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Summary of Contents for Agilent Technologies 6490
- Page 1 Agilent 6400 Series Triple Quadrupole LC/MS System Concepts Guide The Big Picture Agilent Technologies...
- Page 2 Notices Warranty © Agilent Technologies, Inc. 2012 C A U T I O N No part of this manual may be reproduced in The material contained in this docu- any form or by any means (including elec- ment is provided “as is,” and is sub-...
- Page 3 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. Overview Learn how the Agilent 6400 Series Triple Quadrupole helps you do your job.
- Page 4 Agilent 6400 Series Triple Quad LC/MS Concepts Guide...
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Page 5: Table Of Contents
How a single quadrupole mass spectrometer works Triple quadrupole MS operation Design of the Agilent Triple Quadrupole MS Innovative Enhancements in the 6490 Triple Quadrupole Innovative Enhancements in the 6460 Triple Quadrupole Innovative Enhancements in the 6430 Triple Quadrupole Innovative Enhancements in the 6420 Triple Quadrupole... - Page 6 Agilent iFunnel Technology Agilent Jet Stream Technology LC/MS ion sources Front-end ion optics Collision cell Detector Pumping system Agilent MassHunter Workstation Software - Data Acquisition for 6400 Series Triple Quadrupole Tuning Acquisition Agilent 6400 Series Triple Quad LC/MS Concepts Guide...
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Page 7: Overview
What kind of system do you have? Help for applications Help for acquisition Help for data analysis 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... -
Page 8: What Kind Of System Do You Have
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. -
Page 9: Help For Applications
• Femtogram-level limits of detection and quantitation for the 6430 and 6460 • Zeptomole-level limits of detection and quantitation for the 6490 The dwell time is the amount of • Minimized memory effects even at very short dwell times time allotted for analyzing •... -
Page 10: Help For Acquisition
Overview 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 •... -
Page 11: Help For Data Analysis
Overview 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 • Enter and automatically run both individual samples and samples and batches samples organized in a worklist (sequence of samples) (sequences) that you enter •... - Page 12 Overview 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.
- Page 13 Triple quadrupole MS operation Design of the Agilent Triple Quadrupole MS How a triple quadrupole mass spectrometer works Innovative Enhancements in the 6490 Triple Quadrupole Innovative Enhancements in the 6460 Triple Quadrupole Innovative Enhancements in the 6430 Triple Quadrupole How Dynamic MRM works...
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Page 14: Inner Workings - Triple Quadrupole Ms Versus Single Quadrupole Ms
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. -
Page 15: How A Single Quadrupole Mass Spectrometer Works
Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS 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 –... - Page 16 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.
- Page 17 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How a single quadrupole mass spectrometer works Detector API Source Quadrupole Mass Analyzer Ion Guide Figure 3 Single quadrupole: SIM In this example, 1 All of the ions (+, -, and neutrals) are formed in the API source.
- Page 18 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS 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.
- Page 19 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS 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 m/z 325 Analyte Precursor m/z 325 Matrix Precursor m/z 202 Matrix Precursor m/z 184 Product Ion...
- Page 20 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.
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Page 21: Triple Quadrupole Ms Operation
• Can upgrade to a 6460 6460 • Includes Agilent Jet Stream Technology • Includes 3,000 m/z Q1 and Q2 quadrupoles 6490 • Includes iFunnel technology (Agilent Jet Stream, hexabore capillary, and high pressure/low pressure ion funnels) • Adds additional rough pump for ion funnel •... - Page 22 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS Design of the Agilent Triple Quadrupole MS Circled areas indicate enhancements Figure 7 Innovative Enhancements in the 6420, 6430 and 6460 Agilent 6400 Series Triple Quad LC/MS Concepts Guide...
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Page 23: Innovative Enhancements In The 6490 Triple Quadrupole
6460, geometrically it is actually a hexapole filled with nitrogen, the same gas used in the ion source. In the 6490, the collision cell is a hexapole field axial focusing curved collision cell. - Page 24 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS Innovative Enhancements in the 6490 Triple Quadrupole Figure 9 The iFunnel Technology Ions are generated using an electrospray ion source where the analyte is simultaneously ionized and desolvated from the liquid matrix.
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Page 25: Innovative Enhancements In The 6460 Triple Quadrupole
The higher drive frequency produces more ion motion cycles in the quadrupole mass filter. More ion motion cycles gives higher mass resolution. You can tune a 6490 to 0.4 m/z. The higher drive frequency does reduce the mass range to 1400 m/z. - Page 26 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS Innovative Enhancements in the 6460 Triple Quadrupole The desolvated ions then enter the mass spectrometer via an innovative resistive and highly inert capillary transfer tube (denoted as 2 on the 6460 in Figure 7 on page 22) that improves ion transmission and allows virtually instantaneous polarity...
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Page 27: Innovative Enhancements In The 6430 Triple Quadrupole
Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS Innovative Enhancements in the 6430 Triple Quadrupole Innovative Enhancements in the 6430 Triple Quadrupole The 6430 Triple Quadrupole has many of the innovative enhancements that are included in the Agilent 6460 Triple Quadrupole. - Page 28 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How a triple quadrupole mass spectrometer works External Ionization Collision Cell Precursor Quad Filter Product Detector Quad Filter Figure 12 Conceptual model of a triple quadrupole mass spectrometer: With SIM, MS1 (Q1) and MS2 (Q3) are set at a single m/z, while with Full Scan MS/MS, MS1 (Q1) is set at a single m/z and MS2 (Q3) is scanned.
- Page 29 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How a triple quadrupole mass spectrometer works In this example, a precursor ion is selected using the first quadrupole and is sent to the collision cell for fragmentation. The fragments are scanned through the third quadrupole resulting in a product-ion scan MS/MS.
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Page 30: How Dynamic Mrm Works
Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How Dynamic MRM works How Dynamic MRM works Dynamic MRM is a scan type that has a single continuous Time Segment and up to 4000 transitions in the Scan Segments table. You can add a Time segment that sets the divert valve to waste. - Page 31 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How Dynamic MRM works The MassHunter Data Acquisition software, the SmartCard firmware, the Digital Signal Processor and the MS Hardware all are involved in the dynamic MRM algorithm. 1 MassHunter Data Acquisition Software A list of transitions/parameters (up to 4000) are entered by the user.
- Page 32 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How Dynamic MRM works For each individual transition, the DSP sends MS parameters to the hardware in the form of address/data pairs. 4 MS Hardware For each transition, the DSP address/data pair sets the hardware quadrupoles and other parameters.
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Page 33: How Triggered Dynamic Mrm Works
Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How Triggered Dynamic MRM works How Triggered Dynamic MRM works Triggered Dynamic MRM occurs when criteria for primary MRMs trigger confirmatory (secondary) MRMs to be acquired for a compound. If the abundances of the Primary MRMs are higher than the set thresholds and other criteria are met, then the confirmatory (or secondary) MRMs are acquired. - Page 34 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How Triggered Dynamic MRM works that is not marked as a Primary transition but that has the same compound name as a Primary transition is a secondary transition for the compound. You specify a threshold for each Trigger MRM.
- Page 35 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How Triggered Dynamic MRM works Other triggering conditions for each compound Figure 15 Example of Trigger Window Trigger Entrance Delay The Trigger Entrance Delay is the number of scans to skip after the thresholds for the Trigger transitions have been met within the Trigger Window.
- Page 36 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How Triggered Dynamic MRM works Example of Triggered MRM with four compounds Figure 16 Triggered MRM in the Scan Segments table • The Scan type is set to Dynamic MRM and the Triggered check box is marked.
- Page 37 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How Triggered Dynamic MRM works • For sulfachloropyridazine, if the abundance of the primary trigger transition (285 m/z -> 197 m/z) is greater than 800 at scan 80, then because the Trigger Entrance Delay is 2, secondary transitions are acquired starting at scan 83.
- Page 38 Inner Workings – Triple Quadrupole MS versus Single Quadrupole MS How Triggered Dynamic MRM works Agilent 6400 Series Triple Quad LC/MS Concepts Guide...
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Page 39: Agilent Triple Quadrupole Ms And Sensitivity
How each component works to improve sensitivity LC/MS ion sources Front-end ion optics Collision cell Detector Pumping system This chapter shows how the Agilent triple quadrupole mass spectrometer reduces chemical and electronic noise and how each component contributes to enhanced instrument sensitivity. Agilent Technologies... -
Page 40: How The Agilent Triple Quadrupole Ms Improves Sensitivity
Agilent Triple Quadrupole MS and Sensitivity How the Agilent Triple Quadrupole MS improves sensitivity How the Agilent Triple Quadrupole MS improves sensitivity Triple quadrupole mass spectrometers exhibit multiple sources of noise, including noise from all chemical and cluster backgrounds and electronic noise (Figure 17). - Page 41 Agilent Triple Quadrupole MS and Sensitivity Noise reduction Agilent 6430 Agilent 6460 with Agilent Jet Stream Technology Agilent 6490 with Agilent Jet Stream Technology Figure 17 Multiple sources of noise Agilent 6400 Series Triple Quad LC/MS Concepts Guide...
- Page 42 In this case residual frequency hexapole assembly assists with capturing and product ions can interfere with the focusing fragmented ions. For the 6490, the hexapole field product ion spectrum of a axial focusing curved collision cell includes a tapered cell subsequent MRM experiment.
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Page 43: Example Of Chemical Noise Reduction
Agilent Triple Quadrupole MS and Sensitivity Example of chemical noise reduction Example of chemical noise reduction The Agilent 6400 Series Triple Quadrupole mass spectrometer passes through four transitional steps in translating a signal in the MRM process (Figure 18). Figure 18 Multiple reaction monitoring (MRM) Step 1 The spectrum at the far left represents everything that... - Page 44 Agilent Triple Quadrupole MS and Sensitivity Example of chemical noise reduction Step 3 After MS1 (Q1), fragment ions are generated in the collision cell. The corresponding MS/MS spectrum is shown below the collision cell. Step 4 Particular fragment ions can be selected to pass through the MS2 (Q3) quadrupole.
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Page 45: Linearity Of The Agilent 6400 Series Triple Quadrupole Ms
Verapamil results - Calibration Curve 100 Attograms to 100 Picograms on-column, Six orders of magnitude of linear dynamic range (log-log plot) acquired with Agilent 6490 Triple Quadrupole with Agilent Jet Stream Technology Agilent 6400 Series Triple Quad LC/MS Concepts Guide... -
Page 46: How Each Component Works To Improve Sensitivity
17). Agilent iFunnel Technology Agilent’s iFunnel Technology which is available on the Agilent 6490 Triple Quadrupole consists of the Agilent Jet Stream technology, the Hexabore Capillary and the Dual Ion Funnel. The Agilent Jet Stream technology is discussed below. The Hexabore Capillary samples up to 10 times more ion rich gas from the source. -
Page 47: Agilent Jet Stream Technology
Agilent Triple Quadrupole MS and Sensitivity Agilent Jet Stream Technology Figure 21 The Ion Funnel Agilent Jet Stream Technology Agilent Jet Stream Technology enhances analyte desolvation by collimating the nebulizer spray and creating a dramatically “brighter signal.” The addition of a collinear, concentric, super-heated nitrogen sheath gas (Figure 22) to the inlet... - Page 48 Agilent Triple Quadrupole MS and Sensitivity Agilent Jet Stream Technology Figure 22 Electrospray Ion Source with Agilent Jet Stream Technology Agilent Jet Stream thermal gradient focusing consists of a superheated nitrogen sheath gas that is introduced collinear and concentric to the pneumatically assisted electrospray. Thermal energy from the superheated nitrogen sheath gas is focused to the nebulizer spray producing the most efficient desolvation and ion generation possible.
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Page 49: Lc/Ms Ion Sources
Agilent Triple Quadrupole MS and Sensitivity LC/MS ion sources The capillary in the 6460A is a resistive capillary that improves ion transmission and allows virtually instantaneous polarity switching. It is the same, proven capillary that is used in the fast polarity switching version of Agilent's single quadrupole product. - Page 50 Agilent Triple Quadrupole MS and Sensitivity LC/MS ion sources The Agilent 6490 Triple Quadrupole has a Hexabore Capillary which samples up to 10 times more ion rich gas from the source. The capillary in the 6460A, the 6430A and the 6420A is a resistive capillary that improves ion transmission and allows virtually instantaneous polarity switching.
- Page 51 Agilent Triple Quadrupole MS and Sensitivity LC/MS ion sources Figure 24 Agilent G1978B Multimode Source Neutral analytes and ESI charged analytes pass through the divided chamber entering either the APCI Zone or adjacent zone. Analytes are distributed spatially between the two sections. N O T E ESI and APCI are essentially incompatible processes because each needs its own conditions for aerosol drying and electrical...
- Page 52 Agilent Triple Quadrupole MS and Sensitivity LC/MS ion sources Expected When the Multimode source is operated as an ESI or APCI sensitivity source only, no loss in sensitivity is found for the compounds studied. For many compounds run in mixed mode, an equal signal response (compared to single mode operation) or sensitivity gain can be achieved.
- Page 53 Agilent Triple Quadrupole MS and Sensitivity LC/MS ion sources APPI (Atmospheric Pressure Photo Ionization) For analysis of compounds that ionize poorly by ESI and APCI, the atmospheric pressure photoionization (APPI) source provides a useful alternative. It combines Agilent’s proven orthogonal spray nebulization and counterflow drying gas with innovative photoionization from Syagen Technology.
- Page 54 Agilent Triple Quadrupole MS and Sensitivity LC/MS ion sources HPLC-Chip Traditional nanospray mass spectrometry has proven its potential as a cost-effective, sensitive and reproducible technique for the identification of peptides at femtomol to atomol levels. However, connecting nano capillaries, columns and valves frequently is a tedious procedure and requires user skills and routine.
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Page 55: Front-End Ion Optics
Enhancements in the 6490 Triple Quadrupole" on page 23. The 6490 changed the Dielectric capillary to a short hexabore capillary. It has 6 capillary inlets, and it is half as long. It samples up to 10 times more ion rich gas from the source. It captures the majority of the gas from the source region. - Page 56 Agilent Triple Quadrupole MS and Sensitivity Front-end ion optics Quadrupole Lens Quadrupole Lens 2 RF (transmission of higher masses) Hyperbolic quadrupole and post-filter Figure 25 Front-end optics Skimmer Agilent uses a small diameter skimmer orifice with very short distances from the capillary to the skimmer to the octopole. Consequently, more of the ions exiting the capillary are captured by the ion octopole guide, thereby keeping the ion beam very tightly focused.
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Page 57: Collision Cell
Agilent Triple Quadrupole MS and Sensitivity Collision cell Pre-filter The end section of the MS1 (Q1) quadrupole assembly also consists of short hyperbolic rods, but their RF voltages are only high enough to guide ions into the collision cell. A similar set of rods on the exit side of the collision cell are part of the MS2 (Q3) quadrupole. - Page 58 Agilent Triple Quadrupole MS and Sensitivity Collision cell Figure 26 Collision cell technology for the 6460 and 6430 produces higher sensitivity and faster responses without memory or cross-talk effects The components that contribute to this higher sensitivity and faster response are •...
- Page 59 Agilent Triple Quadrupole MS and Sensitivity Collision cell Why a hexapole? The geometry of a hexapole provides advantages in two domains: ion focusing and ion transmission (Figure 27). • The first advantage is in ion focusing where a quadrupole is better than a hexapole, which is better than an octopole, that is, quadrupole >...
- Page 60 Agilent Triple Quadrupole MS and Sensitivity Collision cell Figure 28 Collision cell design A potential difference is always present. This ensures that the precursor ions coming from MS1 (Q1), or fragment ions generated in the collision cell, are transmitted and not allowed to drift around at random.
- Page 61 Agilent Triple Quadrupole MS and Sensitivity Collision cell 5 V Applied Axial Potential 0 V Collision Energy Figure 29 Collision cell clearing profile (500 pg Alprazolam, 20 ms dwell time) The figure shows that the higher the mass the longer it takes to evacuate the collision cell.
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Page 62: Detector
Agilent Triple Quadrupole MS and Sensitivity Detector Detector The High Energy Dynode detector assembly is unique to Agilent (Figure 30). Figure 30 Detector components The two dynodes are orthogonal to the ion beam and neutrals. This orientation reduces the possibility of neutral molecules impacting the detector while at the same time attracting the ions with high voltages. -
Page 63: Pumping System
Pumping system Pumping system 6490 Triple Quadrupole The 6490 has two roughing pumps and a three-stage turbo pump for the first five vacuum stages. The two roughing pumps are needed to decrease the pressure in the Dual Ion Funnel. The... - Page 64 Agilent Triple Quadrupole MS and Sensitivity Pumping system Figure 32 6460 Triple Quadrupole pumping system 6410 or 6420 Triple Quadrupole A single three-stage turbo pump is used for the entire vacuum system. This is achieved by partitioning the turbo pump to create the multiple vacuum stages.
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Page 65: Agilent Masshunter Workstation Software - Data Acquisition For
(Figure 34 on page 66) has the following features: • All LC and MS parameters are immediately visible. • Real-time plots show the instrument at work. • Running multiple samples is easily handled through a worklist—a spreadsheet-like interface. Agilent Technologies... -
Page 66: Series Triple Quadrupole
Agilent MassHunter Workstation Software - Data Acquisition for 6400 Series Triple Quadrupole Figure 34 Data Acquisition program With these windows you can do these operations: • Control and monitor instrument settings • Tune the instrument • Set up acquisition parameters for the LC and the Triple Quadrupole •... -
Page 67: Tuning
(Figure 35). For all models except the 6490, an Autotune can take 15 to 20 minutes for each polarity; the 6490 takes approximately 45 minutes per ionization mode. Everything is automatic since the tuning mix is delivered by the calibrant delivery system (CDS), which is switched on automatically during the tune. - Page 68 Agilent MassHunter Workstation Software - Data Acquisition for 6400 Series Triple Quadrupole Tuning Manual Tune A manual tune of user-defined ion masses with six corresponding profile masses is available. An automated version is also available for the Tuning Mix with Autotune. You can adjust the EMV by clicking the Adjust EMV button on the Detector tab.
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Page 69: Acquisition
Agilent MassHunter Workstation Software - Data Acquisition for 6400 Series Triple Quadrupole Acquisition Acquisition Many Agilent LC modules and the 6400 Series Triple Quadrupole MS can be controlled and monitored (Instrument Status window) from the same program used for entering acquisition settings (Method Editor window) and setting up lists of samples to run (Worklist window)(Figure 34... - Page 70 Agilent MassHunter Workstation Software - Data Acquisition for 6400 Series Triple Quadrupole Acquisition Agilent 6400 Series Triple Quad LC/MS Concepts Guide...
- Page 72 This guide includes concepts for: • Overview • Inner Workings • MS and Sensitivity • Data Acquisition program for 6400 Series Triple Quadrupole © Agilent Technologies, Inc. 2012 Revision A, November 2012 *G3335-90135* G3335-90135 Agilent Technologies...