ultraflex III User Manual
Version 1.0 (September 2006)
Bruker Daltonik GmbH © Copyright 2006 Bruker Daltonik GmbH All Rights Reserved. Reproduction, adaptation, or translation without prior written permission is prohibited, except as allowed under the copyright laws. Document History First edition: March 2004 Printed in Germany. Warranty The information contained in this document is subject to change without notice. Bruker Daltonik GmbH makes no warranty of any kind with regard to this material, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose. Bruker Daltonik GmbH is not liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance or use of this material.
Safety Information Safety Class The ultraflex III with the closed safety cover is a Class I Laser product. It has been designed and tested in accordance with IEC Publication 1010-1 Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use. WARNING
WARNING
Part Numbers
All connections of the instrument must be used in correct way. The instrument should only be used with the wires and cables delivered with the system or otherwise provided by the manufacturer.
In this manual, Bruker Daltonik GmbH part numbers are generally listed in parentheses after the name of the part. A few tools and supplies listed have no part numbers and are not available from Bruker Daltonik GmbH. Most of these can be obtained from laboratory supply companies.
Instrument Identification
Connecting an instrument to a power source that is not equipped with a protective earth contact creates a shock hazard for the operator and can damage the instrument. Likewise, interrupting the protective conductor inside or outside the instrument or disconnecting the protective earth terminal creates a shock hazard for the operator and can damage the instrument.
Each instrument is identified by a serial number. This number is Manual part number: located on the rear of PN # 245596 the instrument.
WARNING
Serial #
When corresponding with Bruker Daltonik GmbH about your instrument, be sure to include the model name and the full serial number. Write the serial number of the instrument here for reference:
The instrument must be disconnected from its power source before any cover is removed or it is opened!
Bruker Daltonik GmbH assumes no responsibility for the use or reliability of its software on equipment that is not furnished by Bruker Daltonik GmbH. The names of actual companies and products mentioned herein may be the trademarks of their respective owners.
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Technical Support If you encounter problems with your system please contact a Bruker representative in your area, or: Bruker Daltonik GmbH
Bruker Daltonik GmbH
Fahrenheitstr. 4
Permoserstr. 15
D-28359 Bremen
D-04318 Leipzig
Germany
Germany
Phone:
+49 (421) 2205 430
Phone:
+49 (341) 2431 395
Fax:
+49 (421) 2205 370
Fax:
+49 (341) 2431 343
E-mail:
[email protected]
E-mail (software support):
[email protected] Internet:
www.bdal.de
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Table of Changes Version 1.0
Date
Changes
Remarks
2006-09-25 ultraflex III User Manual
First edition
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Contents 1
ULTRAFLEX III MASS SPECTROMETER........................................................... 9 1.1 1.2 1.3
2
Weights and Measures of the ultraflex III ...................................................................... 9 Site Preparation Specification...................................................................................... 10 Safety ........................................................................................................................... 10 1.3.1 Safety Symbols.................................................................................................. 10 1.3.2 Manual Conventions and Symbols .................................................................... 11 1.3.3 Operating Precautions ....................................................................................... 12 1.3.4 Electrical Safety ................................................................................................. 13 1.3.5 Environmental Conditions.................................................................................. 14
INSTALLATION AND SETUP ............................................................................ 15 2.1 2.2 2.3
3
Facility and Electrical Requirements............................................................................ 15 Unpacking .................................................................................................................... 16 Setup............................................................................................................................ 17
INSTRUMENT LAYOUT ..................................................................................... 19 3.1
Schematic of the Mass Spectrometer.......................................................................... 19 3.1.1 Instrument Controller ......................................................................................... 20 3.1.2 Rear Panel......................................................................................................... 21 3.1.3 Vacuum System................................................................................................. 24 3.1.4 Scout MTP Ion Source....................................................................................... 25 3.1.4.1 Pulsed Ion Extraction.................................................................................... 26 3.1.4.2 Target Plates ................................................................................................ 27 3.1.4.3 AnchorChip Target .................................................................................... 28 3.1.4.4 Transponder ................................................................................................. 28 3.1.5 TOF/TOF Analyzer ............................................................................................ 29 3.1.5.1 Precursor Ion Selector.................................................................................. 29 3.1.5.2 LIFT 31 3.1.5.3 How Does LIFT Work? ................................................................................. 33 3.1.5.4 Reflector ....................................................................................................... 35 3.1.5.5 Detector ........................................................................................................ 36 3.1.5.6 Digitizer 37 3.1.5.7 Laser System................................................................................................ 37 3.1.5.8 Camera 37 3.2 PC Configuration.......................................................................................................... 38 3.3 Remote Service Capability........................................................................................... 38 3.4 Twister (optional) ......................................................................................................... 41 3.5 Example of the Structure of a Bruker ID ...................................................................... 42
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OPERATION ....................................................................................................... 43 4.1
Locating the Instrument Functions - Turning the Instrument ON or OFF .................... 43 4.1.1 Rear Panel......................................................................................................... 43 4.1.2 Front Panel ........................................................................................................ 45 4.2 Manual Cartridge ......................................................................................................... 47 4.2.1 Checking for Instrument Operational Readiness............................................... 47 4.2.2 Moving Targets In and Out with the Manual Cartridge...................................... 47 4.3 Using the Twister (optional) ......................................................................................... 51 4.3.1 Turning the Instrument On or OFF .................................................................... 51 4.3.2 Checking for Instrument Operational Readiness............................................... 52 4.3.3 Moving Targets In .............................................................................................. 52 4.3.4 Moving Targets Out ........................................................................................... 52 4.3.5 Instrument Control in Analytical Operations ...................................................... 52
5
INSTRUMENT MAINTENANCE ......................................................................... 53
6
INDEX ................................................................................................................. 54
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Safety Instructions The following symbols may be found on or near various components. Before starting the installation, pay attention to these important safety instructions!
Warning Warning attracts attention to procedures that could result in personal injury, if protocols are not followed. This symbol is placed on the product in the area where a hazardous voltage is present. Only trained service personnel should perform work in this area. When the instrument is operating high DC voltages are present, in addition to the 230V AC mains supply. All panels must be fitted prior to operation. Only under these circumstances does the instrument comply with the electromagnetic radiation guidelines (EC certificate).
Laser radiation Indicates that laser light may be present. The autoflex with the closed safety cover is a Class I Laser product. With safety cover opened it turns to a Class IV Laser product.
Caution Caution attracts attention to procedures that could result in damage to the instrument, if protocols are not followed.
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Instrument Classification The autoflex III is an electrical equipment for measurement, control, and laboratory use where the electromagnetic environment is kept under control. That means in such an environment transmitting devices such as mobile phones should not be used in immediate vicinity. EN 61326/A1:1998,
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ultraflex III Mass Spectrometer
ULTRAFLEX III MASS SPECTROMETER
The ultraflex III is a MALDI (Matrix Assisted Laser Desorption/Ionization) tandem mass spectrometer specially designed for automated MS and MS/MS high throughput identification of proteins and peptides. Tandem Mass Spectrometry is a technique that utilizes more than one mass selective stage in a mass spectrometer. The most common form in practice is a two stage arrangement to record MS/ MS or (MS)2 spectra. The incorporated LIFT device allows acquiring full fragment ion spectra within one single scan and replaces traditional measurements of segmented spectra with stepwise-reduced reflector potentials.
1.1 Weights and Measures of the ultraflex III
Figure 1-1
Weights and measures of an ultraflex III instrument (L x W x H = 230 cm x 133,2 cm x 87,4 cm; Weight 550 kg)
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1.2 Site Preparation Specification Before starting the installation of the instrument the site must be properly prepared. Please refer to the Site Preparation Specification document that is sent to any customer prior to the shipment of the instrument. It contains information regarding the device requirements, such as operating environment, gas supply, power, exhaust, venting, grounding, etc. This document has to be verified and returned with your signature to Bruker before a service representative will start the installation.
1.3 Safety Safety considerations for the ultraflex III spectrometer include: • ultraflex III Safety Symbols (chapter 1.3.1) • Manual Conventions and Symbols (chapter 1.3.2) • Operating Precautions (chapter 1.3.3) • Electrical Safety (chapter 1.3.4)
1.3.1 Safety Symbols The following symbols may be found on or near various components of the mass spectrometer: Table 1-1 Symbol
Safety Symbols Description Indicates that an operation or condition on the instrument could cause instrument damage if precautions are not followed. This can be any type of hazard. When this symbol is observed, refer to the safety pages in the manual for further information. Indicates the danger of electric shock due to the presence of high voltage, if precautions are not followed.
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ultraflex III Mass Spectrometer Description Indicates that laser light may be present. The ultraflex III with the closed safety cover is a Class I Laser product. With safety cover opened it turns to a Class IV B Laser product. Indicates that a terminal either receives or delivers alternating current or voltage. Indicates that a protective grounding terminal must be connected to earth ground before any other electrical connections are made to the instrument. Indicates the OFF position of the main power switch. Indicates the ON position of the main power switch.
1.3.2 Manual Conventions and Symbols The following graphic text formats and symbols are used to set apart important safety information: Note:
Indicates important information in a procedure or description.
Caution:
Indicates an operation that could cause instrument damage if precautions are not followed. Indicates that an operation could cause personal injury if precautions are not followed.
Warning:
High Voltage: Indicates that an operation could cause personal injury if precautions are not followed.
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1.3.3 Operating Precautions To protect yourself from harm and prevent system malfunction, observe the following guidelines: •
Before using the instrument, read all warnings presented in the beginning of this manual.
•
Wear appropriate protective clothing, including safety glasses and gloves, when preparing samples and solutions for use with this instrument.
•
Follow the correct safety procedure and the manufacturer's recommendations when using solvents.
•
Clean the exterior surfaces of the instrument with a soft cloth dampened with a mild detergent and water solution. Do not use abrasive cleaners or solvents.
•
Exercise caution when moving as the ultraflex III mass spectrometer weighs 550 kg / 1212 lb. Wear appropriate clothing and use appropriate tools when carrying or moving the instrument.
•
The ultraflex III mass spectrometer contains a Class IV B pulsed ultraviolet laser (100 µJ at 337 nm). Wear appropriate eye protection and never look directly into the laser when any of the protective covers are removed. In operation, laser light is directed onto the analysis chamber sample target. The same interlocks that assure the analysis chamber vacuum integrity, also assure that no laser light escapes through the target loading port. If the vacuum system fails and system allows the load port to be opened, the laser cannot be fired.
Caution: When shipping or transporting the instrument it is critical that the target is in the OUT position. Failure to do so will result in damage to the instrument. Caution: Do not restrict ventilation air intake at the rear of the machine or the exhaust at the back of the machine. To ensure proper operation check the ventilation air filter at the instrument rear every three months and replace, if necessary, as explained in chapter 5 Instrument Maintenance.
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1.3.4 Electrical Safety Electrical safety considerations consist of the following chapters: •
• • •
General Safety: Before installing or operating the ultraflex III mass spectrometer, read the following information concerning hazards and potential hazards. Ensure that anyone involved with installation and operation of the instrument is knowledgeable in both general safety practices for the laboratory and safety practices for the ultraflex III mass spectrometer. Seek advice from your safety engineer, industrial hygienist, environmental engineer, or safety manager before installing and using the instrument. Position the ultraflex III mass spectrometer in a clean area that is free of dust, smoke, vibration, and corrosive fumes, out of direct sunlight, and away from heating units, cooling units, and ducts. Verify that there is an adequate and stable power source for all system components. Verify that the power cord is the correct one for your laboratory and that it meets the national safety agency guidelines for the particular country of use.
Warning:
DO NOT attempt to make adjustments, replacements or repairs to this instrument. Only a Bruker Daltonics Service Representative or similarly trained and authorized person should be permitted to service the instrument.
Warning:
When it is likely that the electrical protection of the ultraflex III mass spectrometer has been impaired: 1. Power off the ultraflex III mass spectrometer. 2. Disconnect the line cord from the electrical outlet. 3. Secure the instrument against any unauthorized operation.
Warning:
Caution:
The ultraflex III mass spectrometer and MALDI-TOF analyses use very high voltages. Under normal operation, the instrument requires NO user access to the inner components of the instrument. NEVER OPERATE the ultraflex III mass spectrometer with the protective cover removed as this exposes the user to risk of severe electrical shock and harmful, invisible, ultraviolet laser radiation. Use only fuses with the required current and voltage ratings and of the specified type for replacement.
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Caution:
Use the instrument according to the instructions provided in this manual. If used otherwise, the instrument-provided protection can be impaired.
Caution:
Connect the instrument to an AC line power outlet that has a protective ground connection. To ensure satisfactory and safe operation of the instrument, it is essential that the protective ground conductor (the green / yellow lead) of the line power cord is connected to true electrical ground. Any interruption of the protective ground conductor, inside or outside the instrument, or disconnection of the protective ground terminal, can impair the instrument-provided protection.
1.3.5 Environmental Conditions The ultraflex III mass spectrometer is designed for indoor use and functions correctly under the following ambient conditions: Table 1-2
Environmental Conditions Operating Conditions
Temperature
10 - 30 °C (50 to 86 °F)
Relative Humidity
15-95% non-condensing 35 °C
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Installation and Setup
INSTALLATION AND SETUP
Please refer to the Bruker Site Preparation Specification document for your mass spectrometer. Installation and setup consists of: •
Facility and Electrical Requirements (chapter 2.1).
•
Unpacking (chapter 2.2).
•
Setup (chapter 2.3).
2.1 Facility and Electrical Requirements The facility must provide: •
100 to 230 V/ 3-1.5 A / 50–60 Hz. The instrument back panel is fitted with an IEC320–C14 mains inlet (Figure 3-3). The instrument comes with a 3 m long IEC320 line cord and a mains plug, suitable for use in your country.
•
The ultraflex III mass spectrometer requires approximately 2 m2 of space on a surface that can safely support the full 550 kg instrument weight.
•
To ensure proper ventilation, and access to the connections and main switch, maintain at least 80 cm of clearance behind the instrument.
Warning:
The main supply must provide adequate grounding.
The system has an exhaust port to accommodate venting. This port is located on the rear of the instrument. Individual facilities may have safety guidelines, which require ventilation. It is the responsibility of the user to adhere to the requirements of their respective facility.
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Installation and Setup
2.2 Unpacking The packing list is created for each order and placed in the crate with the machine. Note: The warranty does NOT cover damage resulting from customer mishandling. Do not open the shipping container unless a BRUKER representative is present. Opening of the container without authorized persons will void the warranty of the instrument. Our service engineers will set up the instrument in customer’s laboratory. The surface on which the instrument is placed must be able to safely support the full 550 kg weight, and computer, monitor and printer as well. Let the machine on the palette and wait for the Bruker representative to move the instrument to its desired location!
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Installation and Setup
2.3 Setup To setup the instrument: 1. At the rear of the instrument, plug in the following cord and cables into the corresponding connections (Figure 3-3. a. Line cord. b. Remote cable. c. Signal cables. 2. Plug the other end of the AC line cord into a suitable AC outlet. Caution: Read and follow all electrical and safety precautions described in chapter 1.3 Safety or chapter 1.3 Safety. 3. Plug the data system end of the signal cable connections into the appropriate connections (Figure 2-1). 4. Plug the data system end of the Remote cable into the COM-1 port on the PC. 5. Turn on the AC mains switch. 6. Ensure that the POWER LED is illuminated (Figure 3-2). After initial power-up it may be as long as 12 hours before the instrument is ready for operation, at which time the Ready LED illuminates (Figure 3-2). Note: If any faults occur within the system the Error LED (Figure 3-2) illuminates and the system goes into standby. Contact your authorized service personnel for help.
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Installation and Setup
Trigger input digitizer
Signal from the linear detector
Signal from the reflector detector
Figure 2-1
Connection at the rear of the PC
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Instrument Layout
INSTRUMENT LAYOUT
Bruker flex systems, such as the ultraflex III are always composed of only two main components: 1. Mass spectrometer (twister optional). 2. Data system (chapter 3.2).
3.1 Schematic of the Mass Spectrometer
IS1 IS1 / IS2 Ground potential
Figure 3-1 shows the principle the ultraflex III mass spectrometer works.
Drift region PLMS
P1+
P2+
P1+
LIFT
Target plate
Figure 3-1
P1+
Linear detector
PCIS
Reflector detector
Reflector
Schematic of the instrument
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Instrument Layout
3.1.1 Instrument Controller When the instrument is ready for operation the three green LED’s Mains, Ready, and Access illuminate green light as shown in Figure 3-2.
Figure 3-2
Control panel at the front of the instrument
1. Load / Eject: Moves the target in or out. 2. Access: a) Permanently lit: When either the tray is inside or outside the instrument in the end position. b) OFF: During the target moves in or out. 3. In Progress: Permanently lit as long as the docking procedure is in progress (Target In/ Out). 4. Error: a) Permanently lit: Malfunction, requires intervention of the user or a service technician details are shown on the PC (flexControl). b) Flashing (combined with system error): Service mode or firmware update. 5. Automode: Permanent lit as long as AutoXecute is in progress. During this run applying the related button cannot retract the target plate. 6. Ready: a) Permanently lit: Ready for acquiring spectra, i.e. any interlock is closed; N2 pressure is correct, sufficient vacuum, HV present, etc. b) OFF: During the docking procedure. 7. Warm up: ultraflex III User Manual, Version 1.0
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a) Permanent lit: Warm-up phase of the laser device (Class IV Laser Product) in progress or the system is not ready for measurement, e.g., the docking procedure in still in progress, HV is off, etc. It takes some time, even when all parameters comply with the requirements. This state is reflected simultaneously on the PC screen providing details via flex Control™. b) Flash: A subordinated malfunction has occurred, which must be verified by the user (less N2 pressure, interlock open). 8. Error: a) Permanent lit: malfunction, requires intervention of the user or a service technician; the tool tips feature on the PC shows details (flexControl™). b) Flashing: (combined with Target Error): Service mode or firmware update. 9. Mains: Permanent lit, when the main circuit breaker is on. Power On / OFF: OFF vents the instrument.
3.1.2 Rear Panel At the rear of the instrument (Figure 3-3), there are the following connections: CID: •
Collision gas inlet for argon or helium (6 bar maximum) for optional Collision Induced Dissociation and flushing the collision cell.
•
Switch to flush the CID-cell (after changing the collision gas).
•
Flushing gas outlet.
Gas supply: •
Compressed air connection to operate the slide valve. Alternatively this port might be externally connected with the nitrogen inlet for the laser to a common N2 supply.
•
Nitrogen inlet to operate a N2-laser (Class IV Laser Product).
•
Gas inlet to vent the instrument.
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Acquisition: •
Three BNC signal connectors (trigger, detector #1 and #2) for the digitizer mounted on the PC.
Control: •
BNC connector to provide the video signal of the sample spot to a video capture card mounted on the PC.
•
Serial port to connect the (optional) Twister.
•
Serial port to connect the PC (HOST).
Pump: •
Vacuum system exhaust 13 mm (1/2 inch) ∅ plastic tubing (Tygon), filter mounted inside. Individual facilities may have safety guidelines, which require ventilation. It is the responsibility of the user to adhere to the requirements of their respective facility.
Service Only: •
Key field for the service to lift the cover.
Mains: •
The instrument is fitted with an IEC320-C14 mains inlet.
At the rear of the machine behind the cover there is the air intake with a filter. Replace the air filter every three month.
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Figure 3-3
Instrument Layout
Schematic of the connections at the rear
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This instrument contains a class IV laser product emitting 337 nm light. The enclosure surrounding the instrument is designed to protect the user from indirect radiation of the invisible light. Operating the instrument with opened covers can expose the user to harmful laser radiation, which may result in blindness. Never look directly into the laser beam. Always wear safety goggles before lifting the cover. Make sure other people cannot expose themselves to the laser light. Avoid bringing highly reflecting parts into the beam (screw drivers, watches, rings) because the reflected visible and invisible light could reach your eye, causing irreparable injuries!
3.1.3 Vacuum System The vacuum system of the ultraflex consists of two high vacuum and three rough vacuum areas. The vacuum lock of the SCOUT-MTP and the exhaust of both turbo molecular pumps are coupled and guided via valves to the vacuum side of the foreline pump. Depending on the system status, e.g., when changing a target plate, each of the rough vacuum lines can be closed separately by computer-controlled valves. The input of the turbo-molecular pumps is attached directly to the high vacuum areas of the ion source housing and the ion flight tube, respectively. The instrument operates at a high vacuum pressure of about 8*10-7 mbar or better. The rough vacuum pressure ranges from atmosphere to about 1*10-2 mbar.
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Instrument Layout
3.1.4 Scout MTP Ion Source Laser beam
Ground potential
Lens arrangement CID cell
Ion flight
P1 Target plate x-ystage
Lighting & camera P2 Electrode
Figure 3-4 Scheme of the time-lag focusing ion source of the “flex”-series The ion source (Figure 3-4) is the part of the mass spectrometer where ions are formed using the MALDI technique. The source consists of three main components. 1. The x-y-table accommodates the target plate, transports it into the ion source, and moves the target inside on x-y-coordinates, according to the selected shot position. 2. The vacuum lock inserts the target from atmosphere to the high vacuum. 3. The ion optics consists of the positively or negatively charged MTP target plate (P1), a second voltage plate (P2) for time lag focusing and a grounded acceleration electrode. When the laser hits the analyte/matrix mixture the formed ions are accelerated by the delayed applied electrical field and focused by a lens system before they leave the source. CID is an acronym for Collision Induced Dissociation. This term stands for the procedure when molecules decay by collisions during a passage through a particular cell that is filled with gas, e.g., Ar. CID has proved to be useful to enhance intensities of fragments in the low fragment mass range. ultraflex III User Manual, Version 1.0
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3.1.4.1 Pulsed Ion Extraction MALDI mass spectrometry is restricted by the energy distribution of the formed ions, which is caused by the ionization process. Because not all the ions are desorbed and ionized at the same time and at the same place inaccuracies occur related to energy, location, and time. Also repulsive electrical forces cause an initial energy distribution of the ions. Summarizing all these inaccuracies which happen during the ion formation process cause that ions of the same mass do not have the same kinetic energy after passing the acceleration field, but leave the source with a certain energy spread resulting in an arrival at slightly different times on the detector. This effects in broadening of the peak width and thus reducing the resolving power. Pulsed Ion Extraction (PIE) is a technique to enhance resolution and sensitivity as well of a TOF mass spectrometer. After desorption ions are delayed extracted (phase 2 and 3, discussed beneath). This method benefits in:
1. A softer acceleration process, because ions do not pass the dense plume of matrix molecules and
2. achieving a time focusing effect provided that the correct parameters are applied. Involved in the PIE process are three components of the ion source, i.e. 1. the target plate(P1) where the analyte is dropped on, 2. the second voltage plate (P2) which is an electrode mounted some mm apart opposite to the sample position, and 3. the following grounded acceleration electrode (Figure 3-1, Figure 3-4). Using PIE this arrangement works as follows: Phase 1: The target plate P1 with the analyte is always connected to potential IS/1. At first the same potential is applied on plate P2. So the analyte is not yet exposed to any exterior effects until to the laser shot. This event is the transition to Phase 2: where molecules and ions are set into motion by laser ionization/desorption with a typical velocity of 700 m/s from P1 towards P2. Meanwhile the analyte is ionized. There is still no electrical potential the ions are exposed to. Only the MALDI process causes their kinetic energy at this moment, where through the evaporation of the analyte surface particles are explosively pushed to that velocity. During the next few hundred nanoseconds the analyte moves further towards P2. ultraflex III User Manual, Version 1.0
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However not all ions start with the initial velocity of 700m/s. Some (the fast ones) fly further than others that fly only a short way (the slow ones). Without using PIE this velocity distribution decreases the resolution on a TOF mass spectrometer in linear mode without using PIE. Then at the end of the delay time begins Phase 3: where potential P2 is pulsed down from IS/1 to IS/2 generating an electrical field whose strength forces all charged particles to move towards P2. That means that the fast higher energetic ions that were able to fly further towards the P2 plate before the voltage was switched are exposed to a less electrical potential than the slow ones in the neighborhood of P1. For this reason the slow ones start on a higher potential than the fast ones. Therefore the slow ones will fly faster in the field free region whereas the fast ones will fly slower. Using the right slope of the potential between P1 and P2 ions of a given mass with different stating velocities will all arrive at the same time on the detector!
3.1.4.2 Target Plates The standard target plate is a SCOUT-MTP plate (MicroTiter Plate) with 384 defined sample positions (Figure 3-5). A video CCD camera and a video capture card inside the PC provide high resolved sample images.
Figure 3-5
Illustration of an microwell plate and a Scout MTP plate
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The micro well plate at the left and the SCOUT-MTP plate (Figure 3-5), are employed as a standard for analyses in the area of biochemistry because of the high multiple sample throughput. Both are of identical dimensions where the arrays of 384 sample spots of each correspond exactly to another. Both plate types may be also stacked in mixed mode.
3.1.4.3 AnchorChip Target AnchorChip targets are SCOUT MALDI MTP targets that are equipped with a special coating improving the crystallization process of MALDI samples. This technique benefits in: •
10 – 100 fold sensitivity increase because of increased analyte concentration.
•
Improved automation because of reduced search for “sweet spots”.
Comprehensive information can be obtained from the manual AnchorChip Technology (PN 215344).
3.1.4.4 Transponder All the Scout MTP targets are equipped with a programmable semiconductor chip, called transponder. The transponder contains information from Bruker, such as the geometry, the part number, and the serial number (Read only data). Further on the chip contains a ”Use Counter”, which identifies the target and counts on ejection how often this one has been used. This information may be useful in the case of AnchorChip Targets that might have to be regenerated after multiple measurements. flexControl must be running on moving targets in for correct results. If flexControl is closed and the target movement is initiated with the corresponding green button on the front panel, the transponder gets no information. The transponder provides three areas (string 1,2,3) for the customer to program. Any string contains 14 ASCII signs maximum. The Bruker TransponderStation RW (manual PN 212308) is a suitable tool to transmit data into the transponder. This feature will be activated on request.
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Instrument Layout
3.1.5 TOF/TOF Analyzer The peculiarity of the ultraflex III analyzer is the LIFT device located in the flight tube, which allows acquiring a full fragment ion spectrum with one single scan.
3.1.5.1 Precursor Ion Selector The precursor ion selector (PCIS) as shown in Figure 3-6 works like a mass filter to separate a particular parent ion and related fragments from all the other ions for MS/MS analysis. It consists of deflector plates arranged in vertical layers below each other. Consecutive electrodes are coupled to a high voltage supply with alternating polarity, according to the Bradbury-Nielsen principle. The potential difference between the plates generates an electrostatic field perpendicular to the ion flight direction. This field deflects all the ions entering this electrode arrangement. Although the plates are coupled to the supply voltage due to the Bradbury-Nielsen principle the ion selector is operated as an improved Barowsky precursor ion selector to take advantage of this selection mode. Just in the moment when the selected ions enter the deflection field the deflection is switched off. The potential between the plates is kept to zero until the ions leave the deflector. In this moment the deflection is switched on again, however with the reverse polarity. This mode results in a compensation of the partial deflection, which occurs in the stray areas at both front ends of the electrodes. This technique allows applying extraordinary short selection times, which contributes to improve resolving power. The ultraflex III is equipped with a second deflection unit PLMS (Post Lift Metastable Suppressor) located between LIFT and the reflector. This assembly can be used to remove daughter ions that are formed after the LIFT procedure by deflecting the parent ion. Now only the fragments formed between the source and the LIFT continue their journey to the reflector where they are separated by mass.
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Instrument Layout
PCIS
+ HV
-
io ct fle De
alternating polarity
n es at Pl
+
Ion flight
+ Ion flight
+ Ion flight
+ Stray field
-
-
Stray field
+HV Ion position
0V -HV
Figure 3-6
Operation principle of the precursor ion selector
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3.1.5.2 LIFT
IS1 / IS2 Ground potential
Drift region PCIS P1+
P1+
P2+ Target plate
Figure 3-7
Parents and fragments
Linear detector
IS1
To understand TOF MS/MS, first it has to be realized that parent ions and fragments formed in the drift region, travel side by side with the same velocity through the TOF. Molecules break after acceleration in the source, since the kinetic energy release is only a few eV. Thus fragments have smaller kinetic energy than parents (Ekin ~m). Our gridless reflector can focus in time and space about 30% of difference in kinetic energy, e.g., from the parent down to fragments with 70% of the parent mass. Ions with less energy do not hit the detector. To obtain a full MS/MS spectrum the reflector voltage has to be stepped down as known for segmented PSD or FAST (Figure 3-7).
P1+ Reflector detector
Reflector
Principle of a conventional PSD measurement
The basic idea of LIFT is to raise the kinetic energies of parent (Ps+) and fragment ions (F+) to a level that the energy difference between parent and smallest fragment does not exceed 30% (Figure 3-9). In that case all the fragments can be simultaneously detected with the parent ion (Figure 3-8). The Pre Cursor Ion Selector (PCIS) is able to pick a parent with the associated fragments out of a mixture, e.g., a digest (chapter 3.1.5.1), as fragments that are produced before arriving at the PCIS assembly pass the unit together with the parent because of their same velocity. .
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IS1 IS1 / IS2 Ground potential
The Post Lift Metastable Suppressor (PLMS) deflects (if switched on) the parent together with those metastable fragments, which are formed after LIFT (Figure 3-1, Figure 3-8). It can be switched off to allow the detection of the parent, e.g., for calibration purposes.
Drift region PLMS
P1+
P2+
P1+
P1+
Linear detector
PCIS
LIFT
Target plate
Reflector detector
Reflector
ion lenses Acceleration potentials
IS1
IS1 / IS2
Ion source (PIE)
Figure 3-8
Ground potential LIFT
Two stage field
Working principle of the TOF/TOF analyzer
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3.1.5.3 How Does LIFT Work? LIFT consists of an arrangement of four electrodes forming three chambers, the LIFT chamber, the Pulsed Acceleration Chamber, and the Acceleration Stage (Figure 3-9). The LIFT device is located just behind the PCIS. This arrangement works as follows: Electrode No.1 and No.2 are always connected in parallel forming a cell that shields from all external effects. Therefore the behavior of this cell can be compared with a Faraday’s cage. During phase 1 when the precursor and related fragments enter the first chamber the four electrodes have the potentials as shown in Figure 3-9: 1. Electrode 1 and 2 are connected to ground. 2. Electrode 3 is connected to 19 kV. 3. Electrode 4 is always connected to ground. Ions leave the ion source with an energy of typically 8 keV. While the ions move inside the first cell, due to phase 2 the electrical potential of the electrodes No.:1 and No.:2 will be raised up from ground potential to typically 19 kV. Because there is no voltage difference and no field between both electrodes the ion motion is influenced in no way. The particles continue moving as before however on this increased electrical potential. This potential jump is necessary for phase 4 to take effect. During phase 3 the ions move from chamber 1 to chamber 2 on the raised potential. Phase 4 begins by pulsing down the electrical potential (2 - 3kV) of electrode 3 while precursor and related fragments are still inside cell 2. Cell 2 acts as the first (delayed) acceleration stage. When entering cell 3 a further acceleration of the particles takes place according to the remaining voltage difference. This pulsed extraction resembles PIE (chapter 3.1.4.1) and makes also use of SVCF (Space-Velocity Correlation Focusing). The two stage acceleration by 19 kV potential difference sufficiently increases the kinetic energies of precursor and fragments that they are reflected properly and directed onto the reflector detector. Some data for illustration: After acceleration by LIFT the energy of the parent ion is raised up to 27 keV (8KeV + 19 keV). A fragment with a molecular weight of 1/20 of the parent ion has got energy of 19,4 keV (8keV/20 + 19 keV). This is only about 28% less energy and allows the ultraflex III User Manual, Version 1.0
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daughter ion to be focused from the reflector together with the parent ion onto the detector. Thus post-acceleration by LIFT may be considered a segmented MALDI FAST measurement using only one voltage segment to record a full daughter spectrum.
LIFT Chamber
Pulsed Acceleration Stage
Acceleration Stage
"Cell 1"
"Cell 2"
"Cell 3"
+ 19 kV
Electrode 4
Electrode 3
Electrode 2
Electrode 1
Ion Flight Direction
Phase 1
Ground + 19 kV
Phase 2
Ground + 19 kV
Phase 3
Ground + 19 kV
Phase 4
Ground
Figure 3-9
Potentials during a LIFT process on the related electrodes
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3.1.5.4 Reflector The main task of a reflector is to compensate flight times of ions with different energies. The metastable decay of ions is caused by an energy excess occurring during the complex MALDI process. Because parents and fragments continue traveling with the same velocity they hit the linear detector simultaneously thus delivering no fragment spectrum but a single peak. For fragment mass separation a reflector can be incorporated into the mass spectrometer to separate metastable ions. Ions of different masses have different kinetic energies and penetrate to different locations into the electrical field before they are reflected to strike the reflector detector at different times. To obtain good mass spectra with a reasonable signal-to-noise ratio the geometry of a reflector has to fulfill specific electrical and size requirements, mainly with respect to the dimensions of the flight tube, and type and size of the used reflector detector. The ultraflex III and other TOF mass spectrometers from the “flex”-series use a gridless reflector with ion lenses (Figure 3-10). Fragments that would arise inside the reflector instead of during their flight in the drift region form an undesired high chemical background. To minimize residence times of ions inside the reflector, a double stage design is employed where the first stage is that one with the stronger field strength. As a result the reflector is shorter and the field free drift length is longer, compared to single stage TOF reflectors. Also this construction deflects all the smaller fragment ions, which would otherwise contribute to the background noise. In addition the gridless entrance lens of the reflector creates a space focusing effect, which increases the sensitivity. The reflector operates at a potential that exceeds the acceleration voltage of the ion source by about two kV.
Acceleration potentials
Acceleration potentials
Ion lenses
Ion source
Figure 3-10
Two stage gridless reflector
Ground potential
Potential distribution in a two stage griddles reflector
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3.1.5.5 Detector The detector converts an ion current into an electrical current, which is then digitized and delivered to the PC. Modern detectors used in TOF mass spectrometers are designed as micro channel plate detectors (Figure 3-11). A micro channel plate is a porous solid core assembly with millions of tiny channels (Ǿ = 5-10µm, length = 0.5-0.8 mm), which are coated inside with a semi-conductive layer. Every one of the microchannels works like an electron multiplier, independent of the adjacent ones. To obtain a maximum electron yield all these channels are electrically connected in parallel. The multiplication process that takes place in a channel is illustrated beneath.
HV
Glass channel wall
Semiconducting layer
ct pa Im
g in
le tic r pa
Secondary electrons
Output electrons Figure 3-11
Principle of a micro channel
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For normal operations a bias of up to 1000 volts is applied across the microchannel. This bias current flowing through the semi-conductive layer supplies the electrons necessary for the avalanching multiplication process. A further performance parameter is the time response of the detector, important to prevent deterioration of the peak resolution. Microchannel plate detectors deliver an output voltage with a rise time (< 1ns) up to ten times faster than other detector configurations, such as channeltrons or discrete dynode multipliers. Each detector of the ultraflex III contains two MCPs coupled in series. The linear detector is equipped with the gating option to be used to avoid saturation effects, which may be caused by the matrix material. Please refer to the flexControl manual. Gating is an additional feature to detect only ions of interest. All the other ions are suppressed inclusive neutrals to avoid saturation of the linear detector.
3.1.5.6 Digitizer Beginning with the laser shot the digitizer records the incoming analog signals from the detectors and converts them into digital information. The digitizer card is able to attain a sample rate up to 2 GS/sec. This assembly is incorporated in the PC. Instrument settings on the Detector page in flexControl control the digitizer.
3.1.5.7 Laser System The laser system operates at a repetition rate of 50 Hz maximum (200 Hz optional). It provides the pulsed laser light to a small spot on the target. The laser system consists of a pulsed UV laser, an attenuator that allows fine adjustment of the laser fluence, a lens system to focus the laser beam and a mirror system to direct the beam into the ion source on the target plate. Standard is a N2 laser with 337 nm wavelength (pulse energy of150µJ) and 3 ns pulse width for use with matrix components absorbing light of this wavelength (class IV B product). Instrument setting are performed in flexControl.
3.1.5.8 Camera The camera delivers an image of the sample spot to the Target Manipulation Segment of the Graphical User Interface.
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3.2 PC Configuration The PC controls the mass spectrometer, and acquires and stores data. This device corresponds to the required configuration on delivery, e.g., 18-inch LCD display, resolution 1280 x 1024, True Color; Laser Printer; Windows 2000 operating system; Software packages flexControl/flexAnalysis.
3.3 Remote Service Capability
http://bdal.webex.com Remote Service
Customer
Daltonics Service Board
Figure 3-12 Operating principle of the remote service To obtain a maximum operating time the ultraflex III is equipped with a remote service capability (Figure 3-12). This feature allows for troubleshooting via the internet. Thus problems can often be solved efficiently with the customer PC being fully controlled by the Daltonics Service Board. For example, both software and firmware update is possible.
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Moreover the service process can be speeded up, as the service engineer can arrive on site with appropriate spare parts, after remote diagnosis. Prerequisite: The customer must have Internet access on the Control PC of the ultraflex III. An example how to obtain Remote Service in Europe: 1. Select http:bdal.webex.com. Figure 3-13 appears. 2. Select Join on the web page. Figure 3-14 appears.
Figure 3-13 Webpage of Bruker Enterprise Note: The “http”-connection changes to a “https”-connection, coded with 128-bit to provide the highest possible level of security!
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Figure 3-14 Enter the required information Please, enter the required information. A call to Bruker Service on +49 421 2205 430, will provide you with the Support Session Number. Press the “Submit” button and follow the instructions to successfully connect the remote service to your ultraflex III.
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3.4 Twister (optional)
Figure 3-15 Twister coupled to the mass spectrometer The Twister (Figure 3-15) is mounted in front of the mass spectrometer (Twister is a trademark of Zymark Corporation). It functions as an automatic sample insertion device to feed the spectrometer with SCOUT MTP MALDI targets. It can store and handle 20 target plates and more. Using the 1536 MTP format more than 30.000 samples / cycle can be unattended processed. The Twister transports target plates from an input rack to the mass spectrometer. After the measurement the unit receives the target and deposits it into the output rack. All these plates are equipped with a transponder. Additionally the customer may furnish the targets with a barcode label, for instance, for preparation with a robot, such as the Bruker MAPII. Both instruments the Twister and the spectrometer are controlled by the controls of the AutoXecute page in flexControl software as shown in Figure 3-16.
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Figure 3-16 Features of the AutoXecute page in flexControl To run the Twister in conjunction with the mass spectrometer select the checkbox “Use Twister”. Select “Barcode”, if a barcode reader is mounted at the lid storage platform for the labeled targets to identify. Barcode Identification has precedence over transponder identification. Target plates without barcodes are identified by the IDs stored in their transponders.
3.5 Example of the Structure of a Bruker ID The ID’s are normally the Bruker Transponder ID. This ID consists of PartNumber.SerialNumber, e.g. 209515.240. This code corresponds to a MTP AnchorChip 400/384 with the serial Number 240.
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Operation
OPERATION
4.1 Locating the Instrument Functions Turning the Instrument ON or OFF 4.1.1 Rear Panel
Figure 4-1
Schematic of the connections at the rear
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At the rear of the instrument are the following connections (Figure 4-1): CID: • • •
Collision gas inlet for argon or helium (6 bar maximum) for optional Collision Induced Dissociation and flushing the collision cell. Switch to flush the CID-cell (after changing the collision gas). Flushing gas outlet.
Gas supply: • • •
Compressed air connection to operate the slide valve. Alternatively this port might be externally connected with the nitrogen inlet for the laser to a common N2 supply. Nitrogen inlet to operate a N2-laser (Class IV Laser Product). Gas inlet to vent the instrument.
Acquisition: •
Three BNC signal connectors (trigger, detector #1 and #2) for the digitizer mounted on the PC.
Control: • • •
BNC connector to provide the video signal of the sample spot to a video capture card mounted on the PC. Serial port to connect the (optional) Twister. Serial port to connect the PC (HOST).
Pump: •
Vacuum system exhaust 13 mm (1/2”) ∅ plastic tubing (Tygon), filter mounted inside. Individual facilities may have safety guidelines, which require ventilation. It is the responsibility of the user to adhere to the requirements of their respective facility.
Service Only: •
Key field for the service to lift the cover.
Mains: •
The instrument is fitted with an IEC320-C14 mains inlet. Instrument power is controlled with the mains switch, where: • 0 = OFF (vents the instrument) • I = ON
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4.1.2 Front Panel The front panel Power LED illuminates when the instrument is properly powered up.
Figure 4-2
Control panel at the front of the instrument
1 LOAD / EJECT: Docking the target in or out of the instrument (in the case of automatic cartridge combined with opening/closing of the cover lid). 2 ACCESS: a) Permanently lit: When either the tray is inside or outside the instrument in the end position. b) OFF: During the target moves in or out. 3 IN PROGRESS: Permanently lit as long as the docking procedure is in progress (Target In/ Out). 4 ERROR: a) Permanently lit: Malfunction, requires intervention of the user or a service technician details are shown on the PC (flexControl). b) Flashing (combined with system error): Service mode or firmware update. 5 AUTOMODE: Permanent lit as long as AutoXecute is in progress. During this run applying the related button cannot retract the target plate.
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6 READY: a)
Permanently lit: Ready for acquiring spectra, i.e. any interlock is closed; N2 pressure is correct, sufficient vacuum, HV present, etc.
b)
OFF: During the docking procedure.
7 WARM UP: a)
Permanent lit: Warm-up phase of the laser device (ClassIIIb/ ClassIV Laser Product) in progress or the system is not ready for measurement, e.g., the docking procedure in still in progress, HV is off, etc. It takes some time, even when all parameters comply with the requirements. This state is reflected simultaneously on the PC screen providing details via flexControl.
b)
Flash: A subordinated malfunction has occurred, which must be verified by the user (less N2 pressure, interlock open).
8 ERROR: c)
Permanent lit: malfunction, requires intervention of the user or a service technician; the tool tips feature on the PC shows details (flexControl).
d)
Flashing: (combined with Target Error): Service mode or firmware update.
9 MAINS: Permanent lit, when the main circuit breaker is on.
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4.2 Manual Cartridge 4.2.1 Checking for Instrument Operational Readiness The front panel Ready LED (Figure 4-2) illuminates when the machine is ready for acquisition. If the Ready LED does not illuminate, either the target is in the out position or the pressure inside the analyzer is too high for proper operation.
4.2.2 Moving Targets In and Out with the Manual Cartridge To change a target of the manual cartridge follow this procedure: 1. Press the “PUSH” button of the cover lid (Figure 4-3).
Figure 4-3
Closed cover lid of the manual cartridge with “PUSH” button
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2. Wait until the cover lid has reached the lower position. Pay attention not to squash the fingers!
Figure 4-4
Open cover lid and free access to the manual cartridge
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3. Open the manual cartridge by pulling the lever with a finger.
Figure 4-5
Opening the cover plate of the manual cartridge
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4. Pull the lever with its target platform until it reaches the horizontal position.
Figure 4-6
Manual cartridge during opening
Figure 4-7
Manual cartridge completely opened
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5. Pull out the target out from the target slide. Caution: Take care not to drop anything into the load port. Doing so can prevent proper operation. 6. Place new target on the target slide and shift it into the load port until the end position. 7. Push up the lever until the cartridge cover is closed. Then push down the lever and lock it into its end-position. 8. Push up the cover lid and close it again. 9. Press the “LOAD/EJECT” button to move the target into the measurement position inside the vacuum system. 10. Wait for the READY LED to illuminate before beginning acquisition. The total time from initiating the target in routine by pushing the button to when the READY LED illuminates should not exceed five minutes and is typically less than two and a half minutes.
4.3 Using the Twister (optional) 4.3.1 Turning the Instrument On or OFF Instrument power is controlled with the mains switch (Figure 3-3), where: • 0 = Off. •
I = On.
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4.3.2 Checking for Instrument Operational Readiness The front panel Ready LED (Figure 3-2) illuminates when the machine is ready for acquisition. If the Ready LED does not illuminate, either the target is in the out position or the pressure inside the analyzer is too high for proper operation.
4.3.3 Moving Targets In To move a target in: 11. Press the “TARGET IN/OUT” button momentarily (Figure 3-2) and wait for the target platform to appear. Place the MTP-target into the target platform. Caution: Take care not to drop any debris into the load port. Doing so can prevent proper operation. 12. Press the “TARGET IN/OUT” button momentarily (Figure 3-2). Wait for the READY LED to illuminate before beginning acquisition. The total time from initiating the target in routine by pushing the button to when the READY LED illuminates should not exceed five minutes and is typically less than two and a half minutes.
4.3.4 Moving Targets Out To move a target out: 1. Press the “TARGET IN/OUT” button momentarily and wait for the target platform to appear. 2. Remove the target, and press the “TARGET IN/OUT” button momentarily a second time.
4.3.5 Instrument Control in Analytical Operations How to control the mass spectrometer in analytical operations is described in the manual flexControl and flexAnalysis.
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Instrument Maintenance
INSTRUMENT MAINTENANCE
Inspect the ventilation air filter every three months. Replace the filter when it is visibly clogged with dust to ensure proper instrument function. The filter must be replaced. Replace the filter by lifting the old filter up and out of the holder and slide in a new one. Note: Replace only the genuine Bruker filter, PN 73121! For service contact please refer to chapter Technical Support.
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Index
INDEX
A Analytical operations AnchorChip Assistance
52 28 iii
B Backpanel of the PC
17
C Camera Checking for Instrument Operational Readiness CID Class IV laser product Counter
37 52 25 24 28
D Deflector voltage/matrix suppression Detectors Digitizer
37 36 37
E Electrical Safety Environmental Conditions Example of a Bruker ID
13 14 42
F Facility and Electrical Requirements flexControl
15 41
G Gas supply Gating Grounding
10 37 10
I Installation and Setup Instrument controller Instrument layout Instrument Maintenance
15 20, 45 19 53
L Laser device Laser system LIFT
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M MALDI Manual Conventions and Symbols Moving targets in Moving targets out O Operating environment Operating Precautions
9 11 47, 52 52 10 12
P PCIS PLMS Power Purpose of the instrument
29, 31 29, 31 10 9
R Remote service capability
39
S Safety symbols Sample throughput Scheme of the ion source SCOUT MTP MALDI target Setup Site preparation Specification
vii, 10 41 25 27 17 10
T Table of Changes Tandem mass spectrometry Targets Transponder Transponder/barcode information Turning the Instrument On or OFF Twister
iv 9 27 28 42 45, 51 41
V Vacuum system Ventilation air filter Venting
24 53 10
24 37 33
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