Agilent 6400 Concepts Guide
Agilent 6400 Series
Triple Quadrupole
LC/MS System
Concepts Guide
The Big Picture
Agilent Technologies
Notices
CAUTION
WARNING
© Agilent Technologies, Inc. 2012
No p art o f this manu al may be re produce d in
any form or by any means (including electronic storage and retrieval or translation
into a foreign language) without prior agreement and written consent from Agilent
Technologies, Inc. as governed by United
States and international copyright laws.
Manual Part Number
G3335-90135
Edition
Revision A, November 2012
Printed in USA
Agilent Technologies, Inc.
5301 Stevens Creek Blvd.
Santa Clara, CA 95051
Microsoft® and Windows® are U.S. registered trademark s of Microsoft Corporation.
Software Revision
This guide applies to the Agilent
MassHunter Workstation Software -- Data
Acquisition for 6400 Series Triple Quadrupole program version B.06.00 or higher until
superseded.
If you have comments about this guide,
please send an e-mail to
feedback_lcms@agilent.com.
.
Warranty
The material contained in this document is provided “as is,” and is subject to being changed, without notice,
in future editions. Further, to the maximum extent permitted by applicable
law, Agilent disclaims all warranties,
either express or implied, with regard
to this manual and any information
contained herein, including but not
limited to the implied warranties of
merchantability and fitness for a particular purpose. Agilent shall not be
liable for errors or for incidental or
consequential damages in connection with the furnishing, use, or performance of this document or of any
information contained herein. Should
Agilent and the user have a separate
written agreement with warranty
terms covering the material in this
document that conflict with these
terms, the warranty terms in the separate agreement shall control.
Technology Licenses
The hardware and/or software described in
this document are furnished under a license
and may be used or copied only in accordance with the terms of such license.
Restricted Rights Legend
U.S. Government Restricted Rights. Software and technical data rights granted to
the federal government include only those
rights customarily provided to end user customers. Agilent provides this customary
commercial license in Software and technical data pursuant to FAR 12.211 (Technical
Data) and 12.212 (Computer Software) and,
for the Department of Defense, DFARS
252.227-7015 (Technical Data - Commercial
Items) and DFARS 227.7202-3 (Rights in
Commercial Computer Software or Computer Software Documentation).Safety
Notices
A CAUTION notice denotes a hazard. It calls attention to an operating procedure, practice, or the like
that, if not correctly performed or
adhered to, could result in damage
to the product or loss of important
data. Do not proceed beyond a
CAUTION notice until the indicated
conditions are fully understood and
met.
A WARNING notice denotes a
hazard. It calls attention to an
operating procedure, practice, or
the like that, if not correctly performed or adhered to, could result
in personal injury or death. Do not
proceed beyond a WARNING
notice until the indicated conditions are fully understood and
met.
2 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
In This Guide...
The Concepts Guide presents “The Big Picture” behind the
operation of the Agilent 6400 Series Triple Quadrupole LC/MS
System by helping you understand how the hardware and
software work.
1 Overview
Learn how the Agilent 6400 Series Triple Quadrupole helps you
do your job.
2 Inner Workings – Triple Quadrupole MS versus Single
Quadrupole MS
Learn the concepts you need to understand how the Agilent
triple quadrupole mass spectrometer works.
3 Agilent Triple Quadrupole MS and Sensitivity
Learn how the Agilent triple quadrupole mass spectrometer
achieves high sensitivity.
4 Agilent MassHunter Workstation Software - Data Acquisition for
6400 Series Triple Quadrupole
Learn concepts behind the design of the Agilent MassHunter
Workstation Software - Data Acquisition for Triple Quadrupole
program.
Agilent 6400 Series Triple Quad LC/MS Concepts Guide 3
4 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Contents
1 Overview 7
What kind of system do you have? 8
Help for applications 9
Help for acquisition 10
Help for data analysis 11
2 Inner Workings – Triple Quadrupole MS versus Single Quadrupole
MS 13
Single quadrupole MS operation 14
Design for a single quadrupole mass spectrometer 14
How a single quadrupole mass spectrometer works 15
Triple quadrupole MS operation 21
Design of the Agilent Triple Quadrupole MS 21
Innovative Enhancements in the 6490 Triple Quadrupole 23
Innovative Enhancements in the 6460 Triple Quadrupole 25
Innovative Enhancements in the 6430 Triple Quadrupole 27
Innovative Enhancements in the 6420 Triple Quadrupole 27
How a triple quadrupole mass spectrometer works 27
How Dynamic MRM works 30
How Triggered Dynamic MRM works 33
3 Agilent Triple Quadrupole MS and Sensitivity 39
How the Agilent Triple Quadrupole MS improves sensitivity 40
Noise reduction 40
Example of chemical noise reduction 43
Linearity of the Agilent 6400 Series Triple Quadrupole MS 45
How each component works to improve sensitivity 46
Agilent 6400 Series Triple Quad LC/MS Concepts Guide 5
Agilent iFunnel Technology 46
Agilent Jet Stream Technology 47
LC/MS ion sources 49
Front-end ion optics 55
Collision cell 57
Detector 62
Pumping system 63
4 Agilent MassHunter Workstation Software - Data Acquisition for
6400 Series Triple Quadrupole 65
Tuning 67
Acquisition 69
6 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Agilent 6400 Series Triple Quadrupole LC/MS System
Concepts Guide
1
Overview
What kind of system do you have? 8
Help for applications 9
Help for acquisition 10
Help for data analysis 11
This chapter provides an overview of the Agilent 6400 Series
Triple Quadrupole LC/MS components and how they help get
the job done.
Agilent Technologies
7
1Overview
What kind of system do you have?
What kind of system do you have?
You can set up a n A g ilent 6400 Series Triple Quadrupole LC/MS in
several configurations:
ESI – Electrospray Ionization
APCI – Atmospheric Pressure
Chemical Ionization
APPI - Atmospheric Pressure
Photo Ionization
HPLC-Chip/MS – High Performance
Liquid Chromatography on a Chip
MMI - Multimode Ionization
• For normal flow LC/MS with a binary pump, quaternary pump,
well-plate sampler (or autosampler or CTC PAL autosampler).
The supported ion sources are ESI, APCI, APPI, and MMI.
• For microflow LC/MS with a capillary pump, micro well-plate
sampler (or CTC PAL micro-plate autosampler) and ESI, APCI or
MMI ion sources
• For nanoflow LC/MS with a nanoflow pump, capillary pump,
micro well-plate sampler and HPLC-Chip/MS interface (used
in place of a standard nanospray source) to increase
reliability and boost performance with narrow peak
dispersion and lower dead volumes.
Each Agilent combination has advantages for different
applications. Each uses the same Data Acquisition program,
Quantitative Analysis program and Qualitative Analysis
program to enable these advantages.
The Agilent 6460 and 6490 Triple Quadrupole LC/MS systems
are the only Triple Quadrupole that can use the Agilent Jet
Stream Technology. This technology utilizes a super-heated
sheath gas to collimate the nebulizer spray which dramatically
increases the number of ions that enter the mass spectrometer.
The Agilent 6490 Triple Quadrupole LC/MS system also utilizes
the iFunnel Technology which includes the Agilent Jet Stream
Technology, shorter desolvation assembly with Hexabore
Capillary, and the Dual Offset Ion Funnel.
8 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Help for applications
You can use one or more of the Agilent 6400 Triple Quadrupole
LC/MS combinations to quantitate trace organic compounds in
complex matrices:
• Food safety studies
• Environmental studies
• Drug discovery
• Toxicology
• Forensics
• Bioanalysis
Paired with Agilent’s 1260 and 1290 Infinity Series LCs, the
6400 Series Triple Quadrupole MS delivers sensitive,
reproducible analyses of target compounds in complex
matrices.
• Femtogram-level limits of detection and quantitation for the
6430 and 6460
• Zeptomole-level limits of detection and quantitation for the
6490
Overview 1
Help for applications
The dwell time is the amount of
time allotted for analyzing
each ion during a scan.
Agilent 6400 Series Triple Quad LC/MS Concepts Guide 9
• Minimized memory effects even at very short dwell times
• Simplified operation with Agilent’s data analysis software
1Overview
Help for acquisition
Help for acquisition
To help you use the Agilent Triple Quadrupole LC/MS for these
applications, the software lets you do these tasks in a single
window with the Data Acquisition Program:
Prepare the instrument
To learn how to install the Agilent
Triple Quadrupole LC/MS, see the
Installation Guide.
To learn how to get started with the
Agilent Triple Quadrupole LC/MS,
see the Quick Start Guide.
To learn more about how to use the
Agilent Triple Quadrupole LC/MS
with real samples and data, see the
Familiarization Guide
To learn how to do individual tasks
with the LC/MS, see the online
Help.
To learn more about an Agilent
1260 or 1290 Infinity LC module,
see the Agilent 1260 or 1290
Infinity LC User’s Guide for the
module.
• Start and stop the instruments from the software
• Download settings to the Agilent 1260 or 1290 Infinity LC
and the Agilent 6400 Series Triple Quadrupole mass
spectrometer in real time to control the instrument
• Evaluate if the MS parameters are within the limits to
produce the specified mass accuracy and resolution with
a
Checktune report
• Optimize MS parameters automatically (Autotune) or
manually through Agilent tuning programs and print an
Autotune report
• Monitor the actual conditions of the instrument
• View the real-time plot for chromatograms and instrument
parameters (both UV/Vis and MS) and print a real-time plot
report
• View the centroided line spectrum of a peak or the mass
range profile spectrum of a peak in real time
Set up acquisition methods
• Enter and save parameter values for all LC modules and the
MS to an acquisition method
• Select and label the total ion chromatograms or extracted ion
chromatograms that you want to appear in the real-time plot
• Set up time segments for each scan type and analysis where
parameters change with the time segment or with the scans
within the time segment
• Print an acquisition method report
10 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Overview 1
Help for data analysis
Acquire data
• Enter sample information and pre- or post-analysis programs
(scripts) and run single samples interactively
A worklist is a list of individual
samples and batches
(sequences) that you enter
and run automatically with
the Data Acquisition program.
Help for data analysis
• Enter and automatically run both individual samples and
samples organized in a
• Set up pre- and post-analysis scripts to run between samples
in a worklist
• Set up and run a worklist to optimize MS acquisition
parameters
• Print a worklist report
• View system events, including start and stop times, run
events and errors and print an event log report
worklist (sequence of samples)
Quantitative Analysis Program
Agilent has designed the quantitative analysis program to help
quantitate very low amounts of material with the following
unique features:
• Imports information directly from the acquisition method
• Provides a curve-fit assistant to test all fits and statistics on
curve quality
• Integrates with an automated, parameter-free integrator that
uses a novel algorithm, optimized for triple quadrupole data
• Presents a Batch-at-a-Glance results window to help you
review and operate on an entire batch of data at once
• Automatically detects outliers
• Provides preconfigured templates for basic reporting and
enables the capability to create custom reports in Microsoft
Excel
Please refer to the Agilent MassHunter Workstation Software -
Quantitative Analysis Familiarization Guide or the online
Help for the Quantitative Analysis program.
Agilent 6400 Series Triple Quad LC/MS Concepts Guide 11
1Overview
Help for data analysis
Qualitative Analysis Program
For fast method development, this software is used to quickly
review the qualitative aspects of the data, such as the optimum
precursor to product ion transitions.
Agilent designed the Qualitative Analysis program to present
large amounts of data for review in one central location. With
the program you can do these operations for any type of mass
spectrometer data that you open:
• Extract chromatograms
• View and extract peak spectra
• Subtract background
• Integrate the chromatogram
• Find compounds
• Identify compounds
• Export results
You can also set up methods to automatically do the tasks in the
list, as well as others, when you open the data files.
Please refer to the Agilent MassHunter Workstation Software -
Qualitative Analysis Familiarization Guide or the online Help
for the Qualitative Analysis program.
12 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Agilent 6400 Series Triple Quadrupole LC/MS System
Concepts Guide
2
Inner Workings – Triple Quadrupole MS
versus Single Quadrupole MS
Single quadrupole MS operation 14
Design for a single quadrupole mass spectrometer 14
How a single quadrupole mass spectrometer works 15
Triple quadrupole MS operation 21
Design of the Agilent Triple Quadrupole MS 21
How a triple quadrupole mass spectrometer works 27
Innovative Enhancements in the 6490 Triple Quadrupole 23
Innovative Enhancements in the 6460 Triple Quadrupole 25
Innovative Enhancements in the 6430 Triple Quadrupole 27
How Dynamic MRM works 30
How Triggered Dynamic MRM works 33
In this chapter you learn about concepts to help you understand
the inner workings of the Agilent 6400 Series Triple Quadrupole
LC/MS.
The foundation for understanding the operation of a triple
quadrupole mass spectrometer is the operation of a single
quadrupole mass spectrometer. Therefore, an explanation of the
workings of a single quadrupole mass spectrometer is presented
first.
Agilent Technologies
13
2 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS
Single quadrupole MS operation
Single quadrupole MS operation
To better understand the specific hardware features of the
Agilent 6400 Series Triple Quadrupole Mass Spectrometer, this
section first reviews the fundamental aspects of the single
quadrupole mass spectrometer.
Design for a single quadrupole mass spectrometer
Mass spectrometry is based on the analysis of ions moving
through a vacuum.
The ionization of a sample occurs in the ion source that is
shown, schematically, on the left. The ions are analyzed by
a mass analyzer (mass filter) that controls the motion of the
ions as they travel to the detector to be converted into actual
signals.
Figure 1 Schematic for single quadrupole mass spectrometer
m/z – mass/charge ratio The quadrupole mass analyzer consists of four parallel rods to
which specific DC and RF voltages are applied. These rods filter
out all ions except those of one or more particular m/z values as
determined by the voltages applied.
14 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS 2
External Ionization Source
Quadrupole Mass Filter
Detector
How a single quadrupole mass spectrometer works
The RF is applied to all four rods, but the negative (–) rods are
180 degrees out of phase with the positive (+) rods. The rods are
labeled + and – in reference to the DC voltages applied to them.
All ions that comprise the sample are generated at the source.
However, when a specific set of voltages is applied, only ions of
the corresponding m/z value may pass through the quadrupole
to reach the detector. As the voltages are increased to other
values, ions with other m/z values are allowed to pass through.
A full MS scan is obtained by increasing the DC and RF voltages
applied to the four rods over an expanded range of values.
How a single quadrupole mass spectrometer works
A diagrammatic model can be used to illustrate the concept of
how a single quadrupole instrument works. See Figure 2.
Agilent 6400 Series Triple Quad LC/MS Concepts Guide 15
Figure 2 Conceptual model of a single quadrupole mass spectrometer
In the model,
• All of the ions contained in a sample are formed in the
external ionization source and collected in a funnel. The balls
of different colors and sizes represent different ions having
different m/z values.
2 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS
How a single quadrupole mass spectrometer works
• The quadrupole mass analyzer is represented by a moving
belt that serves to filter the ions as they pass through
openings of various sizes. The ions pass from the funnel,
through the filter, to the detector. Although in this image,
ions that are smaller might fit through the openings, a
quadrupole mass analyzer filters the ions so that only the
“correct” ions pass through to the detector.
• The detector is represented by the collecting funnel below
the filtering belt.
As the belt (the analyzer) moves, or the voltages on the rods are
changed, ions with different m/z values are filtered through the
mass spectrometer.
As the analyzer moves from a small m/z value to increasingly
larger values, a full MS scan is created.
SIM – Selected Ion Monitoring If the belt does not move, the detector continues to monitor the
same single m/z value over the entire scan period. This type of
analysis is known as SIM. It is the most sensitive operating
mode for a single quadrupole mass spectrometer.
The scan period is selected (fixed) by the user. The user may set
the dwell time to scan a specific mass range (e.g. m/z 50 to
1000) or to remain on one selected ion (SIM) or to move to
several selected ions during the scan period. The quadrupole
mass filter is not scanned in this mode. The required RF/DC
voltages are often set to filter a single mass at one time.
For comparison, see “How a triple quadrupole mass
spectrometer works" on page 27.
Single quadrupole: SIM
To obtain the best sensitivity or quantitation, the single
quadrupole is operated in SIM mode (Figure 3). The duty cycle
is the measure of the instrument’s time actually devoted to
measuring signals. In SIM mode, the single quadrupole analyzes
the signal of a specific m/z ion almost all of the time. This
results in nearly 100% acquisition during the duty cycle.
16 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS 2
API Source
Ion Guide
Detector
Quadrupole Mass Analyzer
How a single quadrupole mass spectrometer works
Figure 3 Single quadrupole: SIM
In this example,
1 All of the ions (+, -, and neutrals) are formed in the API
source.
2 Ion optics guide the ions to the quadrupole mass analyzer.
The Agilent Ion Guide is an octopole filter of eight equally
spaced rods.
3 In the analyzer, only ions of a particular m/z value,
represented by blue balls, are allowed to pass through to the
detector.
4 The detector completes the analysis.
This system has several advantages:
• provides the best sensitivity for quantitation
• increases selectivity
• improves chromatographic specificity
• provides no structural information
Agilent 6400 Series Triple Quad LC/MS Concepts Guide 17
2 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS
API Source
Detector
Quadrupole Mass Analyzer
How a single quadrupole mass spectrometer works
Single quadrupole: Full Scan MS
In a full MS scan, the quadrupole serves as a mass filter over
time, and a scan is carried out by stepping through increasing
DC and RF voltages. This provides filtering through the
corresponding m/z values across a mass spectrum. See Figure 4.
Figure 4 Single quadrupole: Full scan MS
The full scan MS mode is less sensitive because the duty cycle
for each m/z is considerably less than 100%. The quadrupole
mass analyzer scans sequentially, passing each m/z in the
selected mass range to the detector.
A full scan MS is still a useful mode of operation because it
shows all of the ions that are being formed in the ion source.
This is useful for developing SIM acquisitions but also alerts
analysts to other compounds co-eluting with compounds of
interest.
18 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS 2
m/z 325 Analyte Precursor
m/z 325 Matrix Precursor
m/z 202 Matrix Precursor
m/z 184 Product Ion
m/z 124 Product Ion
Detector
API Source
How a single quadrupole mass spectrometer works
What about fragment ions?
Full scans with a single quadrupole instrument can also be used
to study fragment ions. See Figure 5.
Figure 5 Fragment ions with single quadrupole MS
The diagram shows that fragment ions, also known as product
ions, are formed by fragmenting or breaking apart precursor
ions. Precursor ions formed in the ion source travel through the
mass analyzer without change, unless extra energy is applied to
their motion in a region where fragmentation can occur.
This fragmentation or collisionally induced dissociation (CID)
can be carried out in a low pressure region between the ion
source and the mass analyzer. The ion source is under
atmospheric pressure, while the mass analyzer is at a much
lower pressure because it has been evacuated of gas with
a vacuum pump.
On the Agilent single quadrupole mass spectrometer, this region
is between the capillary exit and the skimmer, where the gas
pressure is about 2 Torr, or about three orders of magnitude
below atmosphere pressure (760 Torr). Under normal
operation, a voltage is applied across this region to keep the
Agilent 6400 Series Triple Quad LC/MS Concepts Guide 19
2 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS
How a single quadrupole mass spectrometer works
ions passing through to continue on to the mass analyzer. Even
if these ions collide with the gas molecules in this region, they
usually do not have enough energy to fragment.
CID – Collisionally Induced
Dissociation
However, as the voltage is increased, the ions have more
translational energy. Then, if the ions run into gas molecules,
the collisions convert the translational energy into molecular
vibrations that can cause the ions to fragment (Figure 6). This is
collisionally induced dissociation (CID). Even though this
fragmentation does not occur where the ions are formed at
atmospheric pressure, it’s a tradition to call this type of
fragmentation “In-source CID.”
Figure 6 Ion fragmentation caused by collision-induced dissociation
A single quadrupole mass spectrometer cannot be used to do
MS/MS because all of the ions formed in the ion source are
transferred to the quadrupole whether fragmented or not. At
the end when the mass analyzer filters the ions, it is not
possible to identify which product ions came from which
precursor ions.
A triple quadrupole mass spectrometer can do MS/MS, with
fragmentation within its collision cell as described in the next
section.
20 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS 2
Triple quadrupole MS operation
Be sure to read the previous section on the concepts behind the
operation of a single quadrupole mass spectrometer.
Understanding these concepts helps you understand the
operation of the triple quadrupole mass spectrometer.
Design of the Agilent Triple Quadrupole MS
The triple quadrupole mass spectrometer consists of an ion
source, enhanced desolvation technology, followed by ion optics
that transfer the ions to the first quadrupole positioned to the
right of it. A diagram of some of the current Triple Quadrupole
LC/MS products is shown in Figure 7 on page 22. The Agilent
6430 is shown in Figure 8 on page 23.
Triple quadrupole MS operation
Agilent 6400 Series Triple
Quadrupole System
6420 • Includes one turbo pump and one rough pump
6430 • Adds an additional turbo pump
6460 • Includes Agilent Jet Stream Technology
6490 • Includes iFunnel technology (Agilent Jet
Highlights
• Includes resistive capillary
• Can upgrade to a 6430
• Improves pumping in vacuum stage 2
• Updates collision cell lenses
• Can upgrade to a 6460
• Includes 3,000 m/z Q1 and Q2 quadrupoles
Stream, hexabore capillary, and high
pressure/low pressure ion funnels)
• Adds additional rough pump for ion funnel
• Includes high throughput quadrupole driver
electronics
• Includes curved collision cell assembly
• Has a smaller footprint
Agilent 6400 Series Triple Quad LC/MS Concepts Guide 21
2 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS
Circled areas indicate enhancements
Design of the Agilent Triple Quadrupole MS
Figure 7 Innovative Enhancements in the 6420, 6430 and 6460
22 Agilent 6400 Series Triple Quad LC/MS Concepts Guide
Loading...