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Micromass Quattro Ultima User Manual

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The instrument is marked with this symbol where high voltages are
The instrument is marked with this symbol where hot surfaces are
The instrument is marked with this symbol where the user should refer to
this User's Guide for instructions which may prevent damage to the
Warnings are given throughout this manual where care is required to avoid personal
If the instrument is used in a manner not specified by the manufacturer, the protection
provided by the equipment may be impaired.
This manual is a companion to the MassLynx NT User's Guide supplied with the
All information contained in these manuals is believed to be correct at the time of
publication. The publishers and their agents shall not be liable for errors
contained herein nor for incidental or consequential damages in connection with
the furnishing, performance or use of this material. All product specifications, as
well as the information contained in this manual, are subject to change without
Code: 6666511
Issue 3
© Micromass Ltd.
Tel: +44 161 945 4170 Fax: +44 161 998 8915
Tel: +44 161 282 9666 Fax: +44 161 282 4400
present.
present.
instrument.
injury.
instrument.
notice.
Quattro Ultima
User's Guide
Micromass UK Limited
Floats Road
Wythenshawe
M23 9LZ
Tudor Road
Altrincham
WA14 5RZ
http://www.micromass.co.uk

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  • Page 1 Quattro Ultima User's Guide Micromass UK Limited Floats Road Wythenshawe M23 9LZ Tel: +44 161 945 4170 Fax: +44 161 998 8915 Tudor Road Altrincham WA14 5RZ Tel: +44 161 282 9666 Fax: +44 161 282 4400 http://www.micromass.co.uk The instrument is marked with this symbol where high voltages are present.

  • Page 2: Method

    Quattro Ultima User's Guide...

  • Page 3: Table Of Contents

    Quattro Ultima User's Guide Contents Hardware Specifications Dimensions Weights Lifting and Carrying Power Environment Water Cooling Exhausts Rotary Pumps API Gas Exhaust Nitrogen CID Gas Instrument Description Overview Vacuum System Ionisation Techniques Atmospheric Pressure Chemical Ionisation Electrospray Nanoflow Electrospray Sample Inlet...
  • Page 4 Quattro Ultima User's Guide Rear Panel Connections Event Out Contact Closure In Analog Channels Data System Water Nitrogen Gas In Exhausts CID Gas Power Cord Mains Switch Fuses Rotary Control ESD Earth Facility Internal Layout Electronics Mechanical Components Routine Procedures...
  • Page 5 Quattro Ultima User's Guide Automatic Start up and Shutdown The Shutdown Editor The Auto Control Tasks Page The Shutdown Editor Toolbar Loading Startup and Shutdown Files Saving a Startup or Shutdown File Printing Startup and Shutdown Files Creating Startup and Shutdown Files...
  • Page 6 Quattro Ultima User's Guide Instrument Data Thresholds MaxEnt Profile Data Centroid Data SIR Data Ion Counting Threshold Profile Data - Spike Removal Analog Data System Manager Stopping an Acquisition The Function List Editor Introduction The Function List Editor Toolbar Adding a New Function...
  • Page 7 Quattro Ultima User's Guide Electrospray Introduction Preparing for Calibration Reference Compound Introduction Tuning Instrument Threshold Parameters Calibration Options Selecting the Reference File Removing Current Calibrations Selecting Parameters Automatic Calibration Check Calibration Parameters Mass Measure Parameters Performing a Calibration Acquisition Parameters...
  • Page 8 Quattro Ultima User's Guide Operation Checking the ESI Probe Obtaining an Ion Beam Tuning and Optimisation Megaflow Hints Removing the Probe Sample Analysis and Calibration General Information Typical ES Positive Ion Samples Typical ES Negative Ion Samples Chromatographic Interfacing LC-MS Sensitivity Enhancement...
  • Page 9 Quattro Ultima User's Guide The Vacuum System Vacuum Leaks Pirani Gauge Active Inverted Magnetron Gauge Gas Ballasting and Rotary Pump Oil Recirculation Oil Mist Filter Foreline Trap Rotary Pump Oil The Source Overview Cleaning the Cone Gas Nozzle and Sample Cone...
  • Page 10 Quattro Ultima User's Guide Reference Information Overview Editing a Reference File Positive Ion Horse Heart Myoglobin Polyethylene Glycol PEG + NH4 Sodium Iodide and Caesium Iodide Mixture Sodium Iodide and Rubidium Iodide Mixture Negative Ion Horse Heart Myoglobin Mixture of Sugars...

  • Page 11: Hardware Specifications

    Quattro Ultima User's Guide Hardware Specifications Dimensions 200mm (pumping line) 1325mm 180mm 535mm 120mm (ventilation) 700mm Weights Instrument: 150kg (330lb) Data system (computer, monitor and printer): 60kg (130lb) Rotary pumps E2M28: 40kg (90lb) E1M18: 32kg (72lb) Transformer (optional): 100kg (220lb)

  • Page 12: Lifting And Carrying

    Take action to eliminate the risk. • Plan the operation. • Use trained people. • Refer to local or company guidelines before attempting to lift the instrument. Micromass accept no responsibility for any injuries or damage sustained while lifting the instrument. Hardware Specifications Page 12...

  • Page 13: Power

    Quattro Ultima User's Guide Power Instrument: 230V (+10%, -14%), 13A Data system: 100-120V or 200-240V, 13A Pumps: 230V (+10%, -14%), 13A Environment Ambient temperature: 15-28°C (59-82°F) ≤2°C (≤4°F) Short term variance (1.5 hours): Overall heat dissipation (excluding LC and optional water chiller): 4.2kW maximum...

  • Page 14: Nitrogen

    Quattro Ultima User's Guide Nitrogen A supply of dry, oil-free nitrogen at 6-7 bar (90-100 psi) is required. Caution: The lines supplying nitrogen to the instrument must be clean and dry. If plastic tubing is used it must be made of Teflon. The use of other types of plastic leads to contamination of the instrument.

  • Page 15: Instrument Description

    The transmitted ions are detected by the photomultiplier detection system. The signal is amplified, digitised and presented to the MassLynx NT™ data system. The Micromass Quattro Ultima is a high performance benchtop triple quadrupole mass spectrometer designed for routine LC-MS-MS operation. Quattro Ultima may be coupled to: •...

  • Page 16: Vacuum System

    Quattro Ultima User's Guide Vacuum System Vacuum is achieved using two direct drive rotary pumps, and two turbomolecular pumps. The rotary pumps are mounted on the floor external to the instrument. The E1M18 pumps the ion source block, while the E2M28 pumps the first ion tunnel and also backs the turbomolecular pumps.

  • Page 17: Ionisation Techniques

    Quattro Ultima User's Guide Ionisation Techniques Two atmospheric pressure ionisation techniques are available. Atmospheric Pressure Chemical Ionisation Atmospheric pressure chemical ionisation (APcI) generally produces protonated or deprotonated molecular ions from the sample via a proton transfer (positive ions) or proton abstraction (negative ions) mechanism. The sample is vaporised in a heated nebuliser before emerging into a plasma consisting of solvent ions formed within the atmospheric source by a corona discharge.

  • Page 18: Ms Operating Modes

    Quattro Ultima User's Guide MS Operating Modes Source Collision Cell Detector Collision Cell Resolving RF Only (Pass all masses) RF Only (Pass all masses) Resolving The MS1 mode, in which MS1 is used as the mass filter, is the most common and most sensitive method of performing MS analysis.

  • Page 19: The Daughter Ion Spectrum

    Quattro Ultima User's Guide The Daughter Ion Spectrum This is the most commonly used MS-MS scan mode. Typical applications are: • Structural elucidation (for example peptide sequencing). • Method development for MRM screening studies: Identification of daughter ions for use in MRM “transitions”.

  • Page 20: The Parent Ion Spectrum

    Quattro Ultima User's Guide The Parent Ion Spectrum Typical application: • Structural elucidation. Complementary or confirmatory information (for daughter scan data). Example: Parents of the specific daughter ion at m 195 from reserpine in electrospray positive ion mode. Collision Cell...

  • Page 21: Mrm: Multiple Reaction Monitoring

    Quattro Ultima User's Guide MRM: Multiple Reaction Monitoring This mode is the MS-MS equivalent of SIR (Selected Ion Recording). As both MS1 and MS2 are static, this allows greater “dwell time” on the ions of interest and therefore better sensitivity (~100×) compared to scanning MS-MS.

  • Page 22: The Constant Neutral Loss Spectrum

    Quattro Ultima User's Guide The Constant Neutral Loss Spectrum The loss of a specific neutral fragment or functional group from an unspecified parent or parents. Typical applications: • Screening mixtures, for example during neonatal screening, for a specific class of compound that is characterised by a common fragmentation pathway.

  • Page 23: Front Panel Connections

    Quattro Ultima User's Guide Front Panel Connections CAPILLARY / CORONA STANDBY DESOLVATION ESI / APci NEBULISER C.I.D. GAS CONE GAS VACUUM OPERATE LOAD INJECTOR INJECT Desolvation LC Connection High Voltage Nebuliser Connection Source Connection Desolvation Gas and Probe Nebuliser Gas The PTFE gas lines for the desolvation gas and probe nebuliser gas are connected to the front of the instrument using threaded metal fittings.

  • Page 24: Front Panel Controls And Indicators

    Quattro Ultima User's Guide Front Panel Controls and Indicators CAPILLARY / CORONA STANDBY DESOLVATION ESI / APci NEBULISER C.I.D. GAS CONE GAS VACUUM OPERATE LOAD INJECTOR INJECT Desolvation Gas Control Nebuliser Gas Control Cone Gas CID Gas Control Control Status Display The display on the front panel of the instrument consists of two 3-colour light emitting diodes (LEDs).

  • Page 25: Flow Control Valves

    Quattro Ultima User's Guide Flow Control Valves The Desolvation Gas, Cone Gas and Nebuliser needle valves are five-turn valves. The CID Gas valve is a fifteen-turn valve. The flow increases as the valve is turned counterclockwise. Caution: To prevent damage to the CID Gas valve, take care not to over-tighten when turning the supply off.

  • Page 26: Rear Panel Connections

    Quattro Ultima User's Guide Rear Panel Connections Event Out Four outputs, Out 1 to Out 4, are provided to allow various peripherals to be connected to the instrument. Switches S1 to S4 allow each output to be set to be either a contact closure (upper position) or a voltage output (lower position).

  • Page 27: Mux

    Quattro Ultima User's Guide This 6-way connector connects the instrument to the MUX control base. Data System This RJ45 connector connects the instrument to the data system using the network cable supplied. Instrument Description Page 27...

  • Page 28: Water

    Quattro Ultima User's Guide Electrostatic Discharge Earth (Ground) Power Point Cord Mains Switch Rotary Pump Fuses Control CID Gas ROTARY PUMP 10 AMP (T) CONTROL EARTH ROTARY PUMP 10 AMP (T) FACILITY CONTROL NITROGEN GAS OUTLET INLET WATER Exhaust Source...

  • Page 29: Exhausts

    Quattro Ultima User's Guide Exhausts The exhaust from the rotary pump should be vented to atmosphere outside the laboratory. The gas exhaust, which also contains solvent vapours, should be vented via a separate fume hood, industrial vent or cold trap.

  • Page 30: Internal Layout

    Quattro Ultima User's Guide Internal Layout Electronics High Voltage Power Supplies (4) Transputer Processor Card (TPC) Analogue PCB Control PCB Scan Control PCB RF Control (Upper) PCB RF Control (Lower) PCB Pumping Logic PCB Power Sequence Low Voltage Power Supplies (2)
  • Page 31 Quattro Ultima User's Guide The main electronics modules of the system are: • Two low voltage power supplies. • Four high voltage power supplies, plugged into the backplane below the analyser housing. These supply the detector system and the high voltages for the source and electrospray probe.

  • Page 32: Mechanical Components

    Quattro Ultima User's Guide Mechanical Components Source Analyser Housing Housing Detector Pirani Gauge Active Inverted Magnetron (Penning) Gauge Source Air Filter Turbomolecular (behind cover) Pump The main internal mechanical components of the instrument are: • The source housing, inside which are the ion tunnels.

  • Page 33: Routine Procedures

    Quattro Ultima User's Guide Routine Procedures Start Up Following a Complete Shutdown Preparation If the instrument has been unused for a lengthy period of time, proceed as follows: Check the level of oil in the rotary pump sight Exhaust glass. Refill or replenish as...
  • Page 34 Quattro Ultima User's Guide Check that the rotary pump control box is connected to Rotary Control at the rear of the instrument, and to the rotary pumps. Check that the solenoid valve on the E1M18 rotary pump is connected to the other Rotary Control socket.
  • Page 35 Quattro Ultima User's Guide Switch on the data system. As supplied Windows NT is automatically activated following the start-up sequence whenever the data system is switched on. Windows NT and MassLynx NT can be configured to prevent unauthorised access. Consult the system administrator for any passwords that may be requested.

  • Page 36: Pumping

    Quattro Ultima User's Guide Pumping Caution: To minimise wear to the lubricated components of the rotary pump, the manufacturers recommend that the pump is not started when the oil temperature is below 12°C. Pump down time may be decreased Isolation...

  • Page 37: Measuring The Analyser Pressure

    (monitored by the gas cell Pirani gauge) is always recorded. Using the Instrument Quattro Ultima is now almost ready to use. To complete the start up procedure and prepare for running samples, follow the instructions in Start Up Following Overnight Shutdown in the following pages.

  • Page 38: Preparation For Electrospray Operation

    Quattro Ultima User's Guide Preparation for Electrospray Operation Source Thumb Nuts Glass Tube Probe Thumb Nuts Probe Adjustment Flange If the corona discharge pin is fitted, proceed as follows: Disconnect the gas and electrical connections from the front panel. Unscrew the probe thumb nuts and remove the probe Undo the three thumb screws and remove the probe adjustment flange and glass tube.
  • Page 39 Quattro Ultima User's Guide Exhaust Blanking Corona Liner Cleanable Plug Discharge Baffle Mounting Contact High Voltage Socket Take the electrospray probe and connect its gas line to Nebuliser on the front panel. Connect the liquid flow of a LC system or syringe pump to the probe.

  • Page 40: Preparation For Apci Operation

    Quattro Ultima User's Guide Preparation for APcI Operation Source Thumb Nuts Glass Tube Probe Thumb Nuts Probe Adjustment Flange If the corona discharge pin is not fitted, proceed as follows: Disconnect the gas and electrical connections from the front panel.
  • Page 41 Quattro Ultima User's Guide Corona Blanking Discharge Plug Sample Cone Mounting Contact High Voltage Socket Take the APcI probe and connect its gas line to Nebuliser on the front panel. Connect the liquid flow of a LC system or syringe pump to the probe.

  • Page 42: Operate

    Quattro Ultima User's Guide Operate On the MassLynx top-level window, click on to launch the tune page. The top line of the tune page indicates the current ionisation mode. If necessary, change the ionisation mode using the Ion Mode command.

  • Page 43: Pump Fault

    Quattro Ultima User's Guide The Vacuum LED shows a steady amber. Acquisition continues, although no mass spectral data are recorded. When the pressure recovers, the voltages are restored and the Vacuum and Operate LED’s are steady green. Any further deterioration of the system vacuum results in a pump fault and the system is shut down.

  • Page 44: Tuning

    Quattro Ultima User's Guide Tuning Information concerning the tuning of Quattro Ultima is provided in Tuning later in this document. Refer also to Electrospray, Nanoflow Electrospray and Atmospheric Pressure Chemical Ionisation for tuning information specific to those techniques. Calibration Information concerning the calibration of Quattro Ultima is provided in Mass Calibration later in this document.

  • Page 45: Fragmentation

    Quattro Ultima User's Guide Set up a 1 box display on the tune page and set Function to MS Scan. Observe the candidate parent ion in the display and determine its nominal mass. In this example the reserpine ion at z 609 is used as a model parent.

  • Page 46: Shutdown Procedures

    Quattro Ultima User's Guide Shutdown Procedures Emergency Shutdown In the event of having to shut down the instrument in an emergency, proceed as follows: Switch OFF the power at the wall mounted isolation switch(es), if fitted. If not, switch the power off at the rear of the instrument and switch off all peripherals.

  • Page 47: Complete Shutdown

    Quattro Ultima User's Guide Complete Shutdown If the instrument is to be left unattended for extended periods, proceed as follows: Switch off the LC pumps. On the MassLynx top-level window, click on to launch the tune page. Click on on the tune page.

  • Page 48: Automatic Start Up And Shutdown

    Quattro Ultima User's Guide Automatic Start up and Shutdown MassLynx comes with automatic startup and shutdown files which are run when Startup or Shutdown is selected from the Run menu. These are found in the C:\Masslynx\Shutdown directory and are called ShutDownxxx.acl and StartUpxxx.acl where xxx refers to the instrument...

  • Page 49: The Auto Control Tasks Page

    Quattro Ultima User's Guide There is an option to perform the shutdown tasks immediately after an error occurs or after the time defined in the Shutdown XX.XX minutes after batch or error box. If the Enable Shutdown on error box is checked then the shutdown tasks are performed after the defined time.
  • Page 50 Quattro Ultima User's Guide File Name is selected from the browser displayed when is pressed, or is typed in giving the full path name. To add a task: Select a task from the dropdown task list box. Enter the required parameters.

  • Page 51: The Shutdown Editor Toolbar

    Quattro Ultima User's Guide To change the width of a column: Position the mouse pointer on the heading between two columns until appears. Click the left mouse button and drag until the column is the required width. The Shutdown Editor Toolbar...

  • Page 52: Loading Startup And Shutdown Files

    Quattro Ultima User's Guide Loading Startup and Shutdown Files To open a startup or shutdown file: or select Open from the File menu. Press This displays the open file dialog. Select a data file and press Open. Saving a Startup or Shutdown File To save a startup or shutdown file or select Save or Save As from the File menu.

  • Page 53: Printing Startup And Shutdown Files

    Quattro Ultima User's Guide Printing Startup and Shutdown Files To print a startup or shutdown file: , or select Print from the File menu. Press This displays the print dialog. Select the printer, print range and number of copies and press OK.

  • Page 54: Creating Startup And Shutdown Files

    Quattro Ultima User's Guide Creating Startup and Shutdown Files To create a startup or shutdown file , or select New from the File menu. Press Running Startup and Shutdown Files If Startup or Shutdown is selected from the Run menu, or from the shutdown editor control list menu, then the automatic startup and shutdown files are run.

  • Page 55: Tuning

    Quattro Ultima User's Guide Tuning Overview Before sample data are acquired, the instrument should be tuned and, for the highest mass accuracy, calibrated using a suitable reference compound. • Consult the relevant section of this manual for information concerning source tuning procedures in the chosen mode of operation.

  • Page 56: The Tune Page

    Quattro Ultima User's Guide The Tune Page To display the tune page: Press on the MassLynx screen MS panel. Refer to the fold-out page at the end of this section for details of the tune page layout. Printing Tune Information...
  • Page 57 Quattro Ultima User's Guide If the selected file already exists on disk a warning is displayed. Press Yes to overwrite the existing information or No to enter a different file name. To restore a saved set of parameters: Press , or choose Open from the tune page File menu.

  • Page 58: Modifying The Peak Display

    Quattro Ultima User's Guide Modifying the Peak Display The tune peak display is modified using either the tune peak controls, or the mouse directly on the display. To select peaks: Press , or select Options, Peak Editor. Choose the peaks to be displayed by checking the appropriate boxes.
  • Page 59 Quattro Ultima User's Guide To change the span of a peak: Press the left mouse button at one end of the region of interest and, without releasing the button, drag the mouse horizontally to the other end. As the mouse is dragged a “rubber band” stretches out to indicate the selected range.

  • Page 60: Changing The Display

    Quattro Ultima User's Guide Changing the Display To change the display using the mouse: Click in the peak display area with the right mouse button to display the pop up menu. The display area for each peak can be individually changed, e.g.

  • Page 61: Trace

    Quattro Ultima User's Guide Trace From the pop-up menu: Select the Trace, Outline option to display the peak outline only. Select the Trace, Fill option to fill the trace with the trace fill colour. Select the Trace, Min/Max option to show the minimum and maximum data points only.

  • Page 62: Autotune

    Quattro Ultima User's Guide AutoTune MassLynx can automatically tune the mass spectrometer in both APcI and electrospray ionisation modes. AutoTune ramps the settings for the tuning parameters until they are optimised to give the best intensity, resolution and peak shape.

  • Page 63: Ion Mode

    Quattro Ultima User's Guide Checking that values are defined for all the user controllable instrument parameters and that these are passed to the data system. Checking that readbacks for these parameters are within specified tolerances. • Beam detection • Focus lens tuning •...

  • Page 64: Scope Parameters

    Quattro Ultima User's Guide Scope Parameters Scan Time and Inter Scan Delay control the speed with which the tune peak display is updated. The tuning system behaves more responsively if the scan time and inter scan delay are short. To change the scope parameters: Press , or choose Scope Parameters from the tune page Options menu.

  • Page 65: Resetting The Zero Level

    Quattro Ultima User's Guide To set up a collision energy ramp: Choose Collision Energy Ramp Gradient from the tune page Ramps menu. Two values of collision energy are defined at two particular masses. These values define a gradient for the collision...

  • Page 66: Controlling Readbacks

    Quattro Ultima User's Guide Controlling Readbacks There are three options for displaying system readbacks on the tune page: • Readbacks displayed continuously. • Readbacks hidden. • Readbacks displayed only when differing from their defined values by more than 10%. A number of the readbacks are for diagnostic purposes only, their function being to confirm a voltage is present.

  • Page 69: Data Acquisition

    Quattro Ultima User's Guide Data Acquisition Starting an Acquisition There are two ways of starting an acquisition: • a single sample acquisition from the tune page • a multiple sample one from the MassLynx top level screen. Starting an Acquisition from the Tune Page The easiest way to acquire data is directly from the tune page.

  • Page 70: Parameters

    Quattro Ultima User's Guide Parameters The Data File Name can be up to 128 characters. If the file already exists on disk, a prompt is given to rename the file or to overwrite the existing one. The file is written to the data directory of the current project.

  • Page 71: Multiple Samples

    Quattro Ultima User's Guide Scan Time specifies the duration of each scan in seconds. Inter Scan Time specifies the time in seconds between a scan finishing and the next one starting. During this period no data are stored. Pressing Origin allows additional information about the sample to be analysed to be entered into the following fields: •...

  • Page 72: Process

    Quattro Ultima User's Guide Process The process controls allow processes to be run before and after the acquisition. The Pre-Run control is used to specify the name of a process that is run before acquisition of the files in the sample list.
  • Page 73 Quattro Ultima User's Guide Integrate Samples integrates all the sample data files named in the peak list. Calibrate Standards uses integration results to form quantify calibration curves. Quantify Samples uses integration results and quantify calibration curves to calculate compound concentrations.

  • Page 74: Monitoring An Acquisition

    Quattro Ultima User's Guide Monitoring an Acquisition Acquisition status is also shown on the MassLynx screen. The run time is shown on the MS panel and the scan status, sample number and scan number are shown on the Status bar at the bottom of the page.

  • Page 75: Instrument Data Thresholds

    Quattro Ultima User's Guide When real-time update is on the display is continually updated with spectra from the current acquisition. The actual information displayed is determined by selecting one of the following radio buttons. • Latest scan displays the last acquired scan. This is the default option.

  • Page 76: Maxent

    Quattro Ultima User's Guide MaxEnt The MaxEnt algorithm needs to measure noise accurately within a data file. For this reason Ion Counting Threshold should be set to zero when acquiring data to be analysed using MaxEnt. Profile Data The controls for profile data allow control of the amount of data collected during a continuum data acquisition.

  • Page 77: Ion Counting Threshold

    Quattro Ultima User's Guide Ion Counting Threshold Ion Counting Threshold sets the intensity level below which a data point is ignored. This threshold is applied to all acquisitions, regardless of scanning mode. It is also the most significant of all of the data manipulation variables as it is applied to the raw data first.

  • Page 78: Profile Data - Spike Removal

    Quattro Ultima User's Guide 100% Profile Data - Spike Removal Spikes are distinguished from real data by the fact that they are very narrow and, when compared to their immediate neighbours, very intense. Data points determined to be spikes are removed by setting the value of this data point to the average of its immediate neighbours.

  • Page 79: Analog Data

    Quattro Ultima User's Guide Spike removal involves some additional processing while acquiring and reduces the maximum achievable acquisition rates by approximately 30%. To perform spike removal during an acquisition: Check Use Spike Removal. This is not reflected in the tune page.

  • Page 80: Stopping An Acquisition

    Quattro Ultima User's Guide Stopping an Acquisition To halt the acquisition: From the tune page, press Stop. From the MassLynx screen choose Stop from the Run menu, or press Data acquired up to this point is saved. The Function List Editor...
  • Page 81 Quattro Ultima User's Guide A simple function list is shown above, containing only one function: a centroided mode full scan, between 500 and 1500 amu using ES+ ionisation. Immediately above the function bar display is a time scale that shows from when the function is active, and for how long it runs.

  • Page 82: The Function List Editor Toolbar

    Quattro Ultima User's Guide The Function List Editor Toolbar The toolbar is displayed at the top of the tune window and allows some common operations to be performed with a single click. Create a new function list. Edit the selected function.

  • Page 83: Modifying An Existing Function

    Quattro Ultima User's Guide Modifying an Existing Function To modify an existing function: Select the function in the function list. Press , or double click on the function. This displays the appropriate editor for the function type and allows changes to be made.

  • Page 84: Setting A Solvent Delay

    Quattro Ultima User's Guide Setting a Solvent Delay No data is stored during the solvent delay period, which means that solvent peaks that would normally be seen eluting on the TIC chromatogram are no longer seen. For APcI functions the APcI...

  • Page 85: Saving And Restoring A Function List

    Quattro Ultima User's Guide This description is used on the analog chromatogram dialog as the channel description. See “Chromatogram” in the MassLynx User’s Guide. Enter an Offset to align the external unit with the mass spectrometer. Press OK. Saving and Restoring a Function List To save a function list: Choose Save As from the function list File menu.

  • Page 86: Setting Up A Full Scan Function

    Quattro Ultima User's Guide Setting up a Full Scan Function The full scan function editor, activated by pressing or by selecting MS Scan from the Functions menu, is used to set up centroid, continuum and MCA functions. Mass (m/z) Start Mass and End Mass specify the masses at which the scan starts and stops.

  • Page 87: Scan Duration (Secs)

    Quattro Ultima User's Guide acquisition system as compared to centroided acquisition. Data files tend to be significantly larger than centroided ones and the absolute scanning speed (amu/sec) is slower. It is possible, however, to set a threshold below which the data are not stored.

  • Page 88: Setting Up A Sir Function

    Quattro Ultima User's Guide Setting up a SIR Function The SIR (Selected Ion Recording) technique is typically used in situations where only a few specific masses are to be monitored. Since most of the data acquisition time is spent on these masses, the technique is far more sensitive than full scanning.

  • Page 89: Method

    Quattro Ultima User's Guide Double click on a mass in the list. This displays the values for the selected mass in the edit fields. Change Mass, Dwell or Cone as required. Press Change to update the values in the list.

  • Page 90: Setting Up Ms-Ms Scanning Functions

    Quattro Ultima User's Guide Setting up MS-MS Scanning Functions Many of the fields in the MS-MS editors are similar to those in the full scan editor. Only fields which differ significantly are described below. Mass Daughter This is the most commonly used MS-MS mode and is used to look at fragmentations of a particular ion.
  • Page 91 Quattro Ultima User's Guide It is possible to select the daughter mass to be greater than the parent (precursor) mass. In this case ions which have gained mass in the collision cell, or are of higher mass to charge ratio, are detected. This can occur when a multiply charged ion fragments and loses a charge.

  • Page 92: Collision Energy

    Quattro Ultima User's Guide Collision Energy This specifies the collision energy in electron volts to be used for the collision cell during the scan. When Use Tune Page Settings is selected the collision energy set on the tune page is used. If it is required to adjust the setting during an acquisition then the acquisition must be started from the tune page.

  • Page 93: Setting Up A Mrm Function

    Quattro Ultima User's Guide Setting up a MRM Function Multiple reaction monitoring (MRM) functions are set up in much the same way as SIR functions, but allow a number of MS-MS transitions (fragmentations) between MS1 and MS2 to be monitored.

  • Page 94: Survey And Msms Template Pages

    Quattro Ultima User's Guide Survey and MSMS Template Pages These pages allow the parameters to be set for MS and MS-MS scanning during the survey, and are similar to normal function editor pages. Data Acquisition Page 94...

  • Page 95: Ms To Msms Switching

    Quattro Ultima User's Guide MS to MSMS Switching Switch Criteria MSMS scanning commences: • If TIC is selected, and the TIC of the spectrum rises above the specified Threshold. • If Intensity is selected, and the intensity of the largest...
  • Page 96 Quattro Ultima User's Guide Detected Precursor Inclusion Auto exclude and Always include are not currently available. Include after time, if selected, allows a delay to be incorporated before precursors are included. Data Discard uninteresting survey scans allows only the survey scans that detect precursor ions to be stored.

  • Page 97: Msms To Ms Switching

    Quattro Ultima User's Guide MSMS to MS Switching When MSMS functions have been generated, they are carried out in parallel until the conditions for switching to MS are satisfied. When all MSMS functions have stopped, the MS survey function is again carried out.

  • Page 98: Including And Excluding Masses

    Quattro Ultima User's Guide Including and Excluding Masses Mass ranges and individual masses to be included or excluded from the MS-MS scans are entered in the relevant Range boxes. Masses on the Exclude list are not considered for detection. Ranges take the form massX_massY.

  • Page 99: Monitoring Acquisitions

    Quattro Ultima User's Guide Monitoring Acquisitions When an acquisition is started the automatic switching status dialog is displayed showing the precursors currently running. Data Acquisition Page 99...
  • Page 100 Quattro Ultima User's Guide Data Acquisition Page 100...

  • Page 101: Mass Calibration

    • A brief general overview of the calibration process. • A complete mass calibration of Quattro Ultima using electrospray ionisation with a mixture of sodium iodide and rubidium iodide as the reference compound. • A complete mass calibration of Quattro Ultima using atmospheric pressure chemical ionisation (APcI) with PEG as the reference compound.

  • Page 102: Overview

    A separate mass spectrum of the reference compound is acquired for each selected calibration type. Quattro Ultima requires these three calibrations for both MS1 and MS2, for a maximum of six calibration curves. The table below show which types of calibration are necessary for particular types of experiment.

  • Page 103: The Calibration Process

    Quattro Ultima User's Guide The Calibration Process • Tuning the instrument. • Selecting the appropriate reference file for the reference sample to be used. • Starting an automatic calibration. • Checking the calibration report. Electrospray Introduction When a calibration is completed it is possible to acquire data over any mass range within the calibrated range.

  • Page 104: Preparing For Calibration

    Quattro Ultima User's Guide Preparing for Calibration Reference Compound Introduction The example given here describes an automatic calibration which requires reference compound to be present for several minutes. The introduction of the reference compound is best achieved using a large volume Rheodyne injector loop (50 or 100µl) or an infusion pump (for example, a Hamilton syringe pump).

  • Page 105: Instrument Threshold Parameters

    Quattro Ultima User's Guide Instrument Threshold Parameters Before beginning the calibration procedure, some instrument parameters need to be checked. For most low mass range calibrations, calibration data is acquired in continuum mode. To allow suitable scanning speeds to be used the continuum data parameters need to...

  • Page 106: Calibration Options

    Quattro Ultima User's Guide Calibration Options To access the calibration options: Select Calibration then Calibrate Instrument... on the tune page. Selecting the Reference File To select the appropriate reference file: Click on the arrow in the Reference File box and scroll through the files.

  • Page 107: Selecting Parameters

    Quattro Ultima User's Guide Selecting Parameters A number of parameters needs to be set before a calibration is started. Default parameters are set when the software is initially loaded which usually give a suitable calibration, but under some conditions these may need to be adjusted.

  • Page 108: Calibration Parameters

    Quattro Ultima User's Guide Calibration Parameters These are accessed from Edit, Calibration Parameters..The Peak Match parameters determine the limits within which the acquired data must lie for the software to recognise the calibration masses and result in a successful calibration.

  • Page 109: Mass Measure Parameters

    Quattro Ultima User's Guide Mass Measure Parameters These are accessed through Edit, Mass Measure Parameters..If continuum or MCA data are acquired for calibration then these parameters need to be set before the calibration is carried out. If centroided data are used for calibration then the mass measure parameters are not used.

  • Page 110: Performing A Calibration

    Quattro Ultima User's Guide Performing a Calibration Three types of calibration are available with MassLynx: static calibration, scanning calibration and scan speed compensation. These are selected on the Automatic Calibration dialog box (see below) which is accessed by selecting Start... from the Calibrate dialog box.
  • Page 111 Quattro Ultima User's Guide • If scanning calibration and scan speed compensation are present then the instrument is only calibrated for scanning acquisitions over the same mass range as that used for the calibration, but the scan speed can be changed provided that it remains within the scan speeds used for the two calibrations.

  • Page 112: Acquisition Parameters

    Quattro Ultima User's Guide Acquisition Parameters Selecting Acquisition Parameters... in the Automatic Calibration dialog box brings forward a second box, shown below, where the mass ranges, scan speeds and acquisition mode are set. When this box is first accessed it contains default parameters relevant to the chosen reference file.
  • Page 113 Quattro Ultima User's Guide Static Span sets the size of this small region around each reference peak. A span of 4.0 amu is typical. Static Dwell determines how much time is spent acquiring data across the span. A value of 0.1 second is suitable.

  • Page 114: Starting The Calibration Process

    Quattro Ultima User's Guide Starting the Calibration Process To start the calibration process: Select OK from the Automatic Calibration dialog box. The instrument acquires all of the calibration files in the following order using the data file names shown: MS1 static calibration...
  • Page 115 Quattro Ultima User's Guide ___________________________________________________________________________________ ___________________________________________________________________________________ 28 matches of 28 tested references. SD = 0.0465 MS1 Static Calibration 0.65 -0.15 1000 1500 2000 2500 3000 3500 27 matches of 28 tested references. SD = 0.0459 MS1 Scanning Calibration 0.35 -0.19...

  • Page 116: Checking The Calibration

    Quattro Ultima User's Guide Checking the Calibration The calibration (successful or failed) can be viewed in more detail by selecting Process, Calibration From File... from the Calibrate dialog box. The dialog box which is then displayed (see below) allows the choice of calibration type for viewing.
  • Page 117 Quattro Ultima User's Guide Mass Calibration Page 117...

  • Page 118: Calibration Failure

    Quattro Ultima User's Guide Calibration Failure If the calibration statistics do not meet the requirements then a message is displayed describing at what point and why the calibration failed. This message also states where the attempted calibration data can be viewed so that the exact cause of failure can be determined.
  • Page 119 Quattro Ultima User's Guide In the case of too many consecutive peaks missed: Check the data in the on-screen calibration report to see if the missed peaks are present in the acquired calibration file. If the peaks are not present then the first three reasons above are likely causes.

  • Page 120: Incorrect Calibration

    Quattro Ultima User's Guide Incorrect Calibration If the suggested calibration parameters are used, and providing that good calibration data have been acquired, then the instrument should be calibrated correctly. However in some circumstances it is possible to meet the calibration criteria without matching the correct peaks.

  • Page 121: Manual Editing Of Peak Matching

    Quattro Ultima User's Guide Manual Editing of Peak Matching If an incorrect peak has been matched in the calibration process, this peak can be excluded manually from within the on-screen calibration report. Using the mouse place the cursor over the peak in the acquired spectrum and click with the right mouse button.

  • Page 122: Verification

    Quattro Ultima User's Guide Verification Once a full instrument calibration is in place it is not always necessary to repeat the full calibration procedure when the instrument is next used. Instead a calibration verification can be performed. (There is no benefit in verifying each calibration individually, re-calibration is just as quick.)
  • Page 123 Quattro Ultima User's Guide Set Scan From, Scan To, Run Duration, Data Type, Scan Time and Inter Scan Delay<$]helv_bold> to agree with the acquisition parameters that are to be used for data acquisition. With only the scanning calibration selected all of the other options in this dialog box are unavailable.

  • Page 124: Electrospray Calibration With Peg

    Quattro Ultima User's Guide Electrospray Calibration with PEG Caution should be used when calibrating with PEG in electrospray mode due to the number of peaks which are produced. Although ammonium acetate is added to the PEG reference solution to produce [M+NH...

  • Page 125: Atmospheric Pressure Chemical Ionisation

    User's Guide Atmospheric Pressure Chemical Ionisation Introduction This chapter describes a complete mass calibration of Quattro Ultima using atmospheric pressure chemical ionisation. The procedures described should be followed only after reading the previous chapter in this manual, describing the automated calibration with electrospray ionisation.

  • Page 126: Preparing For Calibration

    Quattro Ultima User's Guide Preparing for Calibration Reference Compound Introduction It is best to use a large volume injection loop (50µl) with a solvent delivery system set up to deliver 0.2 ml/min of 50:50 acetonitrile:water or methanol:water through the injector and into the APcI source. An injection of 50µl of reference solution lasts for approximately 15 seconds, allowing enough time to perform a slow scanning calibration.

  • Page 127: Performing A Calibration

    Quattro Ultima User's Guide Performing a Calibration The three types of calibration (static, scanning and scan speed) must be carried out in single steps. Static Calibration Access the Automatic Calibration dialog box by selecting Start... from the Calibrate page. Check Static Calibration and MS1 in the Types area of the dialog box.
  • Page 128 Quattro Ultima User's Guide When an instrument acquires data for a static calibration it first examines the selected reference file for the expected reference masses. It then acquires data over a small mass span around the expected position of each peak. Thus the acquired data do not contain continuous scans, but each “spectrum”...
  • Page 129 Quattro Ultima User's Guide Manual Calibration To perform a manual calibration using the acquired data: From the chromatogram window call up the calibration file STATMS1. Determine the scan numbers at the beginning and end of the chromatogram peak for the reference solution.
  • Page 130 Quattro Ultima User's Guide If insufficient peaks have been matched, or the wrong peaks have been matched, refer to the section on calibration failure later in this manual. Mass Calibration Page 130...
  • Page 131 Quattro Ultima User's Guide If the correct peaks have been matched then the report can be printed out: Select Print, Print from the report display. ________________________________________________________________________________________________________ Ca l i b r a t i o n R e p o r t...

  • Page 132: Scanning Calibration And Scan Speed Compensation

    Quattro Ultima User's Guide Scanning Calibration and Scan Speed Compensation Acquiring Data To complete the calibration of the instrument two further data files must be acquired. Both files are acquired in scanning mode over the same mass range, one at the slowest speed required for scanning acquisitions and one at the fastest speed.
  • Page 133 Quattro Ultima User's Guide The recommended scan speed for the scan speed compensation is 1000 amu/sec. This is the maximum scan speed permissible when using thresholded continuum data. Although continuum is recommended centroided data may be used. It is possible to scan more quickly in centroided mode, but it is unlikely that a faster acquisition rate would be needed for general use.
  • Page 134 Quattro Ultima User's Guide If the correct peaks have been matched then the calibration report can be printed out: Select Print, Print from the report display. If insufficient peaks have been matched or the wrong peaks have been matched see Calibration Failure later in this chapter.

  • Page 135: Calibration Failure

    Quattro Ultima User's Guide Calibration Failure When calibration is performed manually there is no warning message to show that the calibration has not met the set criteria. This must be judged by viewing the on-screen calibration report and examining the matched peaks and statistics associated with the report.

  • Page 136: Incorrect Calibration

    Quattro Ultima User's Guide In the case of too many consecutive peaks missed: Check the on-screen calibration report to see if the missed peaks are present in the acquired calibration file. If the peaks are not present then the first three reasons above are likely causes.

  • Page 137: Manual Editing Of Peak Matching

    Quattro Ultima User's Guide An alternative cause of calibration failure is from contamination or background peaks. If a contamination or background peak lies within one of the peak matching windows, and is more intense than the reference peak in that window, then the wrong peak is selected.

  • Page 138: Manual Verification

    Quattro Ultima User's Guide Manual Verification Once a full instrument calibration is in place it is not always necessary to repeat the full calibration procedure when the instrument is next used. Instead a calibration verification can be performed. (There is no benefit in verifying each calibration individually, re-calibration is just as quick.)
  • Page 139 Quattro Ultima User's Guide A calibration curve is produced and displayed on the screen in a similar way to when the original calibration was performed. An example is shown above. When OK is selected from this report, unlike the original calibration procedure, the instrument calibration is not changed.
  • Page 140 Quattro Ultima User's Guide Mass Calibration Page 140...

  • Page 141: Electrospray

    Quattro Ultima User's Guide Electrospray Introduction Purge Gas Probe Exhaust Sample Cleanable Exhaust Baffle Liner Sample Cone Gas Cone Isolation Valve Desolvation Nebuliser Analyser Restrictor Tunnel 1 Tunnel 2 Source Enclosure E1M18 E2M28 Turbomolecular Pumps Rotary Pumps The ESI interface consists of the standard Z-spray source fitted with an electrospray probe.
  • Page 142 Quattro Ultima User's Guide The source can be tuned to fragment ions within the ion tunnel 1 vacuum housing. This can provide valuable structural information for low molecular weight analytes. The most common methods of delivering sample to the electrospray source are: •...
  • Page 143 Quattro Ultima User's Guide It is wise periodically to check the flow rate from the solvent delivery system. This can be carried out by filling a syringe barrel or a graduated glass capillary with the liquid emerging from the probe tip and timing a known volume, say 10µl. Once the rate has been measured and set, a note should be made of the back pressure readout on the pump as fluctuation of this reading can indicate problems with the solvent flow.

  • Page 144: Post-Column Splitting

    Quattro Ultima User's Guide Post-column Splitting Although the electrospray source can accommodate flow rates up to 1 ml/min, it is recommended that the flow is split post-column to approximately 200 µl/min. Also, even at lower flow rates, a split may be required for saving valuable samples.

  • Page 145: Megaflow

    Quattro Ultima User's Guide Megaflow Megaflow electrospray enables flow rates from 200 µl/min to 1 ml/min to be accommodated. This allows microbore (2.1mm) or 4.6mm diameter columns to be interfaced without splitting. Changing Between Flow Modes When changing between megaflow and standard electrospray operation, it is essential that the correct tubing is used to connect the probe to the sample injector.

  • Page 146: Operation

    Quattro Ultima User's Guide Operation Exhaust Blanking Corona Liner Cleanable Plug Discharge Baffle Mounting Contact High Voltage Socket Ensure that the source is assembled as described in Maintenance and Fault Finding, and that the instrument is pumped down and prepared for electrospray operation as described in Routine Procedures.

  • Page 147: Checking The Esi Probe

    Quattro Ultima User's Guide Checking the ESI Probe Connect the electrospray probe to a pulse free pump. Solvent should be degassed to prevent beam instabilities caused by bubbles. Connect the PTFE tubing of the electrospray probe to Nebulising Gas on the front panel.

  • Page 148: Obtaining An Ion Beam

    Quattro Ultima User's Guide Obtaining an Ion Beam If necessary, change the ionisation mode using the Ion Mode command. The top line of the tune page indicates the current ionisation mode. Using the needle valve on the front panel, set the desolvation gas flow rate to 300 litres/hour.
  • Page 149 Quattro Ultima User's Guide The position of the probe is adjusted using the probe adjustment collar (in/out) and the adjustment knob (sideways) located to the left of the probe. The two screws can be adjusted singly or simultaneously to optimise the beam. The position for optimum sensitivity and stability for low flow rate work (10 µl/min) is shown.
  • Page 150 Quattro Ultima User's Guide Desolvation Gas Flow Solvent Flow Rate Desolvation Temp Rate µl/min °C litres/hour <10 100 to 120 200 to 250 10 to 20 120 to 250 200 to 400 20 to 50 250 to 350 200 to 400 >50...
  • Page 151 Quattro Ultima User's Guide Restrictor The restrictor is positioned on the opposite side of the source block to the sample cone. Screwing the restrictor into the source block restricts the pumping port and increases the source block pressure. The restrictor is factory adjusted for optimum sensitivity on low molecular weight singly charged species.
  • Page 152 Quattro Ultima User's Guide Source Temperature 100°C is typical for 50:50 CH CN:H O at solvent flow rates up to 50 µl/min. Higher source temperatures, up to 150°C, are necessary for solvents at higher flow rates and higher water content.
  • Page 153 Quattro Ultima User's Guide RF Lens 1 Voltage RF Lens 1 settings between 0 and 50V can be beneficial in removing solvent adducts from certain samples. Values of 50V and above can provide efficient fragmentation of sample ions. As for the Cone, this parameter should be optimised for maximum sensitivity.

  • Page 154: Megaflow Hints

    Quattro Ultima User's Guide Megaflow Hints With this high flow rate technique the setup procedure involves making the following adjustments: • increase Drying Gas flow to approximately 750 litres/hour. • increase Desolvation Temp to 400°C. • increase Source Temp to 150°C.

  • Page 155: Sample Analysis And Calibration

    Quattro Ultima User's Guide Sample Analysis and Calibration General Information Care should be taken to ensure that samples are fully dissolved in a suitable solvent. Any particulates must be filtered to avoid blockage of the transfer line or the probe’s capillary.

  • Page 156: Typical Es Positive Ion Samples

    Quattro Ultima User's Guide Typical ES Positive Ion Samples • Peptides and proteins. • Small polar compounds. • Drugs and their metabolites. • Environmental contaminants (e.g. pesticides / pollutants). • Dye compounds. • Some organometallics. • Small saccharides. Typical ES Negative Ion Samples •...

  • Page 157: Chromatographic Interfacing

    A UV detector may be placed in-line to the Quattro Ultima probe. However, ensure that the volume of the detector does not significantly reduce the chromatographic resolution. Whenever a UV detector is used, the analog output may be input to MassLynx NT for chromatographic processing.

  • Page 158: Lc-Ms Sensitivity Enhancement

    Quattro Ultima User's Guide LC-MS Sensitivity Enhancement The sensitivity of a LC-MS analysis can be increased or optimised in a number of ways, by alterations to both the LC operation and the MS operation. In the LC area some examples include the use of high resolution columns and columns with fully end capped packings.

  • Page 159: Nanoflow Electrospray

    Quattro Ultima User's Guide Nanoflow Electrospray Overview Nano-LC Option Glass Capillary Option Regulator and Injector (Nano-LC option) Three-axis Manipulator Stop Stage Handle Protective Cover The optional nanoflow interface allows electrospray ionisation to be performed in the flow rate range 5 to 1000 nanolitres per minute. There are two options for the spraying capillary, which can be alternately fitted to the interface: •...

  • Page 160: Installing The Interface

    Quattro Ultima User's Guide For a given sample concentration, the ion currents observed in nanoflow are comparable to those seen in normal flow rate electrospray. Great sensitivity gains are therefore observed when similar scan parameters are used, due to the great reductions in sample consumption.
  • Page 161 Quattro Ultima User's Guide Source Thumb Nuts Glass Tube Probe Flange Mounting Pillar Probe Probe Thumb Nuts Adjustment Flange Unscrew the three probe flange mounting pillars, using the holes to obtain the necessary leverage. If the cone gas nozzle is not...
  • Page 162 Quattro Ultima User's Guide If not already in place, attach the microscope or camera brackets using the screw hole and dowels at the top of the bracket. Insert the flexible light guide into the grommet at the base of the perspex cover.

  • Page 163: Operation Of The Camera System

    Quattro Ultima User's Guide Camera Microscope Zoom Lens Objective Lens Grommet Operation of the Camera System Magnification is controlled by the zoom lens. A fine focus can be achieved by rotating the objective lens. Using the Microscope Focusing is adjusted by rotating the top of the microscope.

  • Page 164: Glass Capillary Option

    Quattro Ultima User's Guide Glass Capillary Option Warning: Do not touch the sharp end of the capillary. As well as the risk of injury by a sliver of glass, the needle becomes inoperable. Caution: The capillaries are extremely fragile and must be handled with great care.

  • Page 165: Restarting The Spray

    Quattro Ultima User's Guide Set Capillary between 1 and 1.5kV. Adjust Desolvation Gas using the knob on the front panel of the instrument. An ion beam should now be visible on the tune page. Tune the source voltages, adjust the gas flow and adjust the three-axis manipulator for maximum ion current.

  • Page 166: Nano-Lc Option

    Quattro Ultima User's Guide Nano-LC Option Installation From Injector (or Column Attached Directly) With the sprayer assembly removed from the stage: Cut approximately 25mm of the Valco red stripe peek tubing and, using Ferrule the plug cap and a Valco nut, set a...

  • Page 167: Operation

    Quattro Ultima User's Guide The capillary can now be checked for flow by connecting the output from a Harvard syringe pump to the other side of the union and setting the flow to 1 µl/min, using a micropipette to measure the flow. It is recommended that a syringe with a volume of no more than 50 millilitres is used.

  • Page 168: Changing Options

    Quattro Ultima User's Guide Optimise the ion beam by altering the position of the spray using the controls of the translation stage. The sprayer can now be connected to the HPLC system. The injection valve is plumbed as follows: P from the pump.

  • Page 169: Atmospheric Pressure Chemical Ionisation

    Quattro Ultima User's Guide Atmospheric Pressure Chemical Ionisation Introduction Corona Probe Discharge Pin Exhaust Sample Exhaust Cleanable Liner Baffle Sample C o n e Isolation Valve Nebuliser Desolvation Analyser Restrictor Tunnel 1 Tunnel 2 Source Enclosure E1M18 E2M28 Turbomolecular Rotary Pumps...

  • Page 170: Preparation

    Quattro Ultima User's Guide For APcI operation, the desolvation gas is not heated in the desolvation nozzle. However, it is important that desolvation gas is used throughout. The background spectrum for 50:50 acetonitrile:water is dependent upon the setting of Cone. The main reagent ions for a typical sample voltage of 40V are 83, 101 and 142.

  • Page 171: Checking The Probe

    Quattro Ultima User's Guide Checking the Probe Ensure that the probe heater is off. Unplug the probe from the instrument’s front panel and remove the probe from the source. Connect the PTFE tube to the Nebuliser outlet on the front panel.

  • Page 172: Obtaining A Beam

    Quattro Ultima User's Guide Obtaining a Beam Ensure that the corona discharge pin is fitted and connected as described in Routine Procedures, Preparation for APcI Operation. Ensure that the APcI probe is fitted as described above, that the desolvation gas tube is connected to the front panel, and that the purge gas outlet is plugged.

  • Page 173: Calibration

    Quattro Ultima User's Guide Check that a stable beam of solvent ions is now apparent. Refer to Hints for Sample Analysis later in this chapter for further information on source tuning. Warning: It is normal for the source enclosure, the glass tube and parts of the probe adjustment flange to reach temperatures of up to 60°C during prolonged...

  • Page 174: Hints For Sample Analysis

    Quattro Ultima User's Guide Hints for Sample Analysis Tuning for General Qualitative Analysis Refer to Obtaining a Beam above and tune on solvent ions. Adjust the in/out position of the probe so that it is fully retracted from the source.

  • Page 175: Corona Current

    Quattro Ultima User's Guide Corona Current Corona current can have a significant effect on sensitivity. The corona current required depends upon the polarity of the compound and the polarity of the analytical mobile phase. As recommended above optimization should be done in the presence of the analytical mobile phase.

  • Page 176: Removing The Probe

    Quattro Ultima User's Guide Removing the Probe After a session of APcI operation: Turn off the LC flow. Set APcI Probe Temp to 20°C. Deselect When the probe temperature falls below 100°C: Select Gas, Gas to turn off the nitrogen.

  • Page 177: Maintenance And Fault Finding

    Quattro Ultima User's Guide Maintenance and Fault Finding Introduction Cleanliness and care are of the utmost importance whenever internal assemblies are removed from the instrument. " Always prepare a clear clean area in which to work. " Make sure that any tools or spare parts that may be required are close at hand.

  • Page 178: The Vacuum System

    Quattro Ultima User's Guide The Vacuum System The performance of the mass spectrometer is severely impaired by the lack of a good vacuum in the ion transfer (hexapole) regions or the analyser. • An analyser pressure above 10 mbar results in a general loss in performance indicated by a loss of resolution and an increase in the background noise.

  • Page 179: Vacuum Leaks

    Source components that operate at, or slightly above, atmospheric pressure are not susceptible to vacuum leaks. In the unlikely event of a leak on a feedthrough, then the unit should be replaced or returned to Micromass for repair. Pirani Gauge The Pirani gauge head does not require routine maintenance.

  • Page 180: Gas Ballasting And Rotary Pump Oil Recirculation

    This oil is returned to the rotary pump during gas ballasting. The Quattro Ultima has two rotary pumps; an E1M18 pumping the source block and an E2M28 pumping the first hexapole. Because the source block is maintained at a relatively high pressure, the rate of oil mist being expelled from the E1M18 pump is considerably higher than that from the E2M28 pump.

  • Page 181: Oil Mist Filter

    Quattro Ultima User's Guide Caution: Failure to gas ballast the rotary pump frequently leads to shortened oil lifetime which in turn may shorten rotary pump lifetime. Caution: The instrument should not be vented with the E2M28 manual gas valve open. The E1M18 manual gas ballast valve should remain open at all times.

  • Page 182: Rotary Pump Oil

    Quattro Ultima User's Guide Rotary Pump Oil The oil in the rotary pump should be maintained at the correct level at all times. Check the oil level at weekly intervals, topping up if necessary. It is important to monitor the condition of the oil regularly. Replace the oil when it has changed to a noticeable red colour, or routinely at 4 month intervals (3000 hours operation).

  • Page 183: The Source

    Quattro Ultima User's Guide The Source Overview The Z-spray source is a robust assembly requiring little maintenance. The source consists of three basic parts: • The probe adjustment flange. • The glass tube. • The source flange assembly. The probe adjustment flange and the glass tube can be readily removed, without venting the instrument, to gain access to the source block and sample cone.

  • Page 184: Cleaning The Cone Gas Nozzle And Sample Cone

    Quattro Ultima User's Guide Cleaning the Cone Gas Nozzle and Sample Cone This may be necessary due to lack of sensitivity or fluctuating peak intensity, or if deposited material is visible on the outside of the nozzle or sample cone. Proceed as...
  • Page 185 Quattro Ultima User's Guide Disconnect both electrical connections by pulling back on the plug sleeves to release the plugs from the sockets on the front panel. Undo the two knurled thumb nuts that retain the probe and withdraw it from the source.
  • Page 186 If the components are still not clean, or if the aperture is partially blocked, place the components in an ultrasonic bath containing 50:50 acetonitrile:water or 50:50 methanol:water. To minimise down time fit a spare sample cone and cone gas nozzle, obtainable from Micromass, at this stage. Maintenance and Fault Finding Page 186...
  • Page 187 Quattro Ultima User's Guide Exhaust Liner Cleanable Baffle Sample Cone Gas Cone Nozzle Extraction Tool Gasket Dry the cone and nozzle using nitrogen. If material has built up on the exhaust liner and cleanable baffle: Remove the cleanable baffle and the exhaust liner.

  • Page 188: Removing And Cleaning The Ion Block

    Quattro Ultima User's Guide Removing and Cleaning the Ion Block On the tune page select Other from the menu bar at the top of the tune page. Click on Vent. The rotary pump and the turbomolecular pumps switch off. The turbomolecular pumps are allowed to run down to 50% speed after which a vent valve opens to atmosphere automatically.
  • Page 189 Quattro Ultima User's Guide When the instrument has vented: Remove the four screws, together with the washers, which secure the cover plate to the ion block and remove the cover plate. Warning: The heater supply remains live until the system is fully vented. Do not remove the cover plate until the system has vented.
  • Page 190 Quattro Ultima User's Guide Heater Supply Thermocouple Leads Heater Leads Ion Block Support O Rings Ion Block Restrictor O Ring and Bush PTFE Washer Remove the two screws from the heater connections on the ion block. Carefully straighten the heater supply leads, in such a way that the ion block can later be withdrawn without fouling these leads.
  • Page 191 Quattro Ultima User's Guide Unscrew the restrictor’s outer bush from the ion block, taking care not to disturb the setting of the inner restrictor. Collect the PTFE washer, and ensure that the O ring remains in position on the restrictor shaft.

  • Page 192: Removing And Cleaning The Ion Tunnel Assembly

    Quattro Ultima User's Guide Removing and Cleaning the Ion Tunnel Assembly View from rear Ion Tunnel 1 Ion Block Support O Rings To remove the ion tunnel assembly, proceed as follows: Remove the ion block, as described above. Remove the three screws retaining the ion block support and carefully withdraw it from the pumping block.
  • Page 193 Quattro Ultima User's Guide Differential Aperture Plate To clean the ion tunnel proceed as follows: Immerse the complete assembly in a suitable solvent (100% methanol) and sonicate in an ultrasonic bath. Thoroughly dry the assembly using a flow of nitrogen.

  • Page 194: Reassembling And Checking The Source

    Quattro Ultima User's Guide Reassembling and Checking the Source Check the condition of all O rings. Replace them if necessary. With the two springs in a horizontal plane, feed the hexapole transfer lens into the instrument. Ensure that the O ring is correctly fitted to the differential aperture plate, and that the assembly is pushed fully in.

  • Page 195: The Discharge Pin

    Quattro Ultima User's Guide Insert the thermocouple into its location, and secure it with the clip. Reconnect the heater leads and heater supply leads to the terminal block, carefully bending the supply leads as necessary. Replace the cover plate. Check that the isolation valve is closed.

  • Page 196: The Electrospray Probe

    Quattro Ultima User's Guide The Electrospray Probe Overview Indications that maintenance is required to the electrospray probe include: • An unstable ion beam. Nebulising gas may be escaping from the sides of the probe tip. Ensure that the probe tip O ring is sealing correctly.
  • Page 197 Quattro Ultima User's Guide Blockage of the stainless steel sample capillary may occur if the desolvation heater is left on without liquid flow. This is particularly relevant for samples contained in involatile solvents or high analyte concentrations. To avoid this problem it is good practice to switch off the heater before stopping the liquid flow, and flush the capillary with solvent.

  • Page 198: Replacement Of The Stainless Steel Sample Capillary

    Quattro Ultima User's Guide Replacement of the Stainless Steel Sample Capillary Probe Stainless Steel Capillary Liner Tube Adapter Grub Union Screw GVF/003 Ferrule 0.5mm GVF/16 Finger-tight Ferrule Nut & Ferrule Cover Liner Tube Fused Silica Capillary Liner Tube Rheodyne Nut & Ferrule...
  • Page 199 Quattro Ultima User's Guide Using a GVF/16 ferrule and the adapter nut, connect the sample capillary to the LC union, ensuring that both the liner tube and sample capillary are fully butted into the LC union. Disconnect the LC connection and feed the sample capillary through the probe, ensuring that a 0.3mm graphitised vespel ferrule (GVF/003) is fitted.

  • Page 200: The Apci Probe

    Quattro Ultima User's Guide The APcI Probe Indications that maintenance to the APcI probe is required include: • The probe tip assembly becomes contaminated, for example by involatile samples if the probe temperature is too low during operation (300°C). •...

  • Page 201: Replacing The Probe Tip Heater

    Quattro Ultima User's Guide Select Gas, Gas to turn on the nitrogen. Raise APcI Probe Temp gradually, starting at 100°C and increasing in 50°C intervals to 650°C over a period of 10 minutes. Do not set APcI Probe Temp to 650°C immediately as this may Caution: damage the probe heater.

  • Page 202: Replacing The Fused Silica Capillary

    Quattro Ultima User's Guide Replacing the Fused Silica Capillary Fused Silica Capillary Adapter Filter Grub Screw GVF/004 Ferrule Finger-tight Nut & Ferrule 0.5 to Grub Screw PTFE Tube Rheodyne Nut & Ferrule With the probe removed from the source proceed as follows: Remove the probe tip assembly and the heater, as described in the preceding section.
  • Page 203 Quattro Ultima User's Guide Feed the sample capillary through the probe, ensuring that a 0.4mm graphitised vespel ferrule (GVF/004) is fitted. Using a ceramic capillary cutter, cut the capillary at the nebuliser so that between 0.5 and 1.0mm of capillary is protruding from the nebuliser.

  • Page 204: The Analyser

    User's Guide The Analyser Quattro Ultima is fitted with a pre-filter assembly that is designed to protect the main analyser by absorbing contamination from the ion beam. As a consequence the analyser quadrupoles should never, under normal working conditions, require cleaning.

  • Page 205: Electronics

    DC voltages and mains voltages remain in the power supply units. Caution: Quattro Ultima’s electronic systems contain complex and extremely sensitive components. Any fault finding procedures should be carried out only by Micromass engineers.

  • Page 206: Fault Finding Check List

    In the standby mode (Operate not selected) the high voltages are switched off in the source and analyser assemblies, but high DC voltages and mains voltages remain in the power supply units. Any investigation in the RF generator must be made only by a Micromass engineer. No Beam Refer to the relevant chapters of this manual and check the following: •...

  • Page 207: High Noise Level In Mrm Analyses

    Quattro Ultima User's Guide High Noise Level in MRM Analyses The background noise in MRM analyses can either be electronic or chemical. To distinguish between chemical noise and electronic noise an acquisition should be performed with and without ions being transmitted to the detector. Ions are best prevented from reaching the detector by setting the ion energy 1 (MS1) and ion energy 2 (MS2) fully negative.

  • Page 208: Electronic Noise

    If, after checking the ion counting threshold as above, electronic noise is considered to be the dominant source of noise and has become significantly worse since instrument installation then further investigations should be carried out by a qualified Micromass engineer.

  • Page 209: General Loss Of Performance

    If any of these voltages are absent check that the source and hexapole transfer lens assembly have been correctly reassembled. Further investigation, which require the services of a qualified service engineer, should be left to Micromass personnel. Maintenance and Fault Finding Page 209...

  • Page 210: Cleaning Materials

    Quattro Ultima User's Guide Cleaning Materials It is important when cleaning internal components to maintain the quality of the surface finish. Deep scratches or pits can cause loss of performance. Where no specific cleaning procedure is given, fine abrasives should be used to remove dirt from metal components.

  • Page 211: Preventive Maintenance Check List

    Quattro Ultima User's Guide Preventive Maintenance Check List Avoid venting the instrument when the rotary pump is gas ballasting. Do not gas ballast the rotary pump for more than 2 hours under any circumstances. For full details of the following procedures, consult the relevant sections of this chapter and/or refer to the manufacturer’s literature.
  • Page 212 Quattro Ultima User's Guide Maintenance and Fault Finding Page 212...

  • Page 213: Reference Information

    Quattro Ultima User's Guide Reference Information Overview Calibration reference files consist of two columns of numbers separated by any number of spaces or TAB characters. The first column contains the reference peak masses and the second column contains the reference peak intensities.

  • Page 214: Editing A Reference File

    Quattro Ultima User's Guide Editing a Reference File Calibration reference files can be created or edited using any Windows text editor. To read the currently selected reference file into the Notepad text editor: Press or select Reference File... from the Calibration, Edit menu.

  • Page 215: Positive Ion

    Quattro Ultima User's Guide Positive Ion Ref. File Chemical Name Molecular Uses Name [Sigma Code #] Mass Bovine Ubiquitin 8564.85 650-1500 General [U6253] Human α globin 15126.36 700-1500 Hb analysis [H753] Superoxide dismutase Hb (internal 15591.35 900-1500 [S2515] cal.) Human β globin 15867.22...

  • Page 216: Horse Heart Myoglobin

    Quattro Ultima User's Guide Horse Heart Myoglobin Reference File: myo.ref Molecular Weight: 16951.48 Charge Calculated Charge Calculated Charge Calculated / Value / Value / Value State State State 606.419 808.222 1304.969 616.177 848.583 1413.633 628.841 893.192 1542.053 652.989 942.758 1696.158 679.068...

  • Page 217: Sodium Iodide And Caesium Iodide Mixture

    Quattro Ultima User's Guide Sodium Iodide and Caesium Iodide Mixture Reference File: NAICS Calculated / Value 22.9898 772.4610 1671.8264 2571.1918 3470.5572 132.9054 922.3552 1821.7206 2721.0861 3620.4515 172.8840 1072.2494 1971.6149 2870.9803 3770.3457 322.7782 1222.1437 2121.5091 3020.8745 3920.2400 472.6725 1372.0379 2271.4033 3170.7688 622.5667...

  • Page 218: Negative Ion

    Quattro Ultima User's Guide Negative Ion Ref. File Chemical Name Molecular Uses Name [Sigma Code #] Mass Horse heart myoglobin MYONEG 16951.48 700-2400 General [M1882] Sugar mixture of: maltose [M5885] raffinose [R0250] Low mass SUGNEG 100-1500 maltotetraose range [M8253] corn syrup [M3639]...

  • Page 219: Sodium Iodide And Caesium Iodide (Or Rubidium Iodide) Mixture

    Quattro Ultima User's Guide Sodium Iodide and Caesium Iodide (or Rubidium Iodide) Mixture Reference File: naineg.ref Calculated / Value 126.9045 1026.2699 1925.6353 2825.0008 3724.3662 276.7987 1176.1641 2075.5296 2974.8950 3874.2604 426.6929 1326.0584 2225.4238 3124.7892 576.5872 1475.9526 2375.3180 3274.6835 726.4814 1625.8469 2525.2123 3424.5777...

  • Page 220: Preparation Of Calibration Solutions

    Quattro Ultima User's Guide Preparation of Calibration Solutions PEG + Ammonium Acetate for Positive Ion Electrospray and APcI Prepare a solution of polyethylene glycols at the following concentrations: PEG 200 25 ng/µl PEG 400 50 ng/µl PEG 600 75 ng/µl PEG 1000 250 ng/µl...

  • Page 221: Sodium Iodide Solution For Positive Ion Electrospray

    Quattro Ultima User's Guide Sodium Iodide Solution for Positive Ion Electrospray Method 1 Prepare a solution of sodium iodide at a concentration of 2 µg/µl (micrograms per microlitre) in 50:50 propan-2-ol (IPA):water with no additional acid or buffer. Add caesium iodide to a concentration of 0.05 µg/µl.
  • Page 222 Quattro Ultima User's Guide Reference Information Page 222...
  • Page 223 Quattro Ultima User's Guide Index Acetonitrile Caesium iodide 157, 219, 221, 223 Adducts Calibration 46, 103 Acquisition 46, 71, 130 APcI Parameters 114, 129 Checking Active inverted magnetron gauge 18, 34, 181 Electrospray Air filter Failure 120, 137 Ambient temperature...
  • Page 224 Quattro Ultima User's Guide Data acquisition See: Acquisition Gas ballast 38, 182 Data processing Gas cell Data system 13, 24, 36 Glass capillary (nanoflow) 161, 166 Daughter Gradient elution Daughter ion 21, 46, 47 Grid Desolvation gas 25, 27, 40, 42, 44, 151, 176,...
  • Page 225 Quattro Ultima User's Guide Mains switch Parent Maintenance Parent ion 22, 46 Mass calibration PC link See: Calibration Peak matching 123, 139 Mass measurement See: Polyethylene glycol MaxEnt Penning gauge See: Active inverted magnetron gauge Megaflow 147, 156, 159 Metabolites...
  • Page 226 Quattro Ultima User's Guide Ramp Saccharides Readbacks Sample cone 154, 185, 186 Rear panel 28, 207 Scan control PCB Reciprocating pump 144, 159 Scan duration Reference compound 106, 215 Scan time Reserpine 21, 22, 23, 46 Scope Restrictor Selected ion recording...
  • Page 227 Quattro Ultima User's Guide Target compound analysis UV detector Task 51, 73 UV detector 24, 28, 159 UV photodiode array detector See: Triethylamine Tetrahydrofuran See also: Tetrahydrofuran Vacuum 18, 180 Threshold parameters Leak 45, 181 Thresholds Protection Toxicology Vacuum LED...

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