detector
OnStandby out of Standby mode and turn it On.
2. Ensure that 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 on the Define Scan button to open the Define Scan dialog
box. See Figure 37.
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 of 3 u
(MSn Settings group box, Isolation Width text box, Figure 37).
Click Apply when you are finished entering the values. 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. Verify that your system does not leak.
5. Click on 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 on the On option button to

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

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 on the Unimetrics or Hamilton
syringe click the option button to specify the proper syringe
type.
ii. Click the Volume 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 Finnigan LXQ 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

(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 text box, verify that an isolation width of m/z 3
is being used, per step 3b.
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 The Isolation Width is typically m/z~1-3. After the Isolation
Width is optimized you may 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 spin box, enter 20 to specify an initial
value of 20 for the collision energy. Then, 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.
10. When you have clearly identified a product ion mass-to-charge ratio for
reserpine (or your analyte of interest), click on the Tune button to
display the Tune dialog box.

11. In the Tune dialog box, click on the Collision Energy tab to display the
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 the adjacent box
active. Type 397.2 to specify the product ion at m/z 397.2 for reserpine.
The Finnigan LXQ MS detector can optimize collision energy
automatically by using this product ion of reserpine.

Figure 40. Tune dialog box, showing the Collision Energy page
Optimizing the Collision The optimum relative collision energy is the one that produces the
Energy Automatically for maximum product ion intensity. Optimize the relative collision energy
automatically for the ESI/MS/MS analysis of reserpine (or your analyte of
an MS/MS Experiment interest), as follows:
1. In the Tune dialog box, on the Collision Energy page (Figure 40), click
Start to start the optimization procedure. A message box displays the
following message:
Ensure that the 500 microliter syringe is full.
Ensure that the syringe contains at least 450 mL of the 10 pg/mL

reserpine tuning solution (see Note about reserpine solution at the
beginning of the subtopic “Optimizing the Isolation Width and Setting
Up to Optimize the Collision Energy” on page 82).

2. Click OK to close the message box, and leave the Tune dialog box open.
Your Tune Plus window should now look similar to the one shown in
Figure 41.
3. In the Spectrum view of Tune Plus, observe the MS/MS Full scan
spectrum of reserpine (or that of your analyte of interest).
4. When the collision energy is optimized, the Accept Optimized Value

dialog box appears. See Figure 42.
5. Click Accept to accept the new collision energy value, and return to
Tune Plus. The new value is displayed in the Define Scan dialog box.
6. In the Syringe Pump dialog box, select the Off option button to turn off
the syringe pump. Click Close to close the Syringe Pump dialog box.
7. Click Cancel to close the Tune dialog box.
After you optimize the relative collision energy, the Finnigan LXQ MS
detector is ready to acquire MS/MS data on your analyte of interest.

Figure 41. Tune Plus window, showing MS/MS product ions of reserpine in the Spectrum view. The Tune dialog box from
Figure 40 is not shown.
Figure 42. Accept Optimized Value dialog box

Setting Up to Introduce Sample by Loop Injection into Solvent Flow from an LC
Setting Up to Set up to introduce sample by loop injection into solvent flow from an LC.
Introduce Sample by
To make the plumbing connections for ESI/MS sample introduction by
Loop Injection into loop injection into solvent flow from an LC, do the following:
Solvent Flow from an
LC 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 topic: Installing a New Fused-Silica Sample Tube
and PEEK Safety Sleeve in Finnigan 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 topic: Installing a New
Stainless Steel Needle in the ESI Probe and Installing a New Fused-Silica
Sample Tube and PEEK Safety Sleeve in Finnigan Ion Max API Source
Hardware Manual.
8. Connect an appropriate length of (red) PEEK tubing (transfer line from

the divert/inject valve) from the divert/inject valve to the (stainless steel)
grounding union, as follows. See Figure 43.
a. Connect a length of PEEK tubing fitted with a (stainless steel) nut

and a (stainless steel) ferrule to port 3 of the divert/inject valve.
See Figure 44.
b. Connect the other end of the PEEK tubing with a (natural color)
fingertight fitting and a (natural color) ferrule to the free end of the
grounding union.
9. Connect a 2 μL sample loop with (stainless steel) nuts and (stainless

steel) ferrules to ports 1 and 4 of the divert/inject valve. (See Figure 44)
Figure 43. ESI/MS plumbing connections for the divert/inject valve and
grounding union

To Grounding From To Grounding From

Union LC Union LC
3 3
4 2 4 2
5 1 5 1
Inject Inject
Port 6 Port 6
To Waste To Waste
Load Position Inject Position
Figure 44. Divert/Inject valve, showing the correct setup for analysis by loop
injection and showing the flow of liquid through the valve in the
Load and Inject positions
a. Connect a length of the PEEK tubing with a (stainless steel) nut and
b. Connect the other end of the PEEK tubing with a proper fitting and
a ferrule to the outlet of the LC.
a. Connect a length of PEEK tubing with a (stainless steel) nut and

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

Acquiring MS Data in the SIM Scan Type
Acquiring MS Data in Use the following procedure to acquire a file of reserpine data in the selected
the SIM Scan Type ion monitoring (SIM) type from the Tune Plus window. The Finnigan LXQ
MS detector automatically saves the data you acquire on your hard disk.
1. On the Control/Scan Mode toolbar, click the On/Standby button to

take the MS detector out of Standby mode and turn it On. The MS
OnStandby detector begins scanning, the Finnigan LXQ MS detector applies high
voltage to the ESI probe, and a real-time display shows in the Spectrum
view.
2. Verify that the Centroid data type is selected. (The picture on the
button should be the same as that shown here.)
3. Verify that the scan parameters are defined to acquire SIM data for
reserpine (or your analyte of interest), as follows:
a. Click on the Define Scan button to open the Define Scan dialog
box. See Figure 45.
b. Compare the values in your dialog box to those in Figure 45 and
click OK.
Figure 45. Define Scan dialog box, showing typical settings for acquiring reserpine data in the SIM scan type
4. Turn on the LC pump and specify your flow rate of 400 μL/min. Ensure
that your system is free of leaks.

5. On the Control/Scan Mode toolbar, click Acquire Data to open the

Acquire Data dialog box. See Figure 46.
6. Specify the acquisition parameters, as follows:
a. In the File Name text box, enter the name of the analyte. Enter
reserpine if you are using the reserpine solution mentioned in the
Note at the beginning of the subtopic “Optimizing the Isolation
Width and Setting Up to Optimize the Collision Energy” on
page 82.
b. In the Sample Name text box, enter reserpine to specify the sample
identity. If you are not using reserpine, type the name of your
particular analyte.
c. Type a comment about your experiment. (For example, describe the
scan mode, scan type, ionization mode, sample amount, or method
of sample introduction.). The Xcalibur data system includes the
comment on hard copies of your data.
d. In the Acquire Time group box, select the Continuously option
button to acquire data continuously (until you stop the acquisition).
7. Leave the Acquire Data dialog box open during data acquisition, but
move it to a corner of the Tune Plus window.
8. Click on Start in the Acquire Data dialog box to begin acquiring data.
The Acquisition Status group box displays the following message.
State: Acquiring
Time (min):
9. Click the Divert/Inject Valve to open the Divert/Inject Valve dialog

box. See Figure 47.

Figure 46. Acquire Data dialog box, showing typical settings for acquiring data
10. Select the Load option button, and overfill the 2 μL injector loop with
the 250 fg/μL solution of reserpine (see “Reserpine” on page 138 in
Appendix A, or use a solution of your analyte of interest).
11. Select the Inject option button to inject the reserpine solution into the
ESI source. Leave the Divert/Inject Valve dialog box open.
12. Observe the reserpine peak (m/z 609.2), or that of your analyte of
interest, in the Spectrum view, see Figure 48, right side. Wait about 1
min before you inject again (step 13.b, below).
13. Perform the following repetitive sequence to obtain a total of four

consecutive loop injections of reserpine.
a. Select the Load option button, and overfill the injector loop with
the 125 fg/μL solution of reserpine.
b. Select the Inject option button to inject the reserpine solution into
the ESI source, and then observe the Spectrum view.
c. Wait 1 min before performing the next injection.
d. Perform step 13.a through step 13.c three more times.
14. Click Close in the Divert/Inject Valve dialog box to return to the Tune
Plus window.
15. Click Stop in the Acquire Data dialog box to end the data acquisition.
Click Cancel to close the Acquire Data dialog box.
Review the mass spectrum and chromatogram in the Xcalibur Qual Browser
window. See Figure 48.
For more information about reviewing the data you acquired using the
Finnigan LXQ MS detector with the Xcalibur data system, refer to the
manual: Xcalibur Getting Productive: Qualitative Analysis.

Figure 48. Qual Browser window showing loop injections of reserpine in the Chromatogram view (left) and showing m/z
609 in the Spectrum view (right, centroid data type). Note that the loop injections occur at intervals of
approximately 1 min

Setting Up the Ion Source for
Chapter 6
Acquiring Data in APCI/MS/MS
Mode
This chapter provides information on setting up the ion source for acquiring
data in the APCI/MS/MS mode.
This chapter contains the following sections:
• Removing the ESI Probe
• Removing the Ion Max Ion Source Housing
• Removing the Ion Sweep Cone (optional)
• Installing the Corona Needle
• Installing the Ion Max Ion Source Housing
• Installing the APCI Probe

6 Setting Up the Ion Source for Acquiring Data in APCI/MS/MS Mode
Removing the ESI Probe
Removing the ESI Remove the ESI probe from the Ion Max ion source housing as follows:
Probe
1. Place the LC/MS system in Standby:
a. Stop the flow of solvent to the ESI source:

i. On the Control / Scan Mode toolbar, click Inlet Direct
Control to display the Inlet Direct Control dialog box.
ii. In the Direct Control Panel group box, click on the Pump Off
button to stop the flow of solvent.
b. Click On/Standby on the Control / Scan Mode toolbar to place the
MS detector in Standby.
OnStandby
2. Disconnect the sample transfer tubing from the stainless steel ZDV
fitting (grounding union). See Figure 49.
3. Remove the 8 kV cable from the ESI needle high voltage receptacle as
shown in Figure 49.
a. Unlock the cable by rotating the locking ring counter-clockwise.

b. Unplug the 8 kV cable from the ESI needle high voltage receptacle.

Sample 8 kV
Transfer Cable
Tubing (white)
ESI
Sheath Interlock
Gas Socket (black
Fitting cable)
(blue)
Auxiliary
Gas
Fitting
Grounding (green)
Bar
Probe
Locking
Grounding Nut
Union
Sample
Inlet
Figure 49. Ion Max-S ion source housing with ESI probe installed
4. Disconnect the Auxiliary Gas fitting (green) from the auxiliary gas inlet
(A) on the probe manifold. (Figure 49.)
5. Disconnect the Sheath Gas fitting (blue) from the sheath gas inlet (S)
on the probe manifold.
6. Remove the stainless steel ZDV fitting (Grounding Union) from the
grounding bar on the ion source housing.
7. Unlock the probe locking lever by loosening the probe locking nut.
8. 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. Be

careful not to break the fused-silica sample tube or PEEK safety sleeve.
9. Pull the probe straight out to remove it from the ion source housing.
10. Store the ESI probe in its original shipping container.
The ESI probe is properly removed from the Ion Max ion source housing.

Removing the Ion Max You need to remove the ion source housing to access the ion sweep cone.
Ion Source Housing
Note Disconnect any external liquid lines connected to the ion source
housing before removing the ion source housing.
Remove the Ion Max ion source housing as follows:
1. Remove the drain tube from the ion source housing drain. See
Figure 50.
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 housing in a safe location for temporary storage.
The Ion Max ion source housing is now properly removed. If you want to
remove the ion sweep cone, go to the next topic: Removing the Ion Sweep
Cone. Otherwise, go to the topic “Installing the Corona Needle” on
page 104.

Ion Source
Housing
Locking
Lever(s)
Ion Source Housing

Drain
Figure 50. Ion Max-S ion source housing, detail of components (this component detail also applies to the Ion Max ion
source housing)

Removing the Ion Sweep Cone
Removing the Ion Use of the ion sweep cone is optional. If you do not need to use the ion
Sweep Cone sweep cone, remove the ion sweep cone as follows:
1. Put on a pair of talc-free gloves.
CAUTION AVOID BURNS. At operating temperatures, the ion transfer

capillary 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 capillary when it is exposed.
2. Grasp the outer ridges of the ion sweep cone and pull the cone straight
off of the API cone seal.
3. Store the ion sweep cone in its original shipping container.
The ion sweep cone is now properly removed.
If you need to install the corona needle, go to the next topic: Installing the
Corona Needle. Otherwise, go to the topic: “Installing the Ion Max Ion
Source Housing” on page 105.

Installing the Corona Needle
Installing the Corona Install the corona needle, from the rear of the Ion Max ion source housing,
Needle as follows:

and can puncture your skin. Handle it with care.
1. Using pliers, grasp the needle by the corona needle contact and push the
needle straight into the needle socket in the Ion Max ion source
housing. See Figure 51.
Corona
Needle
Contact
Corona
Needle
(grasp needle with pliers to
install it)
Figure 51. Corona needle, view from inside of the Ion Max housing
2. Make sure the tip of the needle is aligned with the path of travel between
the APCI probe and the ion source interface on the instrument.
The corona needle is now properly installed.

Installing the Ion Max To reinstall the Ion Max ion source housing
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 52 and Figure 53.
Ion Source Housing

Locking Levers
Guide
Pin Holes
Figure 52. Rear view of the Ion Max-S ion source housing

Ion Source
Housing
Guide Pins
Figure 53. 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.
housing and MS detector. Always ensure that liquid in the drain tube is
able to drain to a waste container and that the outlet of the drain tube is
above the level of liquid in the waste container.
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
by pump oil vapor if the API source drain tube and the (blue) forepump
exhaust tubing are connected to the same fume exhaust system.
Your laboratory must be equipped with at least two independent fume

exhaust systems. Route the (blue) forepump exhaust tubing to one of the
dedicated fume exhaust systems. Route the drain tube from the API
source to a waste container. Vent the waste container to the other
dedicated fume exhaust system.

3. Reinstall the ion source drain tube as follows:
a. Connect the 1-in. ID Tygon tubing to the ion source housing drain
fitting.
b. Attach the free end of the hose to a waste container. Ideally, the
waste container should be vented to a fume exhaust system.
The Ion Max ion source housing is now properly installed on the MS
detector.

Installing the APCI Probe
Installing the APCI Install the APCI probe into the Ion Max ion source housing, as follows:
Probe
1. Connect the 8 kV cable to the corona needle high voltage receptacle as
follows:
a. Plug the 8 kV cable into the corona needle high voltage receptacle
on the right side of the top of the ion source housing.
b. Lock the cable by rotating the locking ring clockwise.
2. Be sure to unlock the probe locking lever (widest open position) before
attempting to install the probe.
3. Insert the APCI probe into the port in the ion source housing, align the
guide pin on the probe body at a 45 degree angle from the API interlock
block. See Figure 54
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. Seat the probe all the way down into the alignment notch.

Vaporizer Heater
Cable Socket
Sheath Gas
Inlet (S) Guide Pin
Sample Inlet Auxilary

Fitting Gas Inlet (A)
Figure 54. APCI probe
7. 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.
8. Unplug the vaporizer heater cable from the ESI interlock plug on the
ion source housing.
9. Connect the vaporizer heater cable to the vaporizer heater cable socket
on the APCI probe.
10. Connect the sheath gas fitting (blue) to the sheath gas inlet (S) on the
probe. (See Figure 54)
11. Connect the auxiliary gas fitting (green) to the auxiliary gas inlet (A) on
the probe. (Figure 54)
12. Connect the sample transfer line to the APCI probe inlet.
The APCI probe is now properly installed in the Ion Max ion source
housing.
and MS detector. Always ensure that liquid in the drain tube is able to
drain to a waste container and that the outlet of the drain tube is above
the level of liquid in the waste container.

Leave the LC/MS system in Standby and go to the next chapter: Optimizing
the MS Detector with Your Analyte in APCI/MS Mode.

Optimizing the MS Detector
Chapter 7
with Your Analyte in APCI/MS Mode
This chapter provides information on optimizing the tuning of your MS

detector in the APCI/MS high flow mode. It is not necessary to recalibrate
the MS detector when you switch to APCI/MS operation. You can use the
calibration settings you obtained from the successful automatic calibration
procedure you performed in the ESI/MS mode.
For APCI/MS operation you simply open a default Tune Method located in
your C:\Xcalibur\methods folder, in this case APCIhighflow.LTQTune.
From this starting point, you optimize automatically the tube lens voltage
for your particular analyte. The capillary voltage and ion transfer capillary
temperature may then be optimized manually to enhance ion transmission.
Finnigan LXQ instrument and the Tune Plus window. If you need to
become familiar with your LXQ refer to the Finnigan LXQ online Help,
Finnigan LXQ Getting Connected Manual, and/or the Finnigan LXQ
Hardware Manual.
Ensure that you have performed and completed the procedures in the
topics Tuning and Calibrating Automatically in the ESI/MS Mode and
Setting Up to Acquire Data in the APCI/MS Mode.
This chapter includes the following sections:
• Setting Up the Inlet for Tuning Using High-Flow Infusion
• Setting Up the MS Detector for APCI/MS Operation
• Optimizing the Tune of the MS Detector Automatically in APCI/MS

Mode
• Saving the APCI/MS Tune Method
• Cleaning the MS Detector after Tuning in APCI Mode

7 Optimizing the MS Detector with Your Analyte in APCI/MS Mode
Setting Up the Inlet for Tuning Using High-Flow Infusion
Setting Up the Inlet To make plumbing connections for APCI/MS sample introduction from
for Tuning Using the syringe pump into the LC solvent flow
High-Flow Infusion 1. Connect a 4 cm (1.5 in) segment of Teflon tubing with a fingertight
fitting (natural color) and a ferrule to the (black) LC union. See
Figure 55.
LC Union Fingertight Fitting

(P/N 00101-18202) (P/N 00101-18081)
Ferrule Teflon Tube

(P/N 00101-18196) (P/N 00301-22803)
Figure 55. APCI/MS plumbing connections for the syringe pump
2. Load a clean, 500-μL Unimetrics syringe with 450 μL of a 10 pg/μL

solution of reserpine or your analyte of interest. See “Reserpine” on
page 138 for the procedure to make a 10 pg/μL reserpine tuning
solution (use the dilution at step 9 of the procedure.
3. Insert the needle of a syringe into the segment of Teflon tubing and
place the syringe in the syringe holder of the syringe pump.
4. Connect a fused-silica infusion line from the (black) LC union to the

(black) LC Tee union, as follows. See Figure 56.
a. Connect the infusion line with a fingertight fitting (natural color)

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 ferrule to the side arm of the LC Tee union.

PEEK Tubing LC Tee Union PEEK Tubing Grounding Union

(P/N 00301-22912) (P/N 00101-18204) (P/N 00301-22912) (P/N 00101-18182)
From Divert /
Inject Valve
Stainless Steel
Ferrule
Stainless
Steel Nut Ferrules
(P/N 00101-18196)
Infusion Line
Ferrules Fused-Silica Capillary
(P/N 00101-18120) (P/N 00106-10504)
Fingertight Fingertight
Fitting Fittings
(P/N 00101-18195) (P/N 00101-18081)
From LC Union
Figure 56. APCI/MS plumbing connections for the LC Tee union
Note . To cut the PEEK tubing used to connect your LC to the

divert/inject valve and the divert/inject valve to the APCI source, use a
PEEK tubing cutter. This ensures that the tubing is cut straight. In
addition, make sure your LC fittings, ferrules, and PEEK tubing are
installed properly. By using these precautions, you prevent void (dead)
volumes. The exclusion of void volumes is critical to microbore LC.
Also, void volumes affect the quality of the MS detector signal.
5. Connect a segment of PEEK tubing from the (black) LC Tee union to

the APCI LC inlet, as follows. (See Figure 56.)
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 fingertight fitting and a (brown)
ferrule to a free end of the (black) LC Tee union.
c. Connect the other end of the PEEK tubing with a (red) fingertight
fitting and a ferrule to the LC inlet located on the APCI probe.

divert/inject valve) from the divert/inject valve to the LC Tee union, as
follows. (See Figure 56.)


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.




b. Insert the other end of the PEEK tubing in a suitable waste
container.
The LC plumbing connections are now properly made for APCI/MS
sample introduction from the syringe pump into solvent flow from an LC.

Setting Up the MS Detector for APCI/MS Operation
Setting Up the MS Set up the MS detector for APCI/MS operation

Detector for APCI/MS
1. In Tune Plus, click On/Standby to take the MS detector out of Standby
Operation mode and turn it On. The MS detector begins scanning, and the
Finnigan LXQ MS detector applies high voltage to the corona needle
and shows a real-time display in the Spectrum view.
On Standby
2. Open the APCIhighflow.LTQTune Tune Method, the Tune Method for
high-flow APCI operation, as follows:
a. Choose File > Open to display the Open dialog box.

b. Scroll down until you see the folder C:\Xcalibur\methods. Then,
select the file APCIhighflow.LTQTune.
c. Click OK to open the file. Finnigan LXQ MS detector downloads
the Tune Method parameters to the MS detector.
3. Verify that the Finnigan LXQ MS detector opened the Tune Method, as
follows:
a. On the Instrument Setup toolbar, click API Source to open the

APCI Source dialog box. See Figure 57.
Figure 57. APCI Source dialog box, showing the proper settings for a typical
high flow experiment
b. Verify that the settings in your dialog box are similar to those shown
in Figure 57.

c. Click on OK to close the APCI Source dialog box.
4. Define the scan parameters for tuning the MS detector in the APCI/MS
mode, as follows:
a. On the Control/Scan Mode toolbar, click Define Scan to open the

Define Scan dialog box. Figure 58 (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.)
150 to 2000.
d. In the Scan Type list box, select SIM to specify a selected ion
monitoring scan.
e. In the Scan Time group box, in the 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
Figure 58. Define Scan dialog box, showing typical settings for APCI/MS operation

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

box is clear ( ) to specify that the ion source fragmentation option
is turned off.
h. 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.
i. In the Scan Ranges group box, in the Scan Ranges table, in the
Center Mass text box, type 609.20 to set the center mass for the
scan range to m/z 609.20.
j. In the Width text box, type 2.00 to set the width of the scan range
to m/z 2.00.
k. Verify that the settings in your Define Scan dialog box are the same
as those shown in Figure 58.
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 now completed setting up your MS detector for APCI/MS

operation.

Optimizing the Tune of the MS Detector Automatically in APCI/MS Mode
Optimizing the Tune of You can optimize the tune of the MS detector automatically for APCI
the MS Detector operation.
Automatically in The most important parameters that affect the signal quality during
APCI/MS Mode APCI/MS operation are vaporizer temperature, ion transfer tube
temperature, API gas flows, and solution flow rate. If any one of these
parameters changes, you must re-optimize MS detector parameters. (You
can use the Semi-Automatic tune procedure to tune the MS detector on
individual parameters.)
Use the following procedure to optimize the MS detector automatically on

the reserpine peak at m/z 609.2 at your particular flow rate, for example,
400 μL/min. (See Table 3 on page 12 for guidelines about flow rates and
temperatures.)
Automatically Optimize the MS detector
1. On the Control/Scan Mode toolbar, click Tune to display the

Automatic tuning page. See Figure 59.
3. In the Mass spin box, type 609.2 to specify that you want to tune on the
peak at m/z 609.2.
4. Verify that the Divert/Inject valve is in the Detector position, as follows:
a. Click on the Divert/Inject Valve button to open the Divert/Inject

Valve dialog box.
b. Select the Detector option button, and then click Close to return to
Tune Plus.
5. Start the automatic tuning procedure from the Tune dialog box, as
follows:

Ensure that the syringe pump contains at least 450 mL of

a10 pg/mL reserpine tuning solution. See Appendix A, “Sample
Formulations.” for a procedure for making the reserpine tuning
solution. Use the dilution made in step 9 of the Reserpine Tuning
Solution and Reserpine APCI Sample Solution procedure on
page 138.

b. Click OK to close the message box, and return to the Tune Plus
window.
6. On the File/Display toolbar, click on the Graph View button to display

the view.
box. Your Tune Plus window should now look similar to the one shown
in Figure 60.
You have now successfully tuned the MS detector in APCI/MS mode for the
compound reserpine (or your analyte of interest). Leave the LC pumps on
(with a flow rate of approximately 400 μL/min), and leave the Tune Plus
window open with APCIhighflow.LTQTune to complete the next topic:
Saving the APCI/MS Tune Method.

Figure 60. Tune Plus window with the Tune dialog box, showing the Automatic tuning page

Saving the APCI/MS Tune Method
Saving the APCI/MS You can save the settings you just obtained in a Tune Method specific to
Tune Method your particular analyte and solvent flow rate. (In this case, you save settings
obtained using reserpine.) You can recall the Tune Method and use it as a
starting point for optimizing the MS detector on reserpine at a different
flow rate.
Note Save the Tune Method while the MS detector is still On.
Save the APCI/MS Tune Method
1. Choose File >Save As to display the Save As dialog box. See Figure 61.
3. Click on the File Name text box, and then enter APCImyTune to name
the Tune Method APCImyTune.LTQTune.

Saving the APCI/MS Tune Method
4. Click Save to save the Tune Method and close the Save As dialog box.
Note that the Tune Method is named APCImyTune.LTQTune.
Before you acquire data, go to the next section: Cleaning the MS Detector
after Tuning in APCI Mode.

Cleaning the MS Detector after Tuning in APCI Mode
Cleaning the MS Clean the MS detector after tuning on your analyte of interest
Detector after Tuning
1. Click On/Standby to put the MS detector in Standby mode. When the
in APCI Mode MS detector is in Standby, the Finnigan LXQ MS detector turns off the
vaporizer heater, corona discharge voltage, and syringe pump. The MS
detector stops scanning, and the Finnigan LXQ MS detector freezes the
On Standby 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
Finnigan LXQ MS detector automatically turns 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.)
CAUTION AVOID BURNS. The APCI vaporizer heater can reach

temperatures of 600 ×C. Always allow the APCI probe to cool to
ambient temperature, for approximately 20 min, before handling or
removing the APCI probe from the APCI flange.

and can puncture your skin if you handle it without caution.
4. Remove the Ion Max ion source housing as described in the topic
“Removing the Ion Max Ion Source Housing” on page 31.

5. Flush the sample transfer line, sample tube, and APCI 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 APCI 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 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 APCI probe with
the solution by slowly depressing the syringe plunger. Visually check
that the solution is exiting the tip of the APCI probe on the inside of
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.
6. Repeat step 5 with a solution of 50:50 methanol:water.
7. Reinstall the Ion Max ion source housing as described in “Installing the
Ion Max Ion Source Housing” on page 34.
If you plan to run analytical samples in APCI mode, go to the next chapter:
Acquiring APCI Sample Data Using the Tune Plus Window.
Acquiring APCI Sample Data
Chapter 8
Using the Tune Plus Window
This chapter provides information on acquiring LC/APCI/MS sample data

using the Tune Plus window. This experiment uses reserpine but you can
follow the same procedure with your analyte of interest.
information, refer to the Finnigan LXQ online Help, Finnigan LXQ
Getting Connected, and/or Finnigan LXQ Hardware Manual. Verify that
the procedures in Chapter 3, “Tuning and Calibrating Automatically in
the ESI/MS Mode.” and Chapter 7, “Optimizing the MS Detector with
Your Analyte in APCI/MS Mode.” are completed.
• Setting Up to Introduce Sample by Loop Injection into Solvent Flow

from an LC
• Acquiring APCI Data in the SIM Scan Mode

8 Acquiring APCI Sample Data Using the Tune Plus Window
Setting Up to This topic provides information on how to introduce sample by loop

Introduce Sample by injection into solvent flow from an LC.
Loop Injection into Make the plumbing connections as follows:
Solvent Flow from an
LC 1. Connect an appropriate length of (red) PEEK tubing (transfer line from
the divert/inject valve) from port 3 of the divert/inject valve to the
(stainless steel) sample inlet fitting on the APCI probe. See Figure 62
and Figure 63.
2. Connect a 2 μL sample loop with (stainless steel) set nuts and (stainless
steel) ferrules to ports 1 and 4 of the divert/inject valve.

a. Connect a length of the PEEK tubing with a (stainless steel) nut and
To API From To API From

Source LC Source LC
3 3
4 2 4 2
5 1 5 1
Inject Inject
Port 6 Port 6
To Waste To Waste
Load Position Inject Position
Figure 62. Divert/Inject valve, showing the correct setup for analysis by loop
injection and showing the flow of liquid through the valve in the
Load and Inject positions

Vaporizer Heater
Cable Socket
Sheath Gas
Inlet (S) Guide Pin
Sample Inlet Auxilary

Fitting Gas Inlet (A)
Figure 63. APCI probe

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

b. Insert the other end of the PEEK tubing into a suitable waste
container.
The MS detector is now set up to introduce sample by loop injection into
solvent flow from an LC.

Acquiring APCI Data in the SIM Scan Mode
Acquiring APCI Data Use the following procedure to acquire a file of reserpine data in the selected
in the SIM Scan Mode ion monitoring (SIM) mode. The Finnigan LXQ MS detector automatically
saves the data you acquire on your hard disk.
1. If you have not already done so, in Tune Plus, click On/Standby to take
the MS detector out of Standby mode and turn it On. The MS detector
OnStandby begins scanning, and the Finnigan LXQ MS detector applies high
voltage to the corona needle and shows a real-time display in the
Spectrum view.
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 SIM data for
reserpine (or your analyte of interest), as follows:
a. Click Define Scan to open the Define Scan dialog box. Figure 64
b. Verify that the values in your dialog box are the same as those in
Figure 8-3. Then, click OK.
Figure 64. Define Scan dialog box, showing typical settings for acquiring data in the SIM scan mode
4. Turn on your LC pump, and specify an appropriate flow rate of

400 μL/min, for example. Verify that your system is free of leaks.
5. On the Control/Scan Mode toolbar, click Acquire Data to open the

Acquire Data dialog box. See Figure 65.
6. Specify the acquisition parameters, as follows:

a. In the File Name text box, type reserpine to specify a filename.

b. In the Sample Name text box, type reserpine to specify the sample
identity. If you are not using reserpine, type the name of your
particular analyte.
c. In the Comment text box, you may type a comment about the
experiment. Comments usually specify attributes of the experiment
such as the scan mode, ionization mode, sample amount, and
method of sample introduction (an example would be the entry:
SIM, APCI, 50 pg, loop). Comments may include any other
descriptive information about the experiment. Information entered
in the Comment text box of the Xcalibur data system will be
included on hard copies of the data.
d. In the Acquire Time group box, select the Continuously option
button to specify the continuous acquisition of data (until you stop
the acquisition).
7. Leave the Acquire Data dialog box open during data acquisition, but
move it to a corner of the Tune Plus window.
8. Click Start in the Acquire Data dialog box to begin acquiring data to
the file reserpine3.raw. See Figure 65. The Acquisition Status group box
displays the following message.
State: Acquiring
Time (min):
Figure 65. Acquire Data dialog box, showing the acquisition status of the raw data file

9. Inject the reserpine solution into the APCI source from the Instrument
Setup toolbar, as follows:
a. Click Divert/Inject Valve to display the Divert/Inject Valve dialog

box. See Figure 66.
b. Select the Load option button, and overfill the 2 μL injector loop
with a 250 fg/μL solution of reserpine or a solution of your analyte
of interest. See “Reserpine Tuning Solution and Reserpine APCI
Sample Solution” on page 138 in Appendix A for the 250 fg/μL
reserpine sample solution recipe.
c. Select the Inject option button.
10. Observe the reserpine peak (m/z 609.2), or that of your analyte of
interest, in the Spectrum view (for an example see Figure 67, right side).
11. Perform the following repetitive sequence to obtain a total of four

consecutive loop injections of reserpine in the SIM scan mode. Wait
about 1 min between injections.
a. Select the Load option button to put the divert/inject valve in the
Load position. Overfill the injector loop with the 125 fg/μL
solution of reserpine.
b. Select the Inject option button to inject the reserpine solution into
the APCI source. Then, observe the Spectrum view.
c. Wait 1 min before the next injection.
d. Repeat step 11.a through step 11.c three times
12. Click Stop in the Acquire Data dialog box to end the data acquisition.
Review the mass spectrum and chromatogram in the raw file you just
acquired using the Xcalibur Qual Browser window. See Figure 67.

For more information about reviewing the data you acquire using the
Finnigan LXQ MS detector with the Xcalibur data system, refer to the
Xcalibur Getting Productive: Qualitative Analysis manual.
Note If you want to acquire MS/MS Full scan data in APCI mode, for
information about setting up the Finnigan LXQ MS detector see Setting
Up to Acquire MS/MS Data in the Full Scan Type on page 82 .
Figure 67. Qual Browser window, showing loop injections of reserpine in the Chromatogram view (left) and m/z 609 in the
Spectrum view

Appendix A Sample Formulations
This appendix provides instructions for the preparation of several stock

solutions. These solutions are used for tuning, calibrating, and
demonstrating applications of the APCI / ESI system. Formulations for
sample solutions in this appendix include:
• Caffeine, MRFA, and Ultramark 1621 Stock Solutions

• ESI Calibration Solution: Caffeine, MRFA, Ultramark 1621
• Reserpine
The caffeine, MRFA,Ultramark 1621, and Resperine needed to make the
solutions are supplied with your chemical accessory kit. You can order
replacement chemical accessory kits from Thermo Electron (Thermo
Electron part #97000-62042).

them to examine at any time. Material Safety Data Sheets (MSDS)
provide summarized information on the hazard and toxicity of specific
chemical compounds. MSDSs also provide information on the proper
handling of compounds, first aid for accidental exposure, and procedures
for the remedy of spills or leaks. Producers and suppliers of chemical
compounds are required by law to provide their customers with the most
current health and safety information in the form of an MSDS. Read the
material safety data sheets for each chemical you use.
Potentially hazardous chemicals used in procedures throughout this manual

include:
• Acetic acid
• Acetonitrile
• Methanol
• Reserpine
• Formic Acid

A
Caffeine, MRFA, and Ultramark 1621 Stock Solutions
Caffeine, MRFA, and For tuning and calibrating the ESI system, you use a calibration solution of
Ultramark 1621 Stock caffeine, MRFA, and Ultramark 1621 in an acetonitrile:methanol:water
solution containing 1% acetic acid. You prepare the calibration solution
Solutions from the following stock solutions:
• Caffeine stock solution (prepared stock solution is in the kit)

• MRFA stock solution (you prepare using MRFA supplied in the kit)
• Ultramark 1621 stock solution (you prepare using Ultramark 1621
supplied in the kit)
Note Vials of caffeine and MRFA are included in the API accessory kit.
These compounds are available from Sigma Chemical Co. The Sigma
caffeine Product Number is C6035 (Caffeine Solution, drug standard
1mg/mL±5% in methanol). The Sigma MRFA Product Number is
M1170 (MRFA=Met-Arg-Phe-Ala acetate salt). To order more of these
compounds from Sigma, write or call:
Sigma Chemical Company

P. O. Box 14508
St. Louis, Missouri, USA 63178-9916
(800) 325-3010 (in the USA)
(905) 829-9500 (in Canada)
(314) 771-3750 (outside the USA or Canada)
www.sigmaaldrich.com

A
Note A vial of Ultramark 1621 (neat liquid) is included in the API

accessory kit. This compound is available from Lancaster Synthesis.
The The structure of Ultramark 1621 is ahown below (x is 1, 2, or3).
H(CF2CF2)x H2C O N O CH2(CF2CF2)xH

P P
O CH2(CF2CF2)xH
H(CF2CF2)x H2C O
N N
P
H(CF2CF2)x H2C O O CH2(CF2CF2)xH
The Lancaster Ultramark 1621 Catalog Number is 16698 (Ultramark

1621, Mass Spec Std.). To order more of this compound from Lancaster
Synthesis, write or call:
Lancaster Synthesis, Inc.

P.O. Box 1000
Windham, NH USA 03087-9977
(603) 889-3306, (800)-238-2324 (in the USA & Canada)
+44 (0)1524 36101 (UK & International)
www.lancastersynthesis.com
CAUTION AVOID EXPOSURE TO POTENTIALLY HARMFUL

MATERIALS. Always wear protective gloves when you use solvents or
corrosives. Also contain waste streams, and use proper ventilation. Refer
to your supplier's Material Safety Data Sheet (MSDS) for the proper
handling of a particular solvent. Contain waste streams and use proper
ventilation.
CAUTION AVOID EXPOSURE TO POTENTIALLY HARMFUL

MATERIALS. Always wear safety glasses when you use solvents or
corrosives. Also contain waste streams, and use proper ventilation. Refer
to your supplier's Material Safety Data Sheet (MSDS) for the proper
handling of a particular solvent. Contain waste streams and use proper
ventilation.
Stock Solution: Caffeine A 1 mg/mL stock solution of caffeine in 100% methanol is provided with
your Finnigan LXQ system. This is also the concentration of the Sigma
replacement mentioned in the Note above (Sigma #C6035).

A
Stock Solution: MRFA Prepare a 1.5 mL stock solution of 166.7 pmol/mL MRFA in 50:50
methanol:water as follows:
1. Obtain the vial of L-methionyl-arginyl-phenylalanyl-alanine

acetate•H2O (MRFA) in your accessory kit. In this form, the MRFA
sample has an average molecular weight of 523.7 u. Carefully weigh
2.6 mg of the MRFA sample.
2. Dissolve the MRFA sample in a total volume of 1.0 mL of 50:50

methanol:water.
3. Mix the solution (5.0 nmol/μL) thoroughly.
4. Transfer 50 μL of the 5 nmol/μL solution into a clean polypropylene

tube.
5. Add 1.45 mL of 50:50 methanol:water to the tube.
6. Mix this solution (166.7 pmol/μL) thoroughly.
7. Label the tube MRFA stock solution.
Stock Solution: Ultramark Prepare a 10 mL stock solution of 0.1% Ultramark 1621 in acetonitrile as
1621 follows:
1. Obtain the vial of Ultramark 1621 (neat liquid) in your accessory kit.
2. Using a gas-tight syringe, measure out 10 μL of Ultramark 1621.

Special care in taking the sample may be needed as the Ultramark 1621
has a high viscosity. Dissolve the 10 μL of Ultramark 1621 in 10 mL of
acetonitrile.
3. Mix the solution thoroughly.
4. Label the vial Ultramark 1621 stock solution.

A
ESI Calibration Solution: Caffeine, MRFA, Ultramark 1621
ESI Calibration Prepare 10 mL of the calibration solution, as follows:

Solution: Caffeine,
1. Pipet 200 μL of the stock solution of caffeine into a clean, dry 10 mL
MRFA, Ultramark 1621 volumetric flask.
2. Pipet 100 μL of the stock solution of MRFA into the flask.
3. Pipet 100 μL of the stock solution of Ultramark 1621 into the flask.
Note Use only glass pipets or stainless steel syringes when measuring
glacial acetic acid. Using plastic pipet tips causes contamination of acid
stock solutions that can introduce contaminants in the calibration
solution.
4. Pipet 100 μL of glacial acetic acid into the flask.
5. Pipet 5mL of acetonitrile into the flask.
6. Bring the volume of the solution up to the 10 mL-mark on the flask

with 50:50 methanol:water.
7. Mix the calibration solution thoroughly.
8. Transfer the solution to a clean, dry vial.
9. Label the vial ESI Calibration Solution and store it in a refrigerator until
it is needed.

A
Reserpine
Reserpine Use the following directions to prepare a stock solution of reserpine. Use
serial dilutions of this stock solution to make a sample solution.
Stock Solution: Reserpine Prepare a stock solution of 1 μg/μL reserpine in 1% acetic

acid-methanol
1. Obtain the 1 gram vial of reserpine in your accessory kit. (The average
molecular weight of reserpine is 608.7 u). Weigh out 10 mg of reserpine
and transfer the sample to a 10 mL volumetric flask.
2. Dilute the reserpine up to the 10 mL mark with a solvent of 1% acetic

acid in methanol.
3. Ensure the sample is thoroughly dissolved in solution.
4. Transfer the solution to a clean and dry light resistant vial. Label this vial
as Reserpine Stock Solution (1 μg/μL=1.64nmol/μL). It is best to use the
Reserpine Stock solution as soon as it is made. If it is necessary to store
the solution, keep it in a refrigerator until it is needed.
Reserpine Tuning Solution Prepare 1 mL of either the Reserpine Tuning Solution of 10 pg/μL
and Reserpine APCI (16.4 fmol/μL, step 9) or the Reserpine APCI Sample Solution of
250 fg/μL (411 amol/μL, step 16) in 1% acetic acid-50:50 methanol:water.
Sample Solution
Prepare the appropriate Reserpine solution
1. Pipet 100 μL of the Stock Solution (1 μg/μL) of reserpine into a clean

polypropylene microcentrifuge tube.
2. Add 900 μL of 1% acetic acid in 50:50 methanol:water to the tube.
3. Mix this solution (100 ng/μL=164 pmol/μL) thoroughly.
4. Transfer 10 μL of the 100 ng/μL solution into a clean polypropylene

tube.
6. Mix this solution (1 ng/μL=1.64pmol/μL) thoroughly.
7. Transfer 10 μL of the 1 ng/μL solution into a clean polypropylene tube.

A
Reserpine
9. Mix this solution (10 pg/μL=16.4 fmol/μL) thoroughly. This is the final
dilution for the Reserpine Tuning Solution. If you are stopping at this
dilution, label it as Reserpine Tuning Solution (10 pg/μL). It best to use
this solution immediately after it is made. Keep it in a light resistant
container in a refrigerator if it must be stored. If you are making a
Reserpine APCI Sample Solution, continue with step 10.
10. Transfer 100 μL of the 10 pg/μL solution into a clean polypropylene

tube.
12. Mix this solution (1 pg/μL=1.64 fmol/μL) thoroughly.
13. Transfer 100 μL of the 1 pg/μL solution into a clean polypropylene

tube.
15. Mix this solution (250 fg/μL=411 amol/μL) thoroughly.
16. Label the tube Reserpine APCI Sample Solution (250 fg/μL) . It is best to
use this solution immediately after it is made. Keep it in a light-resistant
container in a refrigerator if it must be stored.

LXQ High Mass Range
Appendix B
Calibration
A high mass range calibration is necessary if you want to use your LXQ to
analyze masses in the 2000 u-4000 u range. This appendix provides
instructions for the high mass range calibration of the LXQ. The
presentation in this Appendix assumes that you are familiar with the
contents of Chapter 3, “Tuning and Calibrating Automatically in the
ESI/MS Mode.” and Appendix A, “Sample Formulations.” .
This Appendix Contains the following topics:
• High Mass Range Calibration Solution
• Normal Mass Range Calibration
• Enter Normal Mass Range Data into Tune Plus
• Tune on m/z 524.3
• High Mass Range Calibration Procedure
• Notes

B
High Mass Range Calibration Solution
High Mass Range The high mass range calibration solution is 70 ng/μL polypropylene glycol
Calibration Solution (PPG) in a solvent of 65/35 methanol/10mM sodium acetate. The PPG
used for the calibration procedure is Aldrich #202347 as specified in the
second Note below.
Note One possible formulation for this solution is 3.5mg of PPG

diluted to 50mL with the 65/35 methanol/10mM sodium acetate
solvent.
Note The procedures that follow assume that the high mass range calibrant
is polypropylene glycol, number average molecular weight about 2700
(Mn~2700), Aldrich product number 202347. To order this compound
from Sigma-Aldrich, write or call:
Sigma Chemical Company

P. O. Box 14508
St. Louis, Missouri, USA 63178-9916
(800) 325-3010 (in the USA)
(905) 829-9500 (in Canada)
(314) 771-3750 (outside the USA or Canada)
www.sigmaaldrich.com

them to examine at any time. Material Safety Data Sheets (MSDS)
provide summarized information on the hazard and toxicity of specific
chemical compounds. MSDSs also provide information on the proper
handling of compounds, first aid for accidental exposure, and procedures
for the remedy of spills or leaks. Producers and suppliers of chemical
compounds are required by law to provide their customers with the most
current health and safety information in the form of an MSDS. Read the
material safety data sheets for each chemical you use.

B
Normal Mass Range Calibration
Normal Mass Range Before calibrating for the high mass range you must calibrate for the normal
Calibration mass range. Use the calibration solution (ESI calibration solution) described
in Appendix A, “Sample Formulations.” and the procedures described in
Chapter 3, “Tuning and Calibrating Automatically in the ESI/MS Mode.”
to perform the normal mass range calibration. The calibration solution
contains caffeine as the low molecular mass component, MRFA as the
mid-molecular mass component, and Ultramark 1621 as the high
molecular mass component. Four pieces of information from this
calibration are needed for the high mass range calibration:
1. The expected low mass (caffeine component=195.10)
2. The expected high mass (1822.00 peak of the Ultramark 1621

component)
3. Observed low mass (observed mass of the caffeine component)
4. Observed high mass (observed mass of the 1822 peak of the Ultramark
1621 component).

B
Enter Normal Mass Range Data into Tune Plus
Enter Normal Mass Enter Nominal Range Data into Tune Plus
Range Data into Tune
1. After carrying out the normal mass range calibration open the Tune Plus
Plus window (Instrument Set Up >LXQ>Start Tune Plus).
2. In the Tune Plus window, open the Diagnostics dialog box

(Diagnostics>Diagnostics) shown in Figure 68.
Figure 68. Tune Plus Diagnostics dialog box, normal mass range data
3. Click Tools in the upper left of the window. Click on Mass Calibration
in the pane below the Tools button as shown in Figure 68.
4. Items mentioned in step 5 through step 7 below are inside of the

Manual Coarse Calibration group box.
5. In the Calibrate Current Scan Type group box, choose User in the Mass
list box. In the Expected Low Mass text box type 195.10 and type
1822.00 in the Expected High Mass text box. Type the Observed Low
Mass and the Observed High Mass values in their respective text boxes.

B
Enter Normal Mass Range Data into Tune Plus
These latter values are obtained as discussed in the preceeding section:

Normal Mass Range Calibration. After entering the observed mass
values click the Execute button.
6. In the Estimate Other Calibration Modes Using Current Full Scan

group box click Execute.
7. Click Save at the bottom right of the Manual Coarse Calibration

window.
8. Click OK at the bottom of the Diagnostics dialog box.

B
Tune on m/z 524.3
Tune on m/z 524.3 Optimize the MS detector Tune on m/z 524.3u
1. On the Control/Scan Mode toolbar, click Tune to display the Tune

dialog box.
2. If necessary, click the Automatic tab to display the Automatic tuning

4. In the Mass spin box, type 524.3 to specify that the Finnigan LXQ MS
detector uses the peak at m/z 524.3 to optimize your tune.
Figure 69. Tune window showing the MS detector Tune on m/z 524.3u
5. Click Start. A message box displays the following message:

B
Tune on m/z 524.3
Ensure that the syringe pump contains at least 450 μL of the ESI
calibration solution as mentioned in the topic “Normal Mass Range
Calibration” on page 143.
6. Click OK to close the message box, and return to the Tune Plus
window.
7. In the File/Display toolbar, click Graph View to display the Graph view.
box.

B
High Mass Range Calibration Procedure
High Mass Range Calibrate the high mass range automatically in ESI mode
Calibration Procedure
1. Load the syringe with PPG2700 solution.
2. From the Tune page go to Define Scan dialog box (see Figure 32 on
page 74). In the Scan Description group box: a) choose High in the
Mass Range spin box, b) choose Normal in the Scan Rate spin box, and
c) choose Full in the Scan Type spin box. In the Scan Ranges box, type
2000 for the First Mass and 4000 for the Last Mass. Click Apply. The
spectrum should look like that shown in Figure 70.
3. Do a two point-mass calibration. Use the Tune Plus Diagnostics dialog

box (Figure 71). Access the Diagnostics dialog box as instructed in
“Enter Normal Mass Range Data into Tune Plus” on page 144. Type the
high mass values as shown in Figure 71. Click Execute, click Save and
close the window.
4. In the Tune Plus window, choose Control>Calibrate to open the

Calibrate dialog box (Figure 72).
Figure 70. PPG2700 mass spectrum

B
Figure 71. Tune Plus Diagnostics dialog box, two point high mass calibration

B
Figure 72. Calibrate dialog box
5. For Mass Range select the High button (upper right of the Calibrate
dialog box). The High Mass Range Calibration tabbed page will appear
(as shown in Figure 72).
6. In the What To Do group box select the Calibrate button. Then enter a
calibration mass list name in the Name list box inside the Calibration
Mass List group box.

B
• If you want to use a previously saved mass list Name in the

Calibration Mass List group box go to step 7.
• If you want to use a new mass list Name in the Calibration Mass List
group box go to step 8.
7. To use a previously saved calibration mass list Name in the Calibration

Mass List group box,
a. Select from the name of a previously saved calibration mass list the
Name box. The calibration masses saved in the selected list appear in
the table below the Name list box (in the Monoisotropic m/z and
the Average m/z columns). The example in Figure 72 shows the
calibration masses saved for the calibration mass list: PPG 2700
(factory).
b. Go to step 9.
8. To assign a new calibration mass list name,
a. You can use the factory default PPG2700 name that is in the Name
list box or you may assign a new name for this mass list.
b. To assign a new name click Save As. The Unique Mass List Name
dialog box appears.
c. Type the new name in the Unique Mass List Name dialog box and
click OK to accept the name. The dialog box closes and the new
name appears in the calibration mass list Name list box.
d. Do not change the masses in the Calibration Mass List table.
9. In the ‘What To Cal/Check’ area, click the Mass for Normal, Mass for
Turbo, and the Mass for Zoom check boxes as shown in Figure 72.
10. Specify the instrument state when the calibration is completed. Use the
‘Set Instrument to Standby When Finished’ check box at the bottom left
of the Calibrate dialog box.
• If you want the LXQ to switch to Standby mode after the

calibration, click the check box so that a check mark is present in the
box.
• If you want the LXQ to remain in the On mode after the

calibration, clear the check box.
11. Start the calibration procedure.

B
a. At the bottom of the Calibrate dialog box, click Start. A message

box appears with the message: Please ensure that the 500 microliter
syringe is full.
b. Verify that the syringe contains at least 420μL of the high mass
range calibration solution.
c. Click OK to close the message box and return to the Calibrate
dialog box. Start the calibration.
12. Observe the Tune Plus window and the Calibrate dialog box. While the
automatic calibration is in progress the Spectrum and Graph views
display a variety of test results and the Status pane in the Calibrate
dialog box displays status messages. The system will automatically use
the average mass values for the Normal and Turbo Scan calibrations.
Monoisotopic masses will automatically be used for the Zoom Scan
calibration.
When all calibration items are completed, your LXQ is properly tuned and
calibrated in the high mass range. At the conclusion of the procedure, you
must clean the syringe and API source (change the tubing and syringe, clean
the API source with acetone to remove the polypropylene glycol).

B
Notes
Notes 1. Two-point mass calibration is available for all high mass range modes
accessed through the Estimate Other Calibration Modes Using Current
Full Scan group box (in the Diagnostics dialog box, Figure 68).
2. Use the Execute button in the box Estimate Other Calibration Modes
Using Current Full Scan to execute estimation for all other calibrations
from the normal mass range-normal scan rate mass calibration. The
other modes for which the calibrations get estimated include Zoom
Scan, Turbo Scan, Enhanced Scan, Isolation Waveform, and High Mass
Isolation Waveform calibrations.
This button changes when the High Mass Scan mode is selected. In this
case, clicking on the Execute button causes the High Mass Isolation to
be estimated from the Current Normal Mass Range Isolation calibration
(assuming that it has been calibrated). This feature has been added to
extrapolate the High Mass Isolation calibration for Initial calibration. As
noted above (item #1), a two point mass calibration is available for all
other scan modes. This makes it possible to get sufficient convergence of
various mass values to allow automatic calibration.
3. In order to calibrate with High Mass Range Zoom Scan it may be

necessary to reduce the Zoom Scan target from 3000 u to 1000u.

Index
A solutions
caution 72
Acquire Data dialog box
preparing 137
displaying 93, 128
spectrum results 53
starting acquisition from 93, 129
syringe pump setup for 43
stopping acquisition from 95, 130
capillary, ion transfer
acquiring data
temperature
prerequisites for, in MS/MS mode 82
setting (figure) 47
SIM scan type 92, 128
voltage
using Tune Plus 81, 125
setting (figure) 47
APCI probe
CAUTIONS
installing 108
avoiding burns
APCI Source dialog box from APCI vaporizer 27, 64, 123
displaying 115
from ion sweep cone 103, 103
APCI vaporizer from ion transfer capillary 33
avoiding burns from 64 avoiding injury from corona discharge needle 29, 104
disabling 38 ensuring nitrogen flow to API source 45
APCI/MS exposing ion source to oxygen 63, 123
setting up Finnigan LXQ for 115 Cautions
using, discussion 5 avoiding contamination of system 14
AutoTune file 47 calibration solution, flow rate limit of 72
solvent waste backing up into ion source housing 35, 106, 109
venting API source 36, 106
B cleaning Finnigan LXQ 63, 123
collision energy
Breadth Focus, in Ion Tree experiment, discussion 23
optimizing 87
buffers, discussion 10
setting, in Define Scan dialog box (note) 82
contamination
caution 14
C note 43
caffeine of solvents 137
preparing calibration solution from 137 corona discharge needle 104
reordering (note) 134 avoiding injury from 29, 104
spectral ion from 51 figure 30
vial of stock solution installing 104
description 135 removing 29
Calibrate dialog box
description 13
displaying 60 D
figure 62
messages in Status box 61 data
acquiring, in SIM scan type 92, 128
calibration
reviewing, in Qual Browser window 96, 131
discussion 13
Data-Dependent Ion Tree
Finnigan LXQ setup 45
description 24
ions 51
figure 23
procedure 60
results 62 Define Scan dialog box 48, 74, 92, 128

Index: E
displaying 47, 73, 92, 116, 128 table 9

MS/MS settings for reserpine 86 operational guidelines 11
Depth Focus, discussion 23 optimizing Tune Method, LC/ESI/MS operation 67
dialog box, ion optics 57 placing in Standby 26
dialog boxes setting up for APCI/MS operation 115
APCI Source 115 setting up for ESI/MS operation 72
Define Scan 48, 74, 83, 92, 128 flow rates
Divert/Inject Valve 77, 130 discussion 11
ESI Source 47, 57 effects of 68
Ion Optics 57
Syringe Pump 50, 84
Tune 54, 76, 119, 119 G
Tune, Collision Energy page 87
Graph view
divert/inject valve 91, 126
displaying 76, 119, 147
Divert/Inject Valve dialog box 77, 130
freezing 63, 123
observing 61
guidelines
E LC/APCI/MS operational 12
ESI probe LC/ESI/MS operational 12
installed view, showing components 99
installing 37
side view 38 I
ESI Source dialog box 47, 57
Instrument Setup window
displaying 47, 51
description 16
figure 47, 57
displaying 26, 45
viewing parameters 55
introducing samples into Finnigan LXQ, discussion 8
ESI/MS
defining scan parameters 48 Ion Max ion source
components 28
discussion 4
components,front view 102
operational guidelines 11
connections 37
optimizing Tune Method 67
discussion 4
preparing calibration solution for 137
ensuring nitrogen flow 45
setting up Finnigan LXQ for 72
ESI probe installed, top view 39
testing Finnigan LXQ 50
exposing to atmosphere 63, 123
experiment types
front view 31
data-dependent 18
installing 105
discussion 16
removing 31, 101
General MS 16
Ion Max ion source housing, rear view 34
Ion Mapping 20
Ion Tree 23 Ion Max ion source, rear view 105
Ion Optics
dialog box, optimized tune 57
Ion Optics dialog box
F displaying 55
Finnigan LXQ ion source mount 35, 106
calibrating 60 ion sweep cone 33
cleaning for normal operation 63, 123 avoiding burns from 103
discussion 2 description 32
ESI/MS mode, testing 50 installing 32
flow rates, discussion 11 removing 103
introducing sample ion transfer capillary
discussion 8 affecting signal quality 14

Index: L
avoiding burns from 33, 64, 103 Normalized Collision Energy parameter 82
cleaning 64
discussion 11, 68
flushing 65
gaining access to 63
O
optimizing temperature of optimizing
discussion 111, 118 auxiliary gas 7
note 68, 77 collision energy 82, 87
reinstalling 64 automatically 86, 87
removing 64 discussion 82
sealing 64 saving value 88
temperature guidelines Finnigan LXQ
APCI operation 12 automatically 45
ESI operation 12 for LC/ESI/MS operation 67
unsealing 65 ion transfer capillary temperature 54
ion transfer capillary, avoid burns 64 isolation width 82, 85
Ion Tree experiment discussion 82
Breadth Focus 23 note 85, 85
Depth Focus 23 performance of data acquisition, discussion 13
isolation width pH of solvent 5
note 85 sweep gas 7
optimizing 82 Tune Method
automatically 75
isolation width, optimizing 85
discussion 54, 67
note 55, 68
L
loop injection 91, 126 P
setup for 90, 126
PEEK tubing, cutting 113
plumbing
divert/inject valve 90
M loop injection 90
MRFA syringe pump 43, 44
preparing calibration solution from 137 syringe pump, Tee to LC 70
preparing stock solution of 136 procedures
reordering (note) 134 acquiring data
spectral ion from 51 SIM scan type 92, 128
MS Detector Setup page using Tune Plus 81, 125
General MS experiment 17 calibrating Finnigan LXQ automatically 60
Triple Play experiment 19 cleaning Finnigan LXQ after tuning and calibrating 63, 123
MS/MS installing
acquiring, data 82 APCI probe 108
discussion 3 corona discharge needle 104
ESI probe 37
Ion Max ion source 105
ion sweep cone 32
N optimizing
nitrogen APCI tune 118
atomic, affect on ion current 6 collision energy 87
sweep gas ESI tune 75
discussion 7 isolation width 82
ensure flow to API source 45 preparing
starting flow of 46 calibration solutions 137

Index: Q
MRFA stock solutions 136 calibration 137

reserpine sample solutions 138 reserpine 138
Ultramark 1621 stock solution 136 Standby, placing Finnigan LXQ in 26
removing sweep gas supply port 32
APCI probe 27 syringe pump
ESI probe 98 description 8
ion sweep cone 103 installing syringe into 43, 69, 112
saving Tune Method 58, 79, 121 locating 43
setting up plumbing 69, 112, 114
Finnigan LXQ analyzing your analyte 72 removing syringe from 63, 123
Finnigan LXQ for APCI/MS operation 115 setting up for tuning and calibrating 43
hardware for tuning and calibrating 25 starting 50, 55, 75, 83, 118, 147
loop injection 90, 126 stopping 63, 88, 123
syringe pump 43, 69 using 13, 14
stopping LC flow 98 Syringe Pump dialog box 84
testing Finnigan LXQ in ESI/MS mode 50 displaying 84
tuning and calibrating
Syringe Pump, dialog box 50
Finnigan LXQ 41
syringe pump, plumbing 43, 44
setting up for 45
tuning Finnigan LXQ automatically
in APCI/MS mode 118
in ESI/MS mode 54 T
using your analyte 67 Tee union, plumbing 70
Total Ion Map page 21
tube lens
Q setting 47
Qual Browser window 96, 131 Tune dialog box
quantitation experiments 16 Collision Energy page 87
showing settings for automatic tuning 54, 76, 119
Tune Methods
opening predefined 46, 72, 115
R saving 58, 79
reserpine Tune Plus window
acquiring, data, loop injection 81, 125 MS/MS spectrum (figure) 89
MS/MS settings for, in Define Scan dialog box 86 opening 26, 45, 72
preparing ESI sample solution 138 placing Finnigan LXQ in Standby from 26
preparing solutions of 138 showing calibration results 62
tuning results 119
using, to acquire data 81, 125
tuning
S automatically 54
sample introduction techniques 9 discussion 13
sample solutions, reserpine, preparing 138 ESI tune files (note) 46
Save As dialog box Finnigan LXQ 55
displaying 58, 79, 121 for LC/ESI/MS operation 67
saving, Tune Method 58, 79 setup for 45, 72
scan parameters, defining 48 syringe pump, setup for 43
sheath gas flow rate 47 voltages 54
signal quality, affect of ion transfer capillary 14
signal quality, ESI 14
SIM scan type, acquiring data in 92, 128 U
solutions, preparing Ultramark 1621

Index: V
cleaning spray shield of 65

contamination from (caution) 14, 72
preparing calibration solution from 137
preparing stock solution of 136
reordering (note) 135
spectral ions from 51
V
voltages
automatic optimization, description 41, 67
ion optics dialog box 57
optimized tune 57
W
Wideband Activation
discussion 3
option 83
X
Xcalibur
displaying Home Page 26, 45, 45, 72
displaying Roadmap view 72
Z
ZoomScan, discussion 4

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Finnigan™

97055-97042 Revision B June 2006

For Research Use Only

© 2006 Thermo Electron Corporation. All rights reserved.

Printing History: Revision B printed in June 2006.

EMC - Directive 89/336/EEC as amended by 92/31/EEC and 93/68/EEC

EN 55011 1998 EN 61000-4-3 2002

EMC issues have been evaluated by UNDERWRITERS LABORATORY, INC (UL)

Safety - Low Voltage Directive 73/23/EEC

FCC Compliance Statement

THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES. OPERATION IS

Notice on Lifting and Handling of

Notice on the Proper Use of

Thermo Electron hat Vereinbarungen getroffen mit Verwertungs-/Entsorgungsanlagen in

Thermo Electron Corporation Finnigan LXQ Getting Started vii

Chapter 3 Tuning and Calibrating Automatically in the ESI/MS Mode .... 41

Chapter 4 Tuning with Your Analyte in LC/ESI/MS Mode........................... 67

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

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

viii Finnigan LXQ Getting Started Thermo Electron Corporation

Optimizing the Tune of the MS Detector Automatically in

Appendix A Sample Formulations......................................................................133

Appendix B LXQ High Mass Range Calibration...............................................141

Thermo Electron Corporation Finnigan LXQ Getting Started ix

To perform analyses in ESI mode, see Chapters 2, 3, 4, and 5. To perform

Related In addition to this guide, Thermo Electron provides the following

• LXQ Preinstallation Guide

• LXQ Getting Connected

• LXQ Hardware Manual

Help is also available from within the software

Safety and special notices include the following:

CAUTION Highlights hazards to humans, property, or the environment.

IMPORTANT Highlights information necessary to avoid damage to

Note Highlights information of general interest.

Thermo Electron Corporation Finnigan LXQ Getting Started xi

Tip Helpful information that can make a task easier.

Contacting Us There are several ways to contact Thermo Electron.

Visit Us on the Web

Contact Technical Support

Find software updates and utilities to download at

Contact Customer Service

In the US and Canada for ordering information:

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

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

xii Finnigan LXQ Getting Started Thermo Electron Corporation

The LXQ™ is a member of the Finnigan™ family of MS detectors. The

• Using the syringe pump (direct infusion)

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

Analysis by direct infusion or flow injection provides no chromatographic

This introduction answers the following questions:

• Why Use the Finnigan LXQ MS Detector?

• Which Ion Source—ESI or APCI—Is Better for Analyzing My Samples?

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

• 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 Finnigan LXQ MS

Thermo Electron Corporation Finnigan LXQ Getting Started 1

• Chromatographic separation and compound detection (non MS