Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System Concepts Guide The Big Picture Agilent Technologies
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In This Guide... The Concepts Guide presents “The Big Picture” behind the Agilent TOF and Q-TOF LC/MS system to help you analyze samples on your Agilent time-of-flight or quadrupole time-of-flight mass spectrometer system. This guide helps you understand how the hardware and software work together. 1 Overview Learn how the Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS system helps you do your job and how the hardware and software work.
Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System Concepts Guide
Contents 1 Overview How does the TOF and Q-TOF system help you do your job? Help for applications 11 Help for data acquisition 11 Help for data analysis 13 10 How do different ion sources work? 16 Electrospray ionization (ESI) and Dual ESI 17 Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI) 21 Atmospheric pressure chemical ionization (APCI) 22 Atmospheric pressure photoionization (APPI) 24 Multimode ionization (MMI) 25 HPLC-Chip 27 How does the Agilent TOF and Q-TOF mass spectrometer work?
Storage and retrieval of tune results and Instrument Mode Tune Set Point Modifications for Medium and Large Proteins 62 Real-time displays 63 Instrument Status Window 63 Real-time parameter values (Actuals) 64 Real-time Chromatogram Plot and Spectral Plot windows System logbook 3 60 66 68 Methods with Acquisition Parameters Parameter entry 72 LC parameter entry 72 TOF and Q-TOF parameter entry 72 Automatic TOF and Q-TOF parameter changes during a run 73 General TOF and Q-TOF parameters 76 Ion source par
Worklist setup 102 Worklist menus 103 Sample entry 104 Script entry 106 Entry of additional sample information (show, add columns) 107 Worklist import 108 Report setup 112 Run setup 113 Estimate of worklist file size 114 Data acquisition for samples and worklists 118 What you can monitor during a run 118 What you can do during a run 119 Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System Concepts Guide 7
Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System Concepts Guide
Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System 1 Overview How does the TOF and Q-TOF system help you do your job? 10 Help for applications 11 Help for data acquisition 11 Help for data analysis 13 How do different ion sources work? 16 Electrospray ionization (ESI) and Dual ESI 17 Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI) 21 Atmospheric pressure chemical ionization (APCI) 22 Atmospheric pressure photoionization (APPI) 24 Multimode ionization (MMI) 25 HPLC-Chip 27 How does th
1 Overview How does the TOF and Q-TOF system help you do your job? How does the TOF and Q-TOF system help you do your job? You can set up an Agilent 6200 Series Time-of-Flight LC/MS (TOF) system and the Agilent 6500 Series Quadrupole Time-of-Flight LC/MS (Q-TOF) system 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 MALDI – Matrix-Assisted La
Overview Help for applications 1 super-heated sheath gas to collimate the nebulizer spray which dramatically increases the number of ions that enter the mass spectrometer. Help for applications You can use one or more of the Agilent 6200 Series TOF or 6500 Series Q-TOF LC/MS systems in the following application areas (for example): • Combinatorial chemistry target compound analysis • Natural products screening • Compound profiling (e.g.
1 Overview Help for data acquisition To help you use the Agilent 6200 Series TOF and the 6500 Series Q-TOF LC/MS systems for these applications, the software lets you perform the following tasks in a single window: Prepare the instrument To learn how to get started with the Agilent 6200 Series TOF and 6500 Series Q-TOF, see the Quick Start Guide. • Start and stop the instruments from the software.
Overview Help for data analysis 1 Set up data acquisition methods • Enter and save parameter values for all LC modules and the Agilent 6200 Series TOF and 6500 Series Q-TOF to a data acquisition method. • Enable reference mass correction and select reference standard masses to correct the mass assignments during a sample run. • Select and label the total ion chromatograms or extracted ion chromatograms that you want to appear in the real-time plot.
1 Overview Help for data analysis Agilent also designed the Qualitative Analysis program to present large amounts of data for review in one central location. With the program you can perform these operations for any type of mass spectrometer data that you open: • Extract and display chromatograms. • View and extract peak spectra. • Subtract background. • Integrate chromatograms. You can also set up methods to automatically do the tasks in the list, as well as others, when you open the data files.
Overview Help for data analysis 1 • Integrates with an automated, parameter-free integrator that uses a novel algorithm • Presents a Batch-at-a-Glance results window to help you review and operate on an entire batch of data at once • Automatically detects and identifies outliers Please refer to the Agilent MassHunter Workstation Quantitative Analysis Software Familiarization Guide or the online Help for the Quantitative Analysis software.
1 Overview How do different ion sources work? How do different ion sources work? The Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS systems operate with the following interchangeable atmospheric pressure ionization (API) sources: • “Electrospray ionization (ESI) and Dual ESI” on page 17 • “Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI)” on page 21 • “Atmospheric pressure chemical ionization (APCI)” on page 22 • “Atmospheric pressure photoionization (APPI)” on page 24 • “Multimode ioni
Overview Electrospray ionization (ESI) and Dual ESI 1 Electrospray ionization (ESI) and Dual ESI You control the spray chamber parameters (nebulizer pressure, drying gas flow and temperature, and capillary voltage) when you set up a method in the Method and Run Control view, described in Chapter 4. Electrospray ionization relies in part on chemistry to generate analyte ions in solution before the analyte reaches the mass spectrometer.
1 Overview Electrospray ionization (ESI) and Dual ESI 1 Formation of ions 2 Nebulization 3 Desolvation 4 Ion evaporation Formation of ions Ion formation in API-electrospray occurs through more than one mechanism. If the chemistry of analyte, solvents, and buffers is correct, ions are generated in solution before nebulization. This results in high analyte ion concentration and good API-electrospray sensitivity. Preformed ions are not always required for ESI.
Overview Electrospray ionization (ESI) and Dual ESI 1 Because the sample solution is not heated when the aerosol is created, ESI does not thermally decompose most analytes. Desolvation and ion evaporation Before the ions can be mass analyzed, solvent must be removed to yield a bare ion. A counter-current of neutral, heated drying gas, typically nitrogen, evaporates the solvent, decreasing the droplet diameter and forcing the predominantly like surface-charges closer together (see Figure 2).
1 Overview Electrospray ionization (ESI) and Dual ESI • Adjust solvent pH according to the polarity of ions desired and the pH of the sample. • To enhance ion desorption, use solvents that have low heats of vaporization and low surface tensions. • Select solvents that do not neutralize ions through gas-phase reactions such as proton transfer or ion pair reactions. • To reduce the buildup of salts in the ion source, select more volatile buffers.
Overview Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI) 1 Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI) With the Dual AJS ESI source, the nebulizing gas for the reference spray can be switched for high flow or low flow applications. The second sprayer improves the reference mass stability over a wide range of LC conditions. Low flow applications are typically less than 200 µL/minute. If the flow is approximately 200 µL/minute, either low or high flow may be appropriate.
1 Overview Atmospheric pressure chemical ionization (APCI) Atmospheric pressure chemical ionization (APCI) APCI is a gas-phase chemical ionization process. The APCI technique passes LC eluent through a nebulizing needle, which creates a fine spray. The spray is passed through a heated ceramic tube, where the droplets are fully vaporized (Figure 4). The resulting gas/vapor mixture is then passed over a corona discharge needle, where the solvent vapor is ionized to create reagent gas ions.
Overview Atmospheric pressure chemical ionization (APCI) 1 APCI is applicable across a wide range of molecular polarities. It rarely results in multiple charging, so it is typically used for molecules less than 1,500 u. Because of this molecular weight limitation and use of high-temperature vaporization, APCI is less well-suited than electrospray for analysis of large biomolecules that may be thermally unstable.
1 Overview Atmospheric pressure photoionization (APPI) Atmospheric pressure photoionization (APPI) With the APPI technique, LC eluent passes through a nebulizing needle to create a fine spray. This spray is passed through a heated ceramic tube, where the droplets are fully vaporized. The resulting gas/vapor mixture passes through the photon beam of a krypton lamp to ionize the sample molecules (Figure 5). The sample ions are then introduced into the capillary.
Overview Multimode ionization (MMI) 1 Multimode ionization (MMI) The multimode source is an ion source that can operate in three different modes: APCI, ESI or simultaneous APCI/ESI. The multimode source incorporates two electrically separated, optimized zones: one for ESI and one for APCI. During simultaneous APCI/ESI, ions from both ionization modes enter the capillary and are analyzed simultaneously by the mass spectrometer.
1 Overview Multimode ionization (MMI) Unlike the APCI and APPI sources where the temperature of the vaporizer is monitored, in the multimode source the actual vapor temperature is monitored. As a result, the vaporizer is typically set to between 200 and 250°C.
Overview HPLC-Chip 1 HPLC-Chip Traditional nanospray mass spectrometry has proven its potential as a cost-effective, sensitive and reproducible technique for the identification of peptides at femtomole to atomol levels. However, connecting nano capillaries, columns and valves frequently is a tedious procedure and requires user skills and routine.
1 Overview How does the Agilent TOF and Q-TOF mass spectrometer work? How does the Agilent TOF and Q-TOF mass spectrometer work? 6200 Series TOF The Agilent TOF is an orthogonal acceleration time-of-flight mass spectrometer (oa-TOF). The acceleration pulse applied to send the ions down the flight tube is orthogonal to the direction that ions are entering the mass analyzer. This geometry minimizes the effect of the entrance velocity on the flight time, leading to higher resolution.
Overview How does the Agilent TOF and Q-TOF mass spectrometer work? 1 Figure 9 shows the complete Agilent 6530 Q-TOF schematic, with major improvements identified. These improvements are described below (“Innovative Enhancements in the 6530 Q-TOF” on page 35).
1 Overview How does the Agilent TOF and Q-TOF mass spectrometer work? Figure 10 shows the complete Agilent 6538/6540 Q-TOF schematic, with major improvements identified. These improvements are described below (“Innovative Enhancements in the 6540 and 6538 Q-TOF” on page 33). Figure 10 30 The Agilent 6540 Q-TOF supports the Agilent Jet Stream Technology. The Agilent 6538 does not.
Overview Innovative Enhancements in the Agilent 6550 iFunnel Q-TOF This figure shows the complete Agilent 6550 iFunnel Q-TOF schematic, with major improvements identified. These improvements are described below (“Innovative Enhancements in the Agilent 6550 iFunnel Q-TOF”). Figure 11 1 The Agilent 6550 iFunnel Q-TOF supports the Agilent Jet Stream ESI and the Dual Agilent Jet Stream ESI.
1 Overview Innovative Enhancements in the Agilent 6550 iFunnel Q-TOF Figure 12 The iFunnel Technology Ions are generated using an electrospray ion source where the analyte is simultaneously ionized and desolvated from the liquid matrix. The iFunnel includes the application of Agilent Jet Stream Technology (first introduced with the 6530) which improves sensitivity via thermal gradient focusing and enhanced desolvation. The next innovative enhancement is the use of a short hexabore capillary.
Overview Innovative Enhancements in the 6540 and 6538 Q-TOF 1 The Dual Ion Funnel (DIF) technology is the next enhancement. The DIF technology removes the gas and neutral noise but captures the ions. It also extends the turbo pump’s lifetime. The Dual Ion Funnel technology can transmit ions efficiently at as high a pressure as possible. The first ion funnel has a pressure between 7 and 14 torr. The second ion funnel is a low pressure ion funnel (1 to 3 torr).
1 Overview Innovative Enhancements in the 6540 and 6538 Q-TOF and pulser region. The narrowed, cooled and condensed beam is a key factor in enabling the gain in mass resolution to 40,000 while maintaining excellent sensitivity. Figure 15 Ion Beam Compression Technology Extended Flight Tube with Enhanced Mirror Technology (EMT) The second improvement is that the flight tube for the 6538/6540 Q-TOF is now five feet long.
Overview Innovative Enhancements in the 6530 Q-TOF Figure 16 1 Fast Bipolar Detector Innovative Enhancements in the 6530 Q-TOF Ions are generated using an electrospray ion source where the analyte is simultaneously ionized and desolvated from the liquid matrix. The first of three (3) innovative Agilent enhancements is found in the application of Agilent Jet Stream Technology (denoted as 1 in Figure 9) which improves sensitivity via thermal gradient focusing and enhanced desolvation.
1 Overview Agilent Jet Stream Thermal Gradient Technology The ions passing through the quadrupole analyzer are then directed through the collision cell where they are fragmented. The collision cell is actually a hexapole filled with nitrogen, the same gas that is used as the drying gas. The collision cell design has axial acceleration for high speed MS/MS analysis.
Overview Agilent Jet Stream Thermal Gradient Technology Figure 17 1 Agilent Jet Stream Electrospray Ion Source Agilent Jet Stream thermal gradient focusing consists of a superheated nitrogen sheath gas that is introduced collinear with 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.
1 Overview Front-end ion optics Front-end ion optics For information on the various ion sources, see “How do different ion sources work?” on page 16 After the API source forms ions, the Agilent 6200 Series TOF or 6500 Series Q-TOF LC/MS system performs the following operations, organized according to the stages of the ion path and the vacuum stages of the TOF or Q-TOF. See Figure 8 on page 28 for details.
Overview Front-end ion optics 1 Ion transport 2 (Vacuum stage 4 for 6220 TOF only) In this fourth vacuum pumping stage, the pressure is now low enough that there are few collisions of the ions with gas molecules. NOTE The following sections are only part of the Q-TOF LC/MS instrument. The next section in the TOF instrument is the Beam shaping (Vacuum stage 4 for both 6220 TOF and Q-TOF) on page 40.
1 Overview Front-end ion optics • Linear axial acceleration • High pressure collision cell • High speed digital electronics The collision cell contains nitrogen, the same gas that is used in the ion source. The small diameter of the hexapole assembly assists in capturing fragmented ions. Why a hexapole? The geometry of a hexapole provides advantages in two domains: ion focusing and ion transmission.
Overview Front-end ion optics 1 which helps in creating a much denser and thinner ion beam that passes through a narrower slit leading into the slicer and pulser region. In the TOF, ions enter a second octopole ion guide of similar design to the first octopole but with a lower direct current potential. This second octopole ion guide accelerates the ions and prepares them for beam shaping. For TOF, the fourth vacuum stage contains Octopole 2 and the beginning of the slicer assembly.
1 Overview Front-end ion optics operation so that ions having different initial velocities still arrive simultaneously at the detector. Because the calculation for the mass of each ion depends on its flight time in the flight tube, the background gas pressure must be very low. Any collision of an ion with residual gas slows the ion on its path to the detector and affects the accuracy of the mass calculation. Ion detection Figure 18 shows a schematic of the Agilent 6500 Series Q-TOF LC/MS detector.
Overview Front-end ion optics 1 ever-increasing cascade of electrons travels to the rear of the plate. Roughly 10 times more electrons exit the MCP than incoming ions contact the surface. These electrons are then focused onto a scintillator, which, when struck by electrons, produces a flash of light. The light from the scintillator is focused through two small lenses onto a photomultiplier tube (PMT), which produces the electrical signal read by the data system.
1 44 Overview Front-end ion optics Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System Concepts Guide
Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System 2 Instrument Preparation LC preparation 46 LC module setup 46 Column equilibration and conditioning 48 TOF and Q-TOF preparation – calibration and tuning 51 TOF mass calibration 51 Tuning choices 53 Tune reports 59 Storage and retrieval of tune results and Instrument Mode 60 Tune Set Point Modifications for Medium and Large Proteins 62 Real-time displays 63 Instrument Status Window 63 Real-time parameter values (Actuals) 64 Real-time Chromatogram P
2 Instrument Preparation LC preparation LC preparation To install, configure and start the LC modules, see the Installation Guide. To prepare the LC for sample runs, you usually do three tasks: • Set up the LC modules for operation • Equilibrate or condition the column • Monitor the plot baseline to assure pump and column stability (See “Real-time displays” on page 63.) See the Quick Start Guide and online Help for instructions on how to prepare the LC for a sample run.
Instrument Preparation LC module setup Table 1 2 Tasks to set up the LC modules If you have this module: And you want to: How: Autosampler Change volumes for the installed syringe Done in the Device Configuration user interface Well-Plate Sampler (WPS) or µWPS or h-ALS or h-ALS-SL or h-ALS-SL+ Change volumes for the installed syringe Done in the Device Configuration user interface Select tray type and its position Right-click the device panel and click Assign Wellplates, Edit Wellplate Types Re
2 Instrument Preparation Column equilibration and conditioning Table 1 Tasks to set up the LC modules (continued) If you have this module: And you want to: How: Thermostat.
Instrument Preparation Column equilibration and conditioning 2 • Interactively You change the loaded method set points to the solvent composition for the run, no volume for the injection, higher than normal flow rates and no data storage. You can then immediately apply these set points to the instrument and interactively stop the run when the column is ready.
2 Instrument Preparation Column equilibration and conditioning • With a method in an interactive run You can save the method with the set points mentioned in the above paragraph, and then do a run. The run uses the method stop time. You can also use a post run time within a sample method to equilibrate the column. For more information on worklists, see Chapter 4, “Data Acquisition”. • With a parameter in a worklist You can set up a blank run in a worklist to use as your equilibration run.
Instrument Preparation TOF and Q-TOF preparation – calibration and tuning 2 TOF and Q-TOF preparation – calibration and tuning See the Installation Guide for instructions on how to install and start the TOF or Q-TOF and perform an initial autotune. After you start the instrument, you calibrate and tune the TOF and Q-TOF. This section presents the background information to help you understand calibration and tuning as they are implemented in the Agilent TOF and Q-TOF LC/MS system.
2 Instrument Preparation TOF mass calibration Before you calibrate the instrument, you have to set the instrument state to the proper instrument mode, mass range and fast polarity switching mode. You set these values on the Instrument State tab. When you change the mass range or enable/disable fast polarity switching on the Instrument State tab, the pulser frequency is changed which results in the DEI pulser warming up or cooling down.
Instrument Preparation Tuning choices 2 Tuning choices You can see the tuning choices available to you on the Autotune tab (Figure 21). Notice that you must tune the quadrupole separately from the TOF for the Q-TOF instrument. Also, starting with the B.02.01 release, not all of the tuning choices are available with all sources. With the ESI, Dual ESI, Dual AJS ESI, Multimode, APPI and APCI ion sources, you can run Check Tune, Quick Tune, and TOF Mass Calibration.
2 Instrument Preparation Tuning choices system into Standard (3200 m/z) mode. All the automatic tuning choices calibrate the TOF using eight to ten masses, except for the 1700 mass range, which calibrates using six masses. If Fast Polarity Switching is enabled on a Q-TOF instrument, the Quadrupole autotune buttons are not available.
Instrument Preparation Tuning choices 2 Initial Autotune See the Installation Guide for instructions on how to do an Initial Autotune When you select this option after installation or major service, the system automatically adjusts all the tunable parameters to optimize signal, resolution and mass axis calibration. Table 2 on page 53 shows all of the sources that you can use to run an Initial Tune. On all instrument models, you can perform an Initial Tune with a Dual ESI source.
2 Instrument Preparation Tuning choices For Q-TOF instruments, after running an Initial Tune (TOF), you need to adjust the Collision Cell Gas pressure. You can find this procedure in the online Help. During the Initial Quad Tune process, the system goes through steps a, c, d and e. Check Tune The Check Tune report lets you know if the mass calibration and optimization limits are met with a Pass or Fail.
Instrument Preparation Tuning choices 2 The Standard Tune (TOF) and Quad Tune do subsets of the Initial Tune (TOF) and Initial Quad Tune actions, and they use the current tune parameters as starting points rather than using the factory defaults. Standard Tune (TOF) takes about 10 to 15 minutes, and Quad Tune takes about 10 to 15 minutes to complete. Quick Tune (TOF) Quick Tune (TOF) automatically adjusts the most commonly required subset of tunable parameters.
2 Instrument Preparation Tuning choices Set Detector Gain Set Detector Gain adjusts the PMT voltage to obtain consistent gain (amplification) of the ion current into electrical current. In the Extended Dynamic Range (2 GHz) mode, it also adjusts the preamp offset values and the time delay between gain channels. This tool is a subset of Initial TOF Tune and Standard Tune. Table 2 on page 53 shows all of the sources that you can use to run a Set Detector Gain.
Instrument Preparation Tune reports 2 Tune reports At the end of every Check Tune, Quick Tune, or Standard Tune, the system generates a printable Tune report in Excel. The TOF and Quad tune reports let you know if optimization limits are met. For a TOF tune, the report also lets you know if the mass calibration is satisfactory or not. To print previous tune reports, you click the Tune Report button in the Autotune tab.
2 Instrument Preparation Storage and retrieval of tune results and Instrument Mode Storage and retrieval of tune results and Instrument Mode You can store the tuning parameters in a single file (*.tun) using the Save or Save As buttons in the Instrument State tab. You can also load tune files in the Instrument State tab (Figure 23). The Mass Range, the Fast Polarity Switching option, the Slicer Mode, and the Instrument Mode are also stored in the tune file.
Instrument Preparation Storage and retrieval of tune results and Instrument Mode 2 Extended Dynamic Range (2 GHz) In this mode, the system acquires data at a 4 GHz ADC rate in dual channel mode. One channel is recorded at a high detector gain while the other channel operates at a low detector gain. The firmware stitches the two channels together to produce a scan with a sampling rate of 2 GHz, which results in a greatly increased dynamic range.
2 Instrument Preparation Tune Set Point Modifications for Medium and Large Proteins Tune Set Point Modifications for Medium and Large Proteins For medium and large proteins, the charge envelope may extend beyond 3200 m/z. It is recommended that you acquire your intact protein data in the Extended Mass Range (1 GHz) mode on the Agilent 6200 Series TOF and Agilent 6520/6530 Q-TOF as this allows the extended mass range needed for larger proteins.
Instrument Preparation Real-time displays 2 Real-time displays Instrument Status Window The Instrument Status window is also called the Dashboard. You can see if a module is On, Off or in Standby by observing the color of the bar in the title for each device pane. The title bar also includes words describing the current state of each device. The Instrument Status Bar appears at the bottom of the Instrument Status window. You can see the overall state of the Instrument in this Instrument Status Bar.
2 Instrument Preparation Real-time parameter values (Actuals) Real-time parameter values (Actuals) What you can display You select parameters and states to monitor for each instrument module in the Actuals window. Figure 25 Actuals Selection dialog box The parameters and states for each module listed below are available for display. See the online Help for descriptions of each of these parameters and states.
Instrument Preparation Real-time parameter values (Actuals) Table 4 Actuals available for display Module Parameter or State WPS, µWPS, h-ALS, h-ALS-SL and h-ALS-SL+ The same as ALS except no Vial and addition of Drawn Volume, Sample Position and Needle Position Binary Pump Solvent Ratio A, Solvent Ratio B, Flow, Pressure, Ripple, Fill A,B, A1, B1, A2, B2, Power, Channel Name A, B, Solvent Selection A, B Capillary Pump Same as binary pump with no Fill A1, A2, B1, B2 and with the addition of Solvent
2 Instrument Preparation Real-time Chromatogram Plot and Spectral Plot windows Real-time Chromatogram Plot and Spectral Plot windows What you can display You can display plots in the Chromatogram Plot window and in the Spectrum Plot window. Table 5 Chromatogram plots available for display Module Plot type Pumps Pressure vs. time Thermostatted Column Compartment Temperature Left, Temperature Right vs. time 35900E ADC signal vs. time DAD Signals A-H vs.
Instrument Preparation Real-time Chromatogram Plot and Spectral Plot windows 2 Profile vs. Centroid spectral displays Centroid spectra for the TOF and Q-TOF display the abundance vs. mass for the calculated centroid of the peak. Profile spectra display the abundance vs. mass over the mass range of the peak. Figure 26 Centroid and profile plots in the Spectrum Plot window The default display is to plot abundance vs. mass, but you can change the x-axis to time.
2 Instrument Preparation System logbook System logbook What you can view in the system logbook The system logbook does not list any changes to a method or worklist.
Instrument Preparation System logbook Table 7 2 Columns available for display in the Logbook Viewer Column Name Description Time Date and time of the event Event Type Normal event or error Event Source The module that produced the event (Worklist, Instr Mgr, App UI, Launcher, DA Mgr) Category More information about the event (e.g.
2 Instrument Preparation System logbook Table 8 Tasks you can perform with the logbook If you want to do this: Click this menu item or icon: View recent events View > Refresh Archive entries File > Save or Shortcut menu > Export Open, close, or save the logbook File menu View method log files (method.log file in the Acq.
Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System 3 Methods with Acquisition Parameters Parameter entry 72 General TOF and Q-TOF parameters 76 TOF and Q-TOF acquisition parameters 79 Ion source parameters 76 Setup of TOF and Q-TOF reference mass correction (recalibration) 86 TOF and Q-TOF chromatogram setup 90 Setting parameters to acquire a data file in All Ions MS/MS mode 91 Method saving, editing and reporting 94 Saving a method with data acquisition parameters 94 Method editing 96 Method repor
3 Methods with Acquisition Parameters Parameter entry Parameter entry LC parameter entry Most of the LC parameter entries are the same as those that you can change with the Agilent 1200 LC control module and with other Agilent software products, such as Agilent ChemStation.
Methods with Acquisition Parameters Automatic TOF and Q-TOF parameter changes during a run Figure 29 3 Q-TOF parameter entry in the Method Editor window Automatic TOF and Q-TOF parameter changes during a run You can set some TOF and Q-TOF method set points to be different at different points in time during the run. The different time points in the method are called time segments. Within each time segment, you can have different sets of parameters, called experiments.
3 Methods with Acquisition Parameters Automatic TOF and Q-TOF parameter changes during a run Figure 30 Location for setup of Time Segments and Experiments Per time segment set points are labeled with (Seg). Per experiment set points are labeled with (Expt). All other set points are per run. These set points can be found in these tabs. • General Tab • Source Tab • Acquisition Tab Acquisition tab You can set up time segments for changing these parameters.
Methods with Acquisition Parameters Automatic TOF and Q-TOF parameter changes during a run 3 • Q-TOF Collision Energy Tab • Q-TOF Targeted List Tab See “TOF and Q-TOF acquisition parameters” on page 79 for a description of the TOF, Auto MS/MS and Targeted MS/MS modes and their parameters.
3 Methods with Acquisition Parameters General TOF and Q-TOF parameters General TOF and Q-TOF parameters You enter general TOF and Q-TOF parameters on the General tab of either the TOF tab or the Q-TOF tab in the Method Editor window. Figure 31 General tab of the Q-TOF Method Editor window Profile vs. centroid spectra You can save mass spectral data as whole peaks over the mass range of the peak, or you can save only the data for the mass whose intensity appears in the “middle” of the peak.
Methods with Acquisition Parameters Ion source parameters 3 • APPI • AP-MALDI • PDF-MALDI • Orthogonal Nanospray • HPLC-Chip/MS interface • Dual Orthogonal Nanospray • Multimode (MMI) • GC-APCI Each of the sources uses different parameters for controlling the ion source. The default parameters are set for an electrospray source. When you select a new ion source in the Method Editor window, you see new parameter options and boxes on the Source tab in the TOF tab or the Q-TOF tab.
3 Methods with Acquisition Parameters Ion source parameters Tips for using the AP-MALDI or PDF-MALDI source For instructions on how to set up and run samples with the AP-MALDI and PDF-MALDI inlet and ion source, see the online Help. For instructions on how to install the AP-MALDI or PDF-MALDI ion source, see the Installation Guide. • Tune the TOF or Q-TOF with an ESI source installed • Make sure the Run Type in the Sample Run window is set to External Start.
Methods with Acquisition Parameters TOF and Q-TOF acquisition parameters 3 TOF and Q-TOF acquisition parameters The Acquisition tab in Figure 33 contains acquisition parameters that you can change with time segments. Figure 33 Acquisition tab in the Method Editor window The acquisition parameters you choose depend on what you are trying to accomplish: • If you want to pass all ions in a specified mass range through the instrument with no fragmentation in the collision cell, click MS (Seg) mode.
3 Methods with Acquisition Parameters TOF and Q-TOF acquisition parameters MS mode In this mode the Q-TOF instrument behaves solely as a TOF instrument with no quad isolation applied. You specify the mass range and the acquisition rate and time to collect spectra. Figure 34 MS (Seg) Mode parameters in the Acquisition tab Transients vs. mass range The mass spectrum resulting from a single pulse of voltage applied to the ion pulser is called a transient.
Methods with Acquisition Parameters TOF and Q-TOF acquisition parameters 3 The transient length is set appropriately for each of these modes. Length of transients – time of red flight Q-TOF Front End detector ion pulser Figure 35 Length of transients is measured from the ion pulser to the detector Targeted MS/MS mode (Q-TOF only) In this mode you specify the precursor ion that you want the quadrupole to select and pass through to the collision cell for fragmentation.
3 Methods with Acquisition Parameters TOF and Q-TOF acquisition parameters Figure 36 The Spectral Parameters tab for Targeted MS/MS mode Spectral Parameters These parameters are the same as those for the TOF mode, but they are applied to both the quad (MS) and the TOF components (MS/MS). Collision Energy You can enter multiple fixed collision energies (each collision energy is used), the slope and offset of a line, or a table of collision energies.
Methods with Acquisition Parameters TOF and Q-TOF acquisition parameters 3 Targeted List For compounds for which you already know the precursor ions, you can place the collision energy in the targeted list for the precursor ion and vary its value to optimize the abundance of the product ion. Figure 38 Targeted List tab for Targeted MS/MS mode The collision energies specified here override the values specified in the Collision Energy tab.
3 Methods with Acquisition Parameters TOF and Q-TOF acquisition parameters The software sorts a list of possible precursor ions whose order depends on the boundary parameters entered: a Passes all ions through (TOF only) and sorts the list from highest abundance to lowest (or by charge then abundance) b Excludes those masses in the specified mass range c Sorts the list based on the priority of charges d Moves preferred ions to the top of list in the order specified e Chooses the top ions on the list based
Methods with Acquisition Parameters TOF and Q-TOF acquisition parameters 3 Varying scan speed based on precursor abundance You can adjust the number of transients as a function of the precursor intensity. Varying the scan speed is very useful when you have a complex sample, such as a protein digest. To use this feature, you mark the Scan speed varied based on precursor abundance check box on the Precursor Selection II tab.
3 Methods with Acquisition Parameters Setup of TOF and Q-TOF reference mass correction (recalibration) Setup of TOF and Q-TOF reference mass correction (recalibration) You must do mass corrections during a run in order to attain the mass accuracy that Agilent specifies for the TOF and Q-TOF. Many applications need as small a deviation of accurate mass as possible. To obtain this accuracy, you recalibrate the mass axis for every spectrum with measurements of known reference masses (i.e “lock masses”).
Methods with Acquisition Parameters Setup of TOF and Q-TOF reference mass correction (recalibration) 3 Enabling reference mass correction You set up and enable reference mass correction in the Ref Mass tab within the TOF tab or the Q-TOF tab of the Method Editor window. Figure 41 Reference Masses tab If you mark the Enable check box, the system uses reference masses of the mass reference standard for automatic recalibration of each acquired spectrum.
3 Methods with Acquisition Parameters Setup of TOF and Q-TOF reference mass correction (recalibration) Number of required reference masses To learn more about the underlying calibration equation and coefficients, see “TOF mass calibration” on page 51. With two unknowns, a minimum of two known values are required to determine both A and to. Practical considerations also come into play.
Methods with Acquisition Parameters Setup of TOF and Q-TOF reference mass correction (recalibration) 3 For example, if a Time Segment contains one Experiment with Fragmentor voltage at 225 V and another Experiment with Fragmentor voltage at 200 V, then a spectral cycle contains two successive spectra, one at 225 V and another at 200 V. Spectra from scans of different fragmentor voltage (or other scan specific parameters) should not be summed and averaged because they yield different masses.
3 Methods with Acquisition Parameters TOF and Q-TOF chromatogram setup TOF and Q-TOF chromatogram setup In the Chromatogram tab, you also select the chromatograms or the set points/actuals that you want to see in the Chromatogram Plot window during the run. Figure 42 Chromatogram tab in the TOF or Q-TOF tab You can select the signal to plot (TIC, EIC, BPC, Set point, Actual), the experiment type, the offset of the baseline for the plot and the valid range of values in counts.
Methods with Acquisition Parameters Setting parameters to acquire a data file in All Ions MS/MS mode 3 Setting parameters to acquire a data file in All Ions MS/MS mode In order to acquire a data file in All Ions MS/MS mode, it is necessary to set up a method that has a Time Segment with at least two and a maximum of four different Experiments containing different Collision Energy values on a Q-TOF or different Fragmentor voltages on a TOF.
3 Methods with Acquisition Parameters Setting parameters to acquire a data file in All Ions MS/MS mode Figure 44 Second Experiment has a Collision Energy of 20 V Since All Ions MS/MS is not an isolation MS/MS experiment, you set the Mode to MS (Seg) on the Acquisition tab. For the acquisition rate, you should attempt to get 8 to 10 data points across the chromatographic peak for each collision energy channel.
Methods with Acquisition Parameters Setting parameters to acquire a data file in All Ions MS/MS mode 3 Setting up an experiment with two or three high energy experiments allows the analysis of a large number of target compounds that span a wide variety of compound stability. While this allows selecting fragment ions with higher signal from an optimized collision energy, it also decreases the time that is spent on the precursor ion in the low energy channel, thereby decreasing its signal.
3 Methods with Acquisition Parameters Method saving, editing and reporting Method saving, editing and reporting Saving a method with data acquisition parameters Adding Pre or Post Run Scripts before saving To learn how to set up scripts, see your Agilent application engineer. Before you save a method, you can enter the pathway for the customized scripts that start before or after a run. You do this on the Properties tab.
Methods with Acquisition Parameters Saving a method with data acquisition parameters Table 10 3 System scripts and the actions they enable Script name Actions the script enable SCP_AcquireCalibrantData Sets “Cal/Ref Mass” to “Cal B” and “LCStream” to “LC->Waste”. Allows you to acquire data for the calibrant solution itself. To be used only as the Pre-run script for a method. The script itself does not do a run and only augments an existing method. Do not use as a standalone script in a worklist run.
3 Methods with Acquisition Parameters Method editing Table 10 System scripts and the actions they enable Script name Actions the script enable SCP_ProcessQuantReport Runs a single sample Quant report. Quantitative Analysis must be installed. Use only as a Post Method script. SCP_CTCReset Resets the CTC autosampler. If a drawer is open, it will be closed. Location of method folders You can save methods to any folder on the system. The default folder is D:\MassHunter\Methods\.
Methods with Acquisition Parameters Method reporting 3 Method reporting You can see the parameters in a method in one of three ways: • You click Acquisition Method from the File > Print menu. • You review the parameters in the Method Editor. • You can see method parameters associated with a data file in the Agilent MassHunter Workstation Qualitative Analysis software.
3 98 Methods with Acquisition Parameters Method reporting Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System Concepts Guide
Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System 4 Data Acquisition Interactive single sample setup 100 Worklist setup 102 Data acquisition for samples and worklists 118 Learn the concepts to help you understand the setup of single samples for interactive data acquisition and the setup of single samples and sequences of samples for automatic data acquisition.
4 Data Acquisition Interactive single sample setup Interactive single sample setup If you want to run just one sample at a time, you enter the information for that sample in the Sample Run window. Sample information The sample information that the system records with the data file is the sample name, vial position and other information in the Sample Run window, such as Sample Type and Injection Volume. Sample information is not part of the method.
Data Acquisition Some of the Additional Information parameters 4 Auto Increment If you want to use the same file name and have the system automatically change the number at the end of the file name, you turn auto-increment on. Then, you type a file name that ends in the number 001, and the system makes sure that a new file is created every time you re-run that sample. Other folders You can save your data to any folder on the system. You must use the browse button to select a different folder.
4 Data Acquisition Worklist setup Worklist setup Agilent developed worklists for the primary purpose of running many samples automatically and then reporting on the compounds found in the samples. The worklist lets you enter sequences of samples—both single and multiple samples—to be run automatically in the order of their listing. The worklist operates as a spreadsheet much like Excel. You can copy, paste, and fill in columns as you would in Excel.
Data Acquisition Worklist menus 4 Worklist menus You find all the tasks to create a worklist in the worklist menus. • Add a single sample one at a time or add multiple single samples all at once • Add scripts before or after the worklist or between samples in a worklist • Add or show more sample information columns • Add, insert or delete rows and columns • Set up to print a worklist report or track a worklist run Each menu has different commands available.
4 Data Acquisition Sample entry Sample entry One-at-a-time entry You may want to do this to equilibrate the system before running a worklist. Multiple sample entry If you want to add several single samples to the worklist at one time, you use the menu selection to add multiple samples. You can add different samples or one sample injected several times. Sample Information When you add multiple samples, you can specify the data folder, method names and injection volume.
Data Acquisition Sample entry Sample Position 4 You can select the sample positions without having to type in their values from the Sample Position tab on the Add Multiple Samples dialog box. Figure 50 Sample Position tab of the Add Multiple Samples dialog box Sample methods You can create a .m method containing either acquisition parameters, data analysis parameters or both. See the Quick Start Guide or the online Help for instructions for creating the method.
4 Data Acquisition Script entry Script entry Scripts are special programs, that execute automatically. Agilent includes scripts with the Agilent MassHunter Workstation Software, and you can write your own scripts. You can enter scripts to be run at the following times: • Before or after samples as part of the method The sample method can include pre- and post-analysis scripts.
Data Acquisition Entry of additional sample information (show, add columns) 4 Entry of additional sample information (show, add columns) The default worklist contains only nine columns for sample information. Figure 51 Default worklist columns You can access these capabilities through the worklist menu.
4 Data Acquisition Worklist import Add sample information columns When you add columns, you can enter sample information and values for compounds, masses and acquisition parameters. You can also enter your own sample information, including empirical formulas. You can add a column for the Molecular Formula which can be used in the Qualitative Analysis program. The Column Type MFC is only available if the Qualitative Analysis program is installed on the same computer.
Data Acquisition Worklist import 4 CSV file mapping You use the Map File Generator program to modify a map file. You start this program by clicking the Map File Generator icon in the Agilent MassHunter Workstation > Acq Tools folder. See the online Help for more information on this program. You can import the csv file to add or insert samples whether the worklist is running or not. You can also import the file in an offline session.
4 Data Acquisition Worklist import Mapping for dynamic worklist columns Some columns in a worklist are dynamic and change from analysis to analysis. The mapping capability in the csv file lets you specify additional columns to be added to the worklist. The name of the added worklist column should use the same name as the csv column specified. You then specify the column type in the worklist, such as Compound, Mass, MS Parameter, User Defined or Custom Parameter.
Data Acquisition Worklist import Table 12 4 Mapping section for csv file [Static Mapping] Acq Method Acquisition Method MyData DataFile Name DA DataAnalysis Method SampPos Sample Position Sample Sample Name [Dynamic Mapping] (//Added column) InternalStdA Compound Caffeine Compound 1 [Data Value Mapping] Sample Unknown QualControl QC Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System Concepts Guide 111
4 Data Acquisition Report setup Report setup After you run the worklist, you can send a worklist report to one or more of these locations: Screen, Printer, Excel File or PDF File. You specify the report destination and the file path in the Worklist Report Options dialog box. You can also specify to print all of the columns that are part of the table or to print only the visible columns, and you can select a different Worklist Report template.
Data Acquisition Run setup 4 Run setup Before you run a worklist you select parameters for the entire worklist. • Start run types and the part of the method to run • Paths for the acquisition method, data analysis method and data file • Whether or not to combine export output when also running a Qualitative Analysis method • Scripts to run before or after the worklist • Free disk threshold The free disk threshold is the amount of disk space in gigabytes that must be available before the worklist starts.
4 Data Acquisition Estimate of worklist file size Overlapped Data Analysis with Acquisition You can choose to start the next data acquisition run when the data analysis is complete, or for higher throughput of samples, while data analysis of the previous sample is still running. This option is selected in the Worklist Run Parameters dialog box, in the Execution for Acquisition-DA list box. To overlap data analysis with acquisition, select Asynchronous.
Data Acquisition Estimate of worklist file size 4 much data when operated in the 2 GHz mode (extended dynamic range or extended mass range) or 4 times as much data when operated in the 4 GHz mode (high resolution). Method parameters that control file size LC data is usually a small fraction of TOF or Q-TOF data.
4 Data Acquisition Estimate of worklist file size given mass range using a typical mass calibration curve. The Agilent 6540 UHD Accurate-Mass Q-TOF has approximately 25% more data points. Note that low masses require more data points than high masses because of the non-linear nature of the time to mass conversion.
Data Acquisition Estimate of worklist file size 4 Storage of method parameters for each run also affects file size but to a lesser degree than the file size of the spectrum. They take up about 10,000 bytes before sample injection starts. For a 10-sample worklist, they take up less than 0.1 MB. Approximate file size for Centroid data Data is not stored as the abundance at evenly spaced ion flight times. Rather, peak centroids are computed first.
4 Data Acquisition Data acquisition for samples and worklists Data acquisition for samples and worklists What you can monitor during a run Tracking sample runs The worklist shortcut menus contain an option called “Track Worklist Run” that you can turn off or on. The default position is on. With Track Worklist Run on, you can see what sample is running at any time during the worklist run.
Data Acquisition What you can do during a run 4 What you can do during a run Locked Mode You can turn Locked Mode on or off using the toolbar icons in the main toolbar. If Locked Mode is turned on, you cannot edit a worklist or method while the data is being acquired. Also, the data file is protected, so you cannot overwrite the data file if you run this method or worklist again. If Locked Mode is turned off, then you can edit the worklist during a run.
4 120 Data Acquisition What you can do during a run Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System Concepts Guide
www.agilent.com In This Book The Concepts Guide presents “The Big Picture” behind the Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS system to help you to understand how to use the TOF and Q-TOF LC/MS system components. This guide includes concepts for: • Inner workings of the TOF and Q-TOF MS • Instrument Preparation • Methods with Acquisition Parameters • Data Acquisition © Agilent Technologies, Inc.
