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Table of ContentsChapter 1. About This Manual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Conventions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1Safety Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2How This Manual Is Organized . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3International Standards Certifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Federal Communications Commission Compliance. . . . . . . . . . . . . . . . . . . . . . . .4Chapter 2. Introduction to the Q Trap LC/MS/MS System . . . . . . . . . . . . . . 5

Triple Quadrupole/Linear Ion Trap Mass Spectrometer . . . . . . . . . . . . . . . . . . . . . . . .6Principles of MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7Principles of MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8Q Trap LC/MS/MS Enhanced Modes of Operation . . . . . . . . . . . . . . . . . . . . . . .10

Chapter 3. Hardware Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13Data System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14Sample Introduction System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

LC Pump or Syringe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15Ion Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

TurboIonSpray Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15Heated Nebulizer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17Source Exhaust System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19Source Exhaust Venturi Gas Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19Gas Connection Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Vacuum System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21Vacuum Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22Vacuum Control System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

Ion Path Chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35Mass Filter Rail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35Quadrupoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35Mass Filters (Q1 and Q3). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35Linear Ion Trap (LIT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36RF-Only Quadrupoles (Q0 and Q2) and Stubbies . . . . . . . . . . . . . . . . . . . . . . . .36Vacuum Feedthroughs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37Collision Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37

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Q Trap LC/MS/MS Hardware Manual

Ion Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Ion Detector and Signal Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Power Distribution Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39AC Power Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40DC Power Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40ARF Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40The System Electronics Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Chapter 4. Operating the Q Trap LC/MS/MS System. . . . . . . . . . . . . . . . . . 41Work Process Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Setting Up Instrument-Specific Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Setting Up Compound-Specific Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Setting Up Source-Specific Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Shutting Down and Powering Up the Q Trap LC/MS/MS System . . . . . . . . . . . . . . 43Powering Up the Q Trap LC/MS/MS System . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Instrument Warmup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Shutting Down the Q Trap LC/MS/MS System . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Pumping Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Pump-Down Sequence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Removing the Instrument Covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Removing the Front Cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Removing the Top Cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Removing the Back Cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Power Distribution Cover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Removing the TurboIonSpray Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Replacing the TurboIonSpray Ion Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Appendix A: Maintenance Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Q Trap LC/MS/MS System Periodic Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Appendix B: PPG Exact Mass Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55Appendix C: Scan Parameter Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Appendix D: Consumables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67Appendix E: Sample Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Small Molecules: EPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Small Molecules: MS3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71Large Molecules: Protein Identification—Tryptic Digest . . . . . . . . . . . . . . . . . . . . . 74

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

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About This Manual
The Q Trap LC/MS/MS Hardware Manual contains a description of the principles of mass spectrometry and an overview of the Q Trap LC/MS/MS system. This manual provides information about the hardware components included in the instrument. You will also find instructions on how to start up and shut down the mass spectrometer, how to remove the instrument covers, how to create sample experiments, and how to troubleshoot instrument problems.

This manual is targeted to users who are familiar with mass spectrometry but are new to the Q Trap LC/MS/MS system.

The Q Trap LC/MS/MS Hardware Manual is part of a set of manuals that includes a standard set of service manuals (Schematics and BOMs, SQ/IQ/OQ, IPV), the Q Trap LC/MS/MS Qualified Maintenance Person’s Manual, the Q Trap Site Planning Guide, and related manuals (Safety Manual, Peripheral Devices Setup Manual, Analyst 1.3 Operator’s Manual).

ConventionsWithin the scope of this manual, the following conventions are used:

WARNING! This symbol indicates a warning of potential injury or damage to the instrument. You should read the warning and follow all precautions before performing any operation described in the manual.

WARNING! This symbol indicates a warning of electrical shock hazard. You should read the warning and follow all precautions before performing any operation described in the manual. Failure to do so can result in serious injury.

WARNING! This symbol indicates a warning of biohazardous materials. You should read the warning and follow all precautions before performing any operation described in the manual.

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About This Manual Q Trap LC/MS/MS Hardware Manual

WARNING! This symbol indicates a warning of high temperatures. The probes and source housing reach high temperatures. Do not remove the probe from the source housing or the housing from the instrument while either is hot. Allow at least ten minutes for it to cool.

CAUTION! Indicates an operation that may cause damage to the instrument if the precautions are not followed.

Safety ConsiderationsThere are a number of important safety considerations you need to review before using the Q Trap LC/MS/MS system.

WARNING! All persons using the Q Trap LC/MS/MS ion source should be aware of potential hazards.

Observe the following safety precautions when operating, maintaining, or servicing the Q Trap LC/MS/MS system.

• Do not override safety interlocks in the mass spectrometer.

• The Q Trap LC/MS/MS system operates with high voltages. Please observe electrical shock hazard safety precautions.

Additional operational information is available in the Analyst online Help or in the user manuals. For detailed information relating to the Q Trap LC/MS/MS system, refer to the following manuals:

• Peripheral Devices Setup Manual

• Q Trap LC/MS/MS TurboIonSpray Ion Source Manual

• Q Trap APCI Heated Nebulizer Ion Source Manual

• Q Trap Flow NanoSpray Ion Source Manual

• Q Trap LC/MS/MS Qualified Maintenance Person’s Manual

Any person using an Applied Biosystems/MDS Sciex mass spectrometer should be fully trained in its safe operation as well as in laboratory procedures. All warnings should be followed implicitly as failure to do so could result in serious injury.

How This Manual Is OrganizedThe information provided in this manual is organized as follows:

Chapter 1: About This ManualThis chapter provides an explanation of the warning symbols in the manual, information about where you can access additional technical support, and a list of international standards the instrument complies with. Also included in the section is a description of the information provided in each chapter of the manual.

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Q Trap LC/MS/MS Hardware Manual About This Manual

Chapter 2: Introduction to the Q Trap LC/MS/MS SystemThis chapter provides an introduction to the Q Trap LC/MS/MS system. The section includes an overview of liquid chromatography (LC), MS, MS/MS, and the linear ion trap (LIT) functionality of the Q Trap LC/MS/MS system.

Chapter 3: Hardware OverviewThis chapter provides information about the sample introduction system, gas and vacuum panel, vacuum system, ion path chamber, and the electronics of the Q Trap LC/MS/MS system.

Chapter 4: Operating the Q Trap LC/MS/MS systemThis chapter provides procedures for powering up and shutting down the Q Trap LC/MS/MS system.

AppendicesAppendix A: Maintenance Checklist

This section provides a list of the regular maintenance procedures you should complete.

Appendix B: PPG Exact Mass Table

This section provides the exact monoisotopic masses and charged species (positive and negative) observed with the polypropylene glycol (PPG) calibration solutions.

Appendix C: Scan Parameters Settings

This section provides a list of the exact monoisotopic masses and charged species (positive and negative) observed with the PPG calibration solutions and Agilent ES Mix.

Appendix D: Consumables

This section provides a list of consumable parts for the Q Trap LC/MS/MS system.

Appendix E: Sample Experiments

This section provides examples of experiments you can perform using the Q Trap LC/MS/MS system.

Technical SupportApplied Biosystems/MDS Sciex and its representatives maintain a staff of fully-trained service and technical specialists located throughout the world. They can answer questions about the API instruments or any technical issues that may arise. For more information, visit the Applied Biosystems/MDS Sciex Web site at:

http://www.appliedbiosystems.com

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About This Manual Q Trap LC/MS/MS Hardware Manual

International Standards Certifications This instrument and its components have been certified by the following international agencies. Applicable labels for these qualifications have been attached to the instrument.

Federal Communications Commission ComplianceThis equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at the user’s own expense. Changes or modifications not expressly approved by the manufacturer could void your authority to operate the equipment.

International ComplianceThe Q Trap LC/MS/MS system is in compliance with the following standards:

• FCC Part 15, Subpart B, Class A

• CISPR publication 11(1997) IEC Standard EN 55011 (1998) Class A

• IEC EN61326-1: 1997

• IEC EN61010-1: 1990

• CE Certificate of Compliance is included with the instrument

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Introduction to the Q Trap LC/MS/MS System
The Q Trap LC/MS/MS system is a hybrid triple quadrupole linear ion trap (LIT) mass spectrometer. The Q3 region can be operated as either a standard quadrupole mass spectrometer or a linear ion trap mass spectrometer. The unique scan modes of both configurations can be linked to provide more and higher quality data than either instrument alone. For example, a precursor ion scan in Transmission mode can be used as a survey scan in order to target specific ions to be used in an enhanced product ion scan (in LIT mode). Conversion between the two modes of operation is rapid, since it involves only the addition or removal of the resolving DC voltages.

The Q Trap LC/MS/MS system retains all of the traditional triple quadrupole scan types such as:

• Q1 MS (Q1)

• Q1 Multiple Ion (Q1 MI)

• Q3 MS (Q3)

• Q3 Multiple Ion (Q3 MI)

• Multiple Reaction Monitoring (MRM)

• Precursor Ion (Prec) (This is not possible with a conventional ion trap.)

• Product Ion (MS2)

• Neutral Loss (NL)

When Q3 operates as an LIT mass spectrometer, a number of new advantages and capabilities are available:

• High sensitivity product ion scanning

• Fast scanning (4000 amu per second)

• High resolution capabilities at reduced scan speeds

• MS/MS/MS capabilities

• Reduced space charge effects

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Introduction to the Q Trap LC/MS/MS System Q Trap LC/MS/MS Hardware Manual

In LIT mode, a pulse of ions is introduced into the ion trap. The main RF fields trap the ions in the radial direction, while DC voltages applied to the lenses at both ends of Q3, trap the ions axially. The trapped ions are allowed to cool for several milliseconds, then the RF voltage is scanned in the presence of a low voltage auxiliary AC applied to the rods. The ions ejected axially toward the detector are counted.

If you configure the mass spectrometer with Q1 operating as a standard quadrupole mass spectrometer and Q3 operated as an LIT mass spectrometer, you can achieve the following enhanced scan types:

• Enhanced MS (EMS)

• Enhanced Resolution (ER)

• Enhanced Product Ion (EPI)

• Enhanced Multi-Charge (EMC)

• Time Delayed Fragmentation (TDF)

• MS/MS/MS (MS3)

In LIT mode, a pulse of ions passes through Q1 operated as a conventional quadrupole mass spectrometer to select the precursor ion of interest. The precursor ions are accelerated into the pressurized Q2 to promote fragmentation. The fragment and residual precursor ions are then trapped in the Q3 linear ion trap. The Q3 RF voltage is ramped and the ions ejected toward the detector are reported. For more information about these enhanced scans, see “Q Trap LC/MS/MS Enhanced Modes of Operation” on page 10.

Triple Quadrupole/Linear Ion Trap Mass Spectrometer

The Q Trap LC/MS/MS system uses a TurboIonSpray, Heated Nebulizer, or Flow Nanospray ion source to produce ions from liquid samples. The term LC/MS/MS, applied to the triple quadrupole series, is a generic label for the combined analytical processes of liquid separation and subsequent mass spectrometric analysis. The instrument is configured to perform complex MS/MS and MS/MS/MS analysis. For less rigorous analytical requirements, it can perform single MS (LC/MS) scans.

The Q Trap LC/MS/MS system allows all modes of MS/MS and MS/MS/MS operation for full characterization of biopharmaceutical compounds and the specificity needed for new drug development. For pharmaceutical and pharmacokinetic samples, MS/MS has the sensitivity and specificity required to analyze hundreds of samples per day without extensive sample preparation.

For peptides and proteins, molecular weights can be determined with accuracies better than 0.01% at 200 kDa.

The major components of the Q Trap LC/MS/MS system are shown in the figure Q Trap LC/MS/MS system components with pump on page 7.

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Q Trap LC/MS/MS Hardware Manual Introduction to the Q Trap LC/MS/MS System

Q Trap LC/MS/MS system components with pump

Principles of MSIn Single Quadrupole mode, the Q Trap LC/MS/MS system separates ions representative of the sample molecular components based on their m/z ratio. Ions of a unique m/z ratio can be separated by the single mass filter quadrupole and counted to provide mass spectra for the sample.

The mass filter quadrupole consists of four cylindrical rods mounted in a ceramic collar surrounding the ion path. Fixing the ratio of RF to DC voltages applied to the quadrupole rods determines the mass of the ions exiting the quadrupole.

Ions of a unique m/z ratio pass unobstructed through the quadrupole as a function of the quadrupole power supply (QPS) voltages applied. Ions of different m/z ratios have unstable oscillations that increase in amplitude until they collide with the quadrupole rods and are removed from the ion stream.

As an example, a sample mixture containing three molecules, R, M, and N, is introduced into the ion source. Soft ionization in the ion source generates R+, M+, and N+ ions(quasi-molecular ions formed typically by attaching one or more protons in the Positive mode, or by removing one or more protons or attaching an electron in the Negative mode).

Isolation of mixture R, M, and N

Additional structural information can sometimes be obtained by fragmenting the precursor ion in a primary collision region between the orifice and the skimmer. This process is often referred to as collision induced dissociation mass spectrometry (CID/MS).

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Isolation of product ions from a sample using the orifice-skimmer technique

The ions generated in the ion source are drawn through a curtain of dry inert gas into the ion optics housed inside the vacuum chamber. The mass filter quadrupole in the vacuum chamber selectively filters the ions based on their m/z ratio. The filtered ions are focused to the detector. As ions collide with the detector, they produce a pulse of electrons. The electron pulse is collected and converted to a digital signal to provide an ion count as a function of ion mass. The acquired data is relayed to the computer where it can be displayed as either full mass spectra, intensity of single or multiple ions versus time, or total ion current versus time.

Principles of MS/MSIn Triple Quadrupole mode, the Q Trap LC/MS/MS system uses two identical mass filter quadrupoles (Q1 and Q3) separated by a collision cell, which encloses an RF-only quadrupole (Q2). The fundamental principle of MS/MS is illustrated in the figure Isolation of product ions from a mixture of R, M and N on page 8.

As an example, a sample mixture containing three molecules, R, M and N, is introduced into the ion source. Soft ionization in the ion source generates R+, M+, and N+ ions (quasi-molecular ions formed typically by attaching one or more protons in the Positive mode, or by removing one or more protons or attaching an electron in the Negative mode).

Isolation of product ions from a mixture of R, M and N

In a Product Ion scan, the first mass filter, Q1, separates or filters ions according to their m/z ratio, and allows only one ion to enter the collision cell (M+). The M+ ion enters Q2 where it is fragmented by collision with neutral gas molecules in a process referred to as collision activated dissociation (CAD). The fragment ions generated are then passed into Q3 and filtered to provide a mass spectrum. The ions created by the source are referred to as precursor ions, the collision products are referred to as product or fragment ions.

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In a Precursor Ion scan, the third quadrupole (Q3) is fixed to the fragment mass of interest and the first quadrupole (Q1) is scanned over a range. The resulting mass spectrum displays the masses of all the compounds that produced the specified fragment mass.

In a Neutral Loss scan, both quadrupoles (Q1 and Q3) are scanned with a constant mass difference between them. The resulting mass spectrum displays the mass of the compounds that have undergone the specified loss. This type of scan is useful in identifying compounds from similar functional groups.

The fragment ions are filtered in Q3 before they are collected at the detector. As ions collide with the detector, they produce a pulse of electrons. The pulse is converted to a digital signal that is counted to provide an ion count. The acquired data is relayed to the computer where it can be displayed as either full mass spectra, intensity of single or multiple ions versus time, or total ion current versus time.

The technique of MS/MS is well suited to mixture analysis because the characteristic fragment ion spectra can be obtained for each component in a mixture without interference from the other components, assuming that the ions have a unique m/z ratio. This analysis can also be used for targeted analysis by monitoring specific precursor/product ions with Q1 and Q3 respectively while the sample is eluting. This type of analysis is more specific than single MS, which only discriminates on the basis of molecular weight.

The MS/MS technique is well suited to structural elucidation studies. The same fragmentation pattern that provides identification of a compound in a complex mixture can also reveal pertinent information regarding the structure of all their precursors.

Additional structural information can sometimes be obtained by fragmenting the precursor ion in a primary collision region between the sampling orifice skimmer. The fragment ions (for example, a second generation fragment ion spectrum), provide structural information on both the original precursor ions and the first generation fragment ions.

Isolation of second generation product ions from mixture M

The triple quadrupole instruments contain the same components as the single quadrupole instruments with the addition of a second mass filter (Q3). The high-pressure region is the same, but the high vacuum region contains the Q1 prefilter (stubbies) and the Q1 and Q3 mass filter quadrupoles that are separated by the collision cell. The collision cell is a ceramic housing enclosing the Q2 RF-only quadrupole, which when pressurized with CAD gas provides a local high-pressure region for ion fragmentation.

Ions pass through the same path as in the single quadrupole instrument until they reach the Q2 RF-only quadrupole. The selected ions arrive at Q2, while those rejected eventually collide with the rods and are lost.

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The Q2 RF-only quadrupole is separated from the Q1 and Q3 mass filters by the interquad lenses IQ2 and IQ3 (or ST3, depending on the triple quadrupole series). The Q2 region has no mass filtering capabilities; it operates in Total Ion mode. If no CAD gas is present to fragment the sample ions, Q2 transports the ions directly into Q3. If CAD gas is present, the ions that enter Q2 collide with the neutral CAD gas molecules. If pressurized, the voltage drop between the entrance lenses and Q2 provides the ions with the energy to induce fragmentation when the ions collide with CAD gas molecules. Through the energetic collisions, the ion translational energy is converted into internal energy that fractures bonds and causes ion fragmentation. After collision, the unfragmented precursor ions and the fragmented ions are transported to Q3 where they are again filtered.

When operating in MS/MS mode, the Q3 mass filter is physically and functionally identical to Q1. The ions, including a mixture of precursor and fragment ions, enter Q3 where they are filtered according to mass. In Single MS Operating mode (Q1 scan type), Q3 acts as an ion transporter (like a Q0 or RF-only quadrupole) with no filtering action.

Terms used to describe this operation are Total Ion mode, RF-only mode, and AC-only mode.

Q Trap LC/MS/MS Enhanced Modes of OperationThe Q Trap LC/MS/MS system has a number of enhanced modes of operation. A common factor of the enhanced modes is that ions are trapped in the Q3 quadrupole region and then scanned out to produce full spectrum data. Many spectra are rapidly collected in a short period of time and are significantly more intense than spectra collected in a comparable standard quadrupole mode of operation. The widths of the peaks in the spectra are usually much narrower than peaks observed in the standard quadrupole mode.

During the collection phase, ions pass through the Q2 collision cell where CAD gas focuses the ions into the Q3 region. The Q3 quadrupole is operated with only the main RF voltage applied. Ions are prevented from passing through the Q3 quadrupole rod set and are reflected back by an exit lens to which a DC barrier voltage is applied. After the fill time elapses (a time defined by the user), a DC barrier voltage is applied to a Q3 entrance lens (IQ3). This confines the collected ions in Q3 and stops further ions from entering. The entrance and exit lens DC voltage barriers and the RF voltage applied to the quadrupole rods confine the ions within Q3.

During the scan out phase, a potential of a few volts is applied to the exit lens to repel the charged ions. An auxiliary AC frequency is applied to the Q3 quadrupole. The main RF voltage amplitude is ramped from low to high values, which sequentially brings masses into resonance with the auxiliary AC frequency. When ions are brought into resonance with the AC frequency, they acquire enough axial velocity to overcome the exit lens barrier and are axially ejected towards the mass spectrometer ion detector. Full spectra data can be acquired from the ions collected in Q3 by rapidly scanning the main RF voltage.

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The enhanced modes of operation are:

• Enhanced MS (EMS): Ions are transferred directly from the ion source and orifice region to the Q3 quadrupole where they are collected. These ions are scanned out of Q3 to produce enhanced single-MS type spectra. Use the EMS mode when you need a rapid enhanced sensitivity survey type scan.

• Enhanced Resolution (ER): This mode is similar to the Enhanced Product Ion mode except that the Q1 precursor ions pass gently through the Q2 collision cell without fragmenting. A small range about the precursor mass is scanned out of Q3 at the slowest scan rate to produce a narrow window of the best-resolved spectra.

• Enhanced Product Ion (EPI): Product ions are generated in the Q2 collision cell by the precursor ions from Q1 colliding with the CAD gas in Q2. These characteristic product ions are transmitted and collected in Q3. These ions are scanned out of Q3 to produce enhanced product ion spectra. Use the EPI mode if you need enhanced resolution and intensity.

• Enhanced Multi-Charge (EMC): This mode operates similarly to the Enhanced MS mode except, before scanning the ions out of Q3, there is a delay period in which low charge state ions (primarily singly charged ions) are allowed to preferentially escape from the Q3 quadrupole. When the retained Q3 ions are scanned out, the multiply charged ion population dominates the resulting spectra.

• Time Delayed Fragmentation (TDF): Product ions are generated and collected in Q3. During the first part of the collection period, the lower mass ions are not collected in Q3. During the second part of the collection period, all masses over the mass range of interest are collected. The resultant enhanced product ion spectra are simplified compared to EPI scan type spectra. The nature of the spectra aids in the interpretation of the structure and fragmentation pathways of the molecule of interest.

• MS/MS/MS (MS3): In MS/MS/MS mode, product ions are generated in the Q2 collision cell by the precursor ions from Q1 colliding with the CAD gas in Q2. These characteristic product ions are transmitted and collected in Q3. Applying the normal mode resolving DC voltages to the Q3 quadrupole isolates a specified mass (m/z) of ion and removes all other ions from Q3. By properly applying a second auxiliary AC frequency to Q3, the specified ion can be resonantly excited. These excited ions collide with the residual nitrogen in Q3 and may fragment, producing a characteristic spectrum of ions. These secondary product ions of the isolated product ion result in MS/MS/MS product spectra.

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3
Hardware Overview
IntroductionThe Q Trap LC/MS/MS system consists of a table-top mounted instrument, an applications computer, and a printer. The user controls the Q Trap LC/MS/MS system through the Analyst software installed on the applications computer (running a Windows operating system).

Q Trap LC/MS/MS system—front view

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Hardware Overview Q Trap LC/MS/MS Hardware Manual

Q Trap LC/MS/MS system—rear view

Data SystemThe Analyst software requires a computer running the Windows operating system. For information on hardware and operating system requirements, refer to the Analyst Laboratory Director’s Guide to Security and Regulatory Compliance. The computer with the associated system software works with the system controller and associated firmware to control the instrument and data acquisition routines. The system controller controls the operation of the main console equipment. When operating the mass spectrometer, the acquired data is relayed to the application software where it can be displayed as either full mass spectra, intensity of single or multiple ions versus time, or total ion current versus time.

Sample Introduction SystemThe sample introduction system for the Q Trap LC/MS/MS system uses one of three removable ion sources (mounted one at a time). This system requires only two or three mechanical adjustments. It provides excellent performance through high sensitivity and low chemical noise.

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Q Trap LC/MS/MS Hardware Manual Hardware Overview

LC Pump or SyringeThe liquid sample stream is pumped to the ion source probe by an optional external pump or syringe drive. Flow rates are determined by the inlet requirements, the chromatography, or the volume of sample available. If introduced by an LC pump, the sample may be injected through a loop injector (flow injection analysis, or FIA) or by a separation column (LC/MS). Samples must be sufficiently prefiltered so that the capillary tubing in the inlets is not blocked by particles, precipitated samples, or salts.

The various optional pumps, autosamplers, and syringe configurations are not described in this manual. For information about a particular pump, autosampler, or syringe configuration, refer to the Peripheral Devices Setup Manual.

Ion SourcesThe Q Trap LC/MS/MS system supports three ion sources:

• TurboIonSpray

• Heated Nebulizer

• Flow Nanospray

The TurboIonSpray source is the standard ion source shipped with the Q Trap LC/MS/MS system. You can, however, install the optional Heated Nebulizer or the Flow Nanospray source.

TurboIonSpray Ion Source TurboIonSpray is ideally suited for LC/MS/MS quantitative analyses. The sensitivity increases that are achieved with this technique are both flow rate and analyte dependent. In the conventional IonSpray source, sensitivity decreases with increased flow rate, while the heated TurboIonSpray process increases ionization efficiency, especially at the higher flow rates. This results in improved sensitivity. Sensitivity is compound dependent and compounds of extremely high polarity and low surface activity usually show the greatest sensitivity increases. The TurboIonSpray technique is mild enough to be used with labile compounds such as peptides, proteins, and thermally labile pharmaceuticals.

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TurboIonSpray ion source

The TurboIonSpray ion source provides the following features:

• Able to function as a conventional IonSpray source when the heater gas is turned off

• Able to function with flow rates from 1 µL/min to 1000 µL/min

• Able to vaporize 100% aqueous to 100% organic solvents

The TurboIonSpray ion source is an atmospheric pressure ion source in which preformed ions in solution are emitted into the gas phase with or without the application of heat. In this way, quasi-molecular ions can be generated from very labile and high molecular weight compounds with no thermal degradation.

The use of an orthogonal heated gas extends the rugged and versatile technique of TurboIonSpray to accept higher flow rates with improved sensitivity. TurboIonSpray will accept flows from 5 to 1000 µL/min of solvent compositions from 100% aqueous to 100% organic, such as acetonitrile, without splitting. This allows the use of 1 mm, 2 mm, and 4.6 mm analytical columns with or without splitting.

A heater probe directs a jet of heated dry gas (up to a maximum of 500 °C) at the mist produced by the sprayer. The gas is sprayed across the orifice at an angle of approximately 45 °C with respect to the curtain plate. The liquid spray emerging from the TurboIonSpray is directed at an angle of about 45° from the opposite direction (or 135°). The TurboIonSpray effluent and the heated dry gas intersect at an angle of approximately 90° near the orifice. This interaction helps focus the TurboIonSpray stream and increases the rate of droplet evaporation resulting in an increased ion signal.

For information about installing the TurboIonSpray, refer to the Q Trap LC/MS/MS TurboIonSpray Ion Source Manual.

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Heated NebulizerThe Heated Nebulizer ion source produces ions by nebulizing the sample in a heated tube and causing the finely dispersed sample drops to vaporize. This process leaves the molecular constituents of the sample intact. The molecules are ionized through the process of atmospheric pressure chemical ionization (APCI), induced by a corona discharge needle, as the molecules pass through the ion source chamber and into the interface region.

The Heated Nebulizer offers an alternative method of introducing samples to the Q Trap LC/MS/MS system. The Heated Nebulizer, much like the standard IonSpray source, generates ions representative of the molecular composition of the sample.

Heated Nebulizer ion source

The Heated Nebulizer ion source provides the following features:

• Able to function with flow rates up to 1.5 mL/min, and can handle the entire flow from a wide bore column without splitting.

• Able to vaporize a 100% aqueous mobile phase.

• Able to handle volatile mobile phase buffers.

• Able to vaporize volatile and labile compounds with minimal thermal decomposition.

• The simple APCI spectra is ideal for MS/MS analysis.

• Capable of being used for rapid sample introduction by flow injection with or without a liquid chromatography (LC) column.

For information about installing the Heated Nebulizer ion source, refer to the Q Trap APCI Heated Nebulizer Ion Source Manual.

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Flow Nanospray Ion SourceThe Flow Nanospray ion source extends the versatility of Q Trap’s ion source techniques to offer lower flow rates with high sensitivity. The Flow Nanospray accommodates low flows up to 500 nL/min of different solvent compositions. Unlike static or discrete flow nanosprays, the Flow Nanospray ion source is intended for continuous flow using an external pump with LC column for pre-separation, providing stable spray at very low flow rates. The Flow Nanospray ion source can be connected to a variety of autosamplers and pumps to provide automated capillary LC/MS/MS applications.

In nanospray applications, potential is placed on the sample as it passes through a steel union into a fine-tipped needle. The sample is ionized by ion evaporation and the resulting ions enter the mass spectrometer for filtering and detection. As a “soft” ionization technique, nanospray is particularly useful for analyzing biological samples such as proteins while using very small sample amounts, and for taking full advantage of capillary chromatography.

Flow Nanospray ion source

The Flow Nanospray ion source provides the following features:

• Low flow rates up to 500 nL/min

• Near 100% sample utilization

• Minimal sample amount requirement

• Good sensitivity, as the ionization technique results in fine droplets for ionization

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• Ease of installation and exchange for other ion sources with the Q Trap LC/MS/MS system

• CCTV monitors for positioning the emitter accurately and for observing spray

For information about installing the Flow Nanospray ion source, refer to the Q Trap Flow Nanospray Ion Source Manual.

Source Exhaust SystemAll of the ion sources produce both sample and solvent vapors. These vapors are a potential hazard to the laboratory environment. The source exhaust system is designed to safely remove and allow for the appropriate handling of the ion source exhaust products.

The source exhaust system is a venturi system that uses a flow of gas through a venturi tube to draw the exhaust away from the ion source. The exhaust, along with the air used to drive the venturi, is delivered to the gas connections panel at the rear of the instrument where an external connection is available to remove the exhaust from the laboratory. The exhaust gas can be connected to a fume hood (or to some other means) to remove the gas safely from the laboratory.

WARNING! Ensure the source exhaust system and laboratory exhaust systems are operating correctly to ensure the safe disposal of the source exhaust gases.

The TurboIonSpray, Heated Nebulizer, and Flow Nanospray ion sources produce large volumes of exhaust products because all three ion sources use additional volumes of gas and heat to produce ions. As a result, the source exhaust system is an essential component of these ion sources. In fact, when the TurboIonSpray, Heated Nebulizer, or the Flow Nanospray source is installed, the firmware will not enable the instrument’s electronics unless the source exhaust system is operating.

WARNING! If you are analyzing gases containing toxic or highly volatile chemicals or solvents, ensure the source exhaust system and laboratory exhaust systems are operating correctly.

Note: The source exhaust system slightly reduces the pressure in the ion source. The reduction in pressure has proven to be beneficial for the ionization performance of the TurboIonSpray, Heated Nebulizer, and Flow Nanospray ion sources.

Source Exhaust Venturi Gas SupplyThe source exhaust pump is a venturi system that uses a flow of gas through a venturi tube to draw the vapors and liquid from the drain chamber. Clean compressed air from an external source is needed to drive the source exhaust gas flow. The flow of source exhaust (venturi) gas and effluent creates a negative pressure on the input side of the pump, drawing air into the drain chamber to dilute the gases.

The effluent output of the source exhaust pump connects through a fitting on the gas and vacuum panel with a drain line to a 10-liter (3-gallon) drain vessel. The drain vessel can be placed under a fume hood, or the gas output hose from the lid of the vessel can be loosely coupled to an exhaust vent system, to remove the exhaust gases from the laboratory.

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Q Trap LC/MS/MS source exhaust system

The figure Q Trap LC/MS/MS source exhaust system on page 20 shows the gas flows through the venturi system. A solenoid valve is operated by 24 VDC controlled by the system controller that switches power to the solenoid valve whenever a valid ion source is installed. When the power is switched to the solenoid valve, it opens, enabling the gas flow through the venturi tube.

A pressure switch attached to the source exhaust line is monitored by the system controller. The switch status indicates the operational status of the source exhaust system. Should the pressure in the line rise above the trip point (0.1 in. water), the system controller assumes that the source exhaust system is off. If this occurs when the Heated Nebulizer source is installed, the system controller interrupts the Power Supply Enable signal and shuts down the instrument’s electronics.

WARNING! With the TurboIonSpray source attached, the instrument will operate if the pressure in the source exhaust line exceeds the trip point. The pressure switch will be tripped, however, the Power Supply Enable signal will not be disrupted. It is important that the source exhaust system be left on at all times.

Note: When the pressure in the exhaust line falls below the set point of the instrument’s electronics, the system controller automatically restores the Power Supply Enable signal, and the instrument’s electronics are activated.

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Whenever an instrument is switched on and an ion source is installed, power to the source exhaust solenoid is switched on, initiating the venturi gas flow. The solenoid valve has no manual or software control.

Note: Closing the valve causes pressure to rise and can trip the pressure switch. Depending on the ion source attached, this can disable the instrument’s electronics and interrupt any ongoing data acquisitions. Ensure that you open the valve when the pump is operating normally.

Note: The Analyst software must be running before the source exhaust pump will turn on. The pump will remain on after the Analyst software stops running. To shut down the pump, click Standby from the Acquire menu.

Gas Connection PanelThe gas connection panel is located at the rear right-hand corner of the chassis. The vacuum lines to the roughing pump are connected through the gas connection panel. The panel also houses, on the left-hand side, the Gas 1/Gas 2 and curtain gas supply connections, the CAD gas adjustment valve, and the external connections for the source exhaust system.

Gas connection panel

Vacuum SystemThe vacuum system consists of the vacuum interface, vacuum control system, and vacuum chamber. The vacuum interface includes the gas curtain plate, orifice plate, and skimmer cone. The vacuum control system includes the turbo pump, vacuum gauge solenoid gas controller, and analog gas controllers. The vacuum chamber or ion path chamber includes ion optics, quadrupoles, collision cell, and the detector.

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Vacuum InterfaceThe vacuum interface separates the low-pressure vacuum chamber from the atmospheric pressure in the ion source. The purpose of the vacuum interface is to allow the transfer of ions from the ion source to the mass spectrometer while restricting sample, solvent, and ambient air from entering the vacuum chamber. This is accomplished using a “gas curtain” of dry nitrogen.

The vacuum interface, as shown in the figure Vacuum interface—side view on page 22 comprises two distinct pressure chambers: the gas curtain interface and the differentially pumped interface. The two interface regions are separated by an orifice plate containing a 0.010'' orifice through which the ions and a small volume of curtain gas must pass before entering the vacuum chamber.

Vacuum interface—side view

Ions are transferred from the ion source through the vacuum interface into the vacuum chamber by the potential gradient across the vacuum interface. The operator can adjust the ion flow by varying the voltages applied to the orifice plate. The curtain plate voltage is fixed and varies only in the polarity of ions to be analyzed.

The vacuum interface is bolted to the main body of the vacuum chamber. For easy access to the interface, the operator can remove the ion source housing without using tools.

Gas Curtain InterfaceThe gas curtain interface is a small volume chamber between the curtain plate and the orifice plate. It operates at atmospheric pressure and is flushed with a pure, inert curtain gas (99.999% nitrogen). The figure Vacuum interface—front view on page 23 shows the curtain plate with the ion source removed.

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Vacuum interface—front view

Approximately 600 mL/min of curtain gas flows through the orifice into the differentially pumped interface. The remaining gas flows back into the ion source through the aperture in the curtain plate.

The gas curtain interface provides a region for ion declustering. In the interface, sample ions collide with the gas molecules. The collision energy assists in breaking apart ion clusters and separating the sample ions from solvent molecules. The controlled inert atmosphere in the interface helps to retain the stable ion-molecule products from the ion source.

The curtain gas flow rate is set from the acquisition computer and is physically controlled by a variable orifice valve controller. The gas line is connected to the gas curtain interface through a connection on the bottom of the vacuum interface.

To protect the sensitive components of the instrument, the curtain gas flow is interlocked to the pumping system and ion optics. If the curtain gas pressure is more than 5 psig from the required pressure, the system controller disables the high-voltage supplies, sets the ion optics voltage to zero, and turns off the turbo pump. When the gas flow is restored, the system controller automatically restarts the turbo pump and attempts to recover the operating conditions.

Differentially Pumped InterfaceThe differentially pumped interface is the first low-pressure stage in the transition from the atmospheric pressure ion source to the low-pressure vacuum chamber. The pressure in the interface is maintained below 1.4 torr by the roughing pump located outside the main console.

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Curtain gas and ions are drawn from the curtain gas interface into the differentially pumped interface by the pressure differential across the orifice plate. The ions are further drawn through the differentially pumped interface by the voltage difference (for example, declustering voltage) between the orifice plate and the skimmer. The ions enter the vacuum chamber through the aperture in the skimmer.

Vacuum lines connect the interface pump to the port underneath the differentially pumped interface. The pump is interlocked to the ion optics and the pumping system by a pressure switch connected to the vacuum port. If the pressure in the interface rises sharply, the switch trips, notifying the system controller of an Interface Pump fault.

In the event of a pump fault, the system controller disables the high-voltage power supplies, sets the ion optics voltages to zero, and turns off the turbo pump until the pressure in the differentially pumped interface is restored.

Entrance OpticsThe entrance optics consist of the curtain plate and the orifice plate. The voltages applied to these elements control the ion flow through the vacuum interface. The curtain plate voltage is set automatically depending on the polarity of the ions (determined by the user from the application computer).

The Entrance Optics Functions table lists the curtain and orifice plate functions.

The power supplies used to generate the voltages applied to the curtain plate and the orifice plate are located on the lens power supply board inside the system electronics box.

Vacuum Control SystemThe vacuum system is controlled transparently by the system controller. When the mass spectrometer is switched on, the system controller automatically attempts to pump down the vacuum chamber. Only after reaching a stable operating pressure does the system controller enable the instrument’s analytical components.

The pressure inside the vacuum chamber is monitored using a hot cathode vacuum gauge. The system controller continually monitors the vacuum gauge output and several physical interlocks to determine the vacuum status. If the vacuum integrity is breached, the system

Entrance Optics Functions

Optic Element Function

Curtain Plate • Separates the sample flow from the curtain gas flow.

• Is electrically isolated from the vacuum housing so that the ions are not constrained to pass through ground potential at this point.

• Ensures the voltage is controlled by the computer.Orifice Plate • Provides a division between atmosphere and the

approximately 1.4 torr pressure of the differentially pumped interface.

• Contains the 0.010" orifice.

• Is electrically isolated.

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controller shuts down the instrument’s high voltages until the vacuum operating conditions are restored.

Vacuum Off SequenceWhen the Vacuum Off sequence is initiated, the turbo pump, ion optics, and vacuum gauge are disabled, and the gas flows are set to the values in the Pump-Down state. The sequence then recycles to the beginning of the Pump-Down sequence. If the sequence fails in three attempts to restore a stable operating pressure, a hard fault results and the system exits the Pump-Down sequence.

Pumping SystemThe pumping system uses a staged combination of the turbo and roughing pump to maintain the high vacuum pressure in the vacuum chamber. The turbo pump maintains the Q0 region of the vacuum chamber at 8 × 10–3 torr, and the high vacuum Q1 region is maintained at about 1 × 10–5 torr. A roughing pump maintains the differentially pumped interface at a pressure below 1.4 torr.

Turbo PumpThe turbo pump is clamped horizontally to the side flanges at the back of the vacuum chamber. The turbo pump is not connected directly to the system electronics, but is controlled by a separate controller. The turbo pump and its controller are maintenance-free.

Turbo Pump Controller The turbo pump controller (as well as the gas flow controller assembly for the gas control) is mounted on the chassis on the bracket at the inlet end of the main console (see the figure Turbo pump controller on page 26).

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Turbo pump controller

The controller is a frequency converter that converts the single-phase AC power into the three-phase, variable frequency power required by the turbo pump’s induction motors. The converter is controlled remotely by the system controller as part of the vacuum control system.

The turbo pump controller has four LEDs labelled power, acceleration, normal and failure. These indicate the pump’s operational status.

Upon receiving a signal from the system controller, the turbo pump controller initiates a startup procedure for the turbo pump. This includes a self-diagnostic routine during which the controller outputs are turned off and the four indicator lights on the controller’s front panel are illuminated. If the diagnostics routine completes successfully, the indicator lights, with the exception of the power indicator, are shut off. The turbo pump is then started and the acceleration light illuminates as the turbomolecular fans accelerate. The pump is in normal operating mode when it reaches its rated rotational speeds. In normal operating mode, the power and normal indicators are illuminated.

During normal operation, the controller monitors the turbo pump for significant changes in turbo speed, operating parameter temperature, and load faults. Should a fault occur, the controller shuts off the pump, and the controller’s failure indicator is illuminated.

The instrument’s firmware detects the change in the turbo pump’s status and attempts to reestablish operating vacuum conditions. If the turbo pump fails to stabilize after three attempts within a set time-out period, a hard fault is registered and the operator must restart the instrument.

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Roughing PumpThe second stage of the pumping system utilizes a roughing pump. The roughing pump is connected to the exhaust ports of the turbo pump, and acts in support of the turbo pump.

The roughing pump eases the initial start up load on the turbo pump by reducing the pressure in the vacuum chamber to about 0.3 torr. It also creates a pressure differential across the turbo pump’s exhaust ports, ensuring that a back pressure does not overload the pumps.

The intake port of the roughing pump is linked to a vacuum line connecting the turbo pump exhaust ports through the vacuum pump bulkhead.

The roughing pump is housed outside the instrument’s main console and is not controlled by the system firmware or the applications computer. It requires its own external 230 VAC, 50/60 Hz power supply, and is operated manually using switches mounted on the pump.

The operational status of the pumps is monitored using pressure switch interlocks. The pump must maintain a pressure low enough to satisfy the interlocks before the system controller will initiate the turbo pump. If the pressure in the pump’s intake line rises sufficiently to trip the interlocks, the system controller disables the turbo pump and the ion optics power supplies.

The roughing pump features an anti-suckback valve and a gas ballast valve (see “Anti-Suckback Valve” on page 28 and “Gas Ballast Valve” on page 29). An optional mist eliminator is strongly recommended if the pump is operated in a closed environment. The pumps require periodic maintenance that includes changing the pump oil and, if the mist eliminator is installed, replacing the mist eliminator’s coalescing element.

WARNING! If hazardous, biohazardous, or radioactive material is injected into the instrument, all appropriate safety precautions should be taken when handling the pump’s oil and coalescing filter. The oil will be contaminated and should be handled according to hazardous material safety regulations in the country of use. (For example, WHMIS, in Canada.)

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Roughing Pump

Roughing pump example

Anti-Suckback ValveThe roughing pump has a built-in anti-suckback valve that prevents any pump oil vapors from migrating into the vacuum chamber. The valve is triggered automatically when the pump is shut off or there is a power failure. As the main shaft rotation slows, a valve opens. This causes the pumping chamber to vent and the anti-suckback valve to close. When closed, the valve seals the pump intake, isolating the pump chamber from the instrument.

Interface VacuumVacuum for the interface is developed by the external roughing pump. An outlet on the gas and vacuum panel is internally connected to the vacuum flange on the bottom of the vacuum interface. From the interface, the roughing pump withdraws curtain gas, which is

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drawn through the orifice by the pressure difference between the gas curtain interface and the differentially pumped interface.

Gas Ballast ValveThe roughing pump includes a manually controlled gas ballast valve to eliminate water vapor and other condensable gases. Condensation degrades the pump oil, limiting the pump's performance and life expectancy.

When opened, the gas ballast valve permits a controlled volume of air to enter the pump chamber. This lowers the partial pressure of condensable vapors in the pump and causes the pump temperature to rise. Both these factors hinder condensation. However, operating a roughing pump with the gas ballast valve open raises the pump’s ultimate pressure, increases pump oil consumption, and increases the amount of pump oil in the exhaust.

Given the controlled, dry atmosphere in the vacuum interface and the vacuum chamber, condensation is seldom a problem.

The gas ballast valve is controlled by the black knob on the top of the oil casing. It is closed when set to zero.

CAUTION! Under normal instrument operating conditions, the roughing pump should be operated with the gas ballast valve closed.

Mist EliminatorUnless there is an oil exhaust system available, and the instrument is operated in a closed environment, it is highly recommended that a mist eliminator be installed on the exhaust port of the roughing pump. This will prevent the emission of oil vapors into the environment.

Vacuum GaugeA hot cathode vacuum gauge is used to monitor the pressure inside the vacuum chamber. The gauge is connected directly to a port on the rear of the vacuum chamber near the turbo pump. The gauge is controlled by the vacuum gauge controller.

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Vacuum gauge

The vacuum gauge produces and measures an ion current proportional to the pressure inside the vacuum chamber. Electrons produced by a controlled current flowing through the filament inside the vacuum gauge accelerate toward the grid electrode, which is held at a potential of +150 V. The electrons collide with gas molecules inside the vacuum gauge tube creating positive ions. These ions are attracted to the collector electrode, which is held at -13 V.

The ion current measured at the collector is directly proportional to the vacuum pressure. The electron emission current flowing between the filament and the grid is the gauge sensitivity factor. By regulating the electron emission current, the pressure inside the vacuum chamber can be directly determined from the ion current measured at the collector.

Vacuum Gauge ControllerThe vacuum gauge controller is located inside the system electronics box. It performs the following functions:

• Enables power to the vacuum gauge filament in response to the Gauge Enable signal from the system controller. This occurs after the turbo pump reaches normal operating condition (usually below 10-3 torr).

• Regulates the voltage applied to the vacuum gauge filament to ensure a consistent electron emission current of 0.1 mA.

• Provides the Vacuum Ready signal to the system controller to enable the ion path voltages once the pressure reaches 10-4 torr.

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• Monitors the ion current signal at the vacuum gauge collector to ensure the pressure inside the vacuum chamber remains below 5.0 X 10-5 torr.

If the pressure inside the vacuum chamber rises above 10–4 torr, the vacuum gauge controller sends a digital signal to the system controller. The system controller then initiates the Vacuum Off sequence, disables power to the high-voltage power supplies and the turbo pump. The system controller then sets the ion path voltages to zero and instructs the vacuum gauge controller to turn off the vacuum gauge (to protect the filament). After the Vacuum Off sequence completes, the system controller restarts the Vacuum Pump On sequence. The system controller attempts to recover the vacuum integrity automatically without operator intervention.

Gas Control SystemFour gas flows are required for the instrument:

• CAD gas

• Curtain gas

• Nebulizer gas (Gas 1)

• Heater gas (Gas 2)

Gas Flow ControllerThe controller circuit works by sensing the pressure in the volume of gas between a variable inlet and a fixed orifice outlet. It continually adjusts the pressure by varying the inlet to match the sensed pressure with the set point pressure. If the pressure is too high, the inlet closes, allowing the pressure to drop. If the pressure is too low, the inlet opens to raise the pressure. As the measured pressure reaches the required set point, the analog valve closes to the point where it keeps the flow through the controller the same as the flow through the orifice, thereby keeping the pressure constant at the outlet.

CAD Gas FlowThe CAD gas is the target gas in the collision cell. Collisions between the ions speeding along the ion path and the CAD gas molecules in the collision cell provide the energy for ion dissociation.

The CAD gas is tapped from the curtain gas input, and pressure is relieved through a metering valve into the roughing pump exhaust manifold. The gas is then directed through the solenoid gas flow controller to the face plate at the right end of the vacuum chamber. The CAD gas is routed along the mass filter rail and fed to the collision cell through the hollow locating pin that locates the Q2 rod set.

Curtain Gas FlowThe curtain gas is used to isolate the ion source from the vacuum chamber. The gas acts as its name suggests, like a curtain restricting the flow of air, sample, and solvent into the vacuum chamber.

The curtain gas is connected through the gas connection panel on the chassis to the gas flow controller assembly. For the gas flow controller, the curtain gas is connected to the gas curtain interface through the vacuum interface housing. The flow is interlocked to the

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vacuum control system and the ion optics by a pressure switch connected to the gas flow controller’s intake manifold.

Nebulizer Gas (Gas 1) FlowNebulizer gas is used to optimize the signal’s stability and sensitivity. Typically, a value of 10 to 90 psig is used as applied by the applications computer.

Heater Gas (Gas 2) FlowHeater gas aids in the evaporation of the solvent that aids in increasing the ionization of the sample. The higher the liquid flow, or the higher the aqueous composition of the solvent, the higher the heater gas temperature and gas flow required. However, a temperature that is too high can cause premature vaporization of the solvent, and result in a high chemical background noise. A heater gas flow that is too high can produce a noisy or unstable signal.

Gas 1/Gas 2 control connection

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Gas Control SequenceWhen the vacuum chamber pressure is stable below the Vacuum Ready set point of 1 X 10-4 torr, the three gas flows are controlled by the operator at the applications computer. However, when the instrument is either pumping down or venting, the curtain gas flow rates are set by the system controller independent of the software setting at the applications computer. By overriding the software gas flow controller settings, the system controller ensures the consistent, predictable behavior of the instrument when pumping down or venting.

During the pumping down or venting process, the curtain gas is set to its maximum flow. In the Pump-Down sequence (see “Pumping Down” on page 46), the vacuum gauge controller informs the system controller that the vacuum is ready when the vacuum pressure surpasses the setpoint (1 X 10-4 torr). Upon receiving the Vacuum Ready signal, the system controller releases the gas control to the software.

After the curtain gas flow is returned to software control, the vacuum pressure typically drops rapidly from the Vacuum Ready set point at 1 x 10-4 torr to the operating pressure specification at 1 X 10-5 torr. This rapid decrease in vacuum pressure occurs because the curtain gas software setting is generally considerably lower than the maximum flow set by the system controller.

Safety InterlocksThe vacuum control system has safety interlocks to protect the instrument’s sensitive electronic components. These interlocks prevent the normal operation of the instrument if certain operating parameters outside the direct control of the system circuitry are not maintained.

The two interlocks that directly affect the pumping sequence are:

• Curtain gas flow

• Roughing pump pressure

When an interlock is triggered, the Vacuum Off sequence is initiated The turbo pump is disabled and the ion optics voltages are set to zero. When the interlocks are recovered, the system automatically attempts the Pump-Down sequence.

A set of interlocks prevents the instrument from switching to Analysis mode if a valid ion source is not properly installed. These interlocks do not, however, affect the pumping system.

Curtain Gas InterlocksCurtain gas flow is essential to the consistent and safe operation of the instrument. Without a significant flow of curtain gas, the vacuum chamber draws ambient air from the ion source, the moisture and composition of which can negatively affect the operation of the instrument. Even though small amounts of curtain gas enter the vacuum chamber with the sample, the operation of the instrument is not detrimentally affected because the quantity and the composition is controlled.

A pressure switch connected to the curtain gas flow between the gas cylinder and the gas flow controller is triggered if the pressure drops below a set point that corresponds to a flow rate of 0.7 L/min. If the interlock is tripped, the Vacuum Off sequence is initiated and

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the turbo pump and the ion optics voltages are disabled. The instrument automatically attempts to pump down when the curtain gas flow interlock is satisfied.

Roughing Pump InterlockA pressure switch attached to the vacuum line connects the exhaust port of the turbo pump to the roughing pump and acts as the indicator of the roughing pump’s operational status. If the pressure in the vacuum line rises significantly and triggers the interlock switch, the Vacuum Off sequence is initiated. The turbo pump shuts down and the ion optics are disabled.

The instrument automatically attempts to pump down when the vacuum pressure in the roughing pump line is regained and the pressure switch interlock closes.

Vacuum Control SequenceOn power-up, the instrument goes directly into Pump-Down mode. The Pump-Down sequence is controlled by the firmware independent of the applications computer. This means that the Pump-Down sequence, once initiated, is transparent to the user.

When a stable operating vacuum pressure is established and the required safety interlocks are satisfied, the instrument switches directly to Analysis mode. In Analysis mode, the instrument is ready for spectrographic analysis; the ion path voltages, gas flows, and the other operating parameters are controlled by the operator at the applications computer.

Operating modes

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Ion Path ChamberThe vacuum chamber is a single aluminum extrusion housing the ion optics, collision cell, and the detector. The quadrupoles, collision cell, and the associated ion optics are assembled on the mass filter rail and inserted into the vacuum chamber as a single unit. The ion detector, housed in the ion detector module, is installed inside the vacuum chamber after the mass filter rail is in position.

A seal formed by the front bulkhead on the mass filter rail divides the vacuum chamber into two distinct regions. The Q0 region contains the Q0 rod set. It is located between the vacuum interface and the front bulkhead on the mass filter rail. This region is maintained at 8 X 10-3 torr by the turbo pump.

The high vacuum region contains the three remaining rod sets and the associated ion optics. It is maintained at about 1 X 10-5 torr by the turbo pump. The Q2 quadrupole rod set is contained in the collision cell that forms part of the high vacuum region. The Q1 and Q3 quadrupoles are located on either side of the collision cell and are open for free pumping by the turbo pump.

Note: The vacuum chamber is safety interlocked such that if the pressure inside the high vacuum region reaches 1 X 10 -4 torr or greater, all ion path voltages are set to zero.

Mass Filter RailThe quadrupole rod sets and ion optics are installed, aligned, and wired on the mass filter rail before the rail is inserted into the vacuum chamber. The front end of the mass fIlter rail is supported by the front bulkhead. The other end of the mass filter rail is bolted to the rear flange that seals the detector end of the vacuum chamber. The front bulkhead can be accessed through the vacuum interface to easily remove the mass filter rail.

All gas lines and internal wires are routed along the mass filter rail. The external ion optics and the gas connections are made through vacuum connectors on the rear flange.

Vacuum feedthroughs are used to connect the RF and DC voltages for the Q1 and Q3 mass filter quadrupoles through the bottom of the vacuum chamber. The leads are connected after the mass filter rail has been installed.

QuadrupolesThe four quadrupoles are mounted on the mass filter rail inside the vacuum chamber. The Q1 and Q3 rod sets are mass filters that selectively filter ions based on their mass-to-charge ratio (m/z). The Q0 and Q2 rod sets are RF-only quadrupoles that have no filtering effect.

Mass Filters (Q1 and Q3)In the Q Trap LC/MS/MS system, the Q1 and Q3 quadrupoles consist of four cylindrical electrodes (rods) to which precise DC and RF voltages are applied. The Q1 and Q3 rods are enclosed by ceramic collars and positioned accurately on the mass filter rail using the locating pins.

35


The Q1 and Q3 rod sets have very high mechanical precision necessary for achieving high transmission and high resolution. The normal trade-off between high ion transmission and narrow peak width must be optimized for each particular application. The Q1 and Q3 rod sets normally operate at a constant mass width that is independent of the ion mass (M). Hence, the resolution in this mode of operation is directly proportional to the mass being observed.

The Q3 rod set is also capable of being operated in Total Ion mode in which only RF voltage is applied to the quadrupole rods (other terms are RF-only mode and AC-only mode). This essentially allows ions of all masses present in Q3 to be transmitted to the ion detector.

Linear Ion Trap (LIT)The Q Trap LC/MS/MS system has enhanced modes of operation. In any of the linear ion trap modes, a pulse of ions is introduced into the linear ion trap (Q3). The main RF fields trap the ions in the radial direction and DC voltages applied to the lenses at both ends of the linear ion trap are used to trap the ions axially. The trapped ions are allowed to cool for several milliseconds, and then the RF voltage is scanned in the presence of a low-voltage auxiliary AC applied to the rods. The ions that are ejected axially toward the detector are counted.

During the collection phase, ions pass through the Q2 collision cell where CAD gas focuses the ions into the Q3 region. The Q3 quadrupole is operated with only the main RF voltage applied. Ions are prevented from passing through the Q3 quadrupole rod set and are reflected back by an exit lens to which a DC barrier voltage is applied. After the fill time (a time defined by the user), a DC barrier voltage is applied to a Q3 entrance lens. This confines the collected ions in Q3 and stops more ions from entering. The entrance and exit lens DC voltage barriers and the RF voltage applied to the quadrupole rods confine the ions within Q3.

During the scan out phase, a potential of a few volts is applied to the exit lens to repel the charged ions. An auxiliary AC frequency is applied to the Q3 quadrupole. The main RF voltage amplitude is ramped from low to high values, which sequentially brings masses into resonance with the auxiliary AC frequency. When ions are brought into resonance with the AC frequency, they acquire enough axial velocity to overcome the exit lens barrier and are axially ejected towards the mass spectrometer ion detector.

RF-Only Quadrupoles (Q0 and Q2) and StubbiesAn RF-only quadrupole is similar in construction to a quadrupole mass filter, but is only capable of being operated in Total Ion mode (only RF voltage is applied to the rods). In the Q Trap LC/MS/MS system, both Q0 and Q2 are RF-only quadrupoles.

The Q0 rod set is mounted in the front bulkhead of the mass filter rail. The Q0 rod set focuses and transfers ions from the vacuum interface through the interquad lens Q1 into the stubbies and in the high vacuum region. The stubbies prefilter and transfer the ions into the Q1 mass filter. To optimize ion transfer, both Q0 and the stubbies are electrically connected to the Q1 RF voltage. The RF voltage applied to Q0 and the stubbies is a consistent fraction of the RF voltage applied to Q1.

M∆( )M M∆⁄( )

36


The Q2 rod set is housed inside the collision cell that is mounted between Q1 and Q3 on the mass filter rail. It transmits ions through the collision cell into Q3. Similar to Q0, the Q2 RF voltage is capacitively coupled to the Q3 RF voltage. The Q3 RF voltage is capacitively coupled to the Q2 voltage so that the Q2 RF voltage is ramped in a constant ratio with respect to that of Q3.

Vacuum FeedthroughsThe amplified RF and DC voltages for Q1 and Q3 are connected through the bottom of the vacuum chamber through the vacuum feedthroughs. There are four feedthroughs: two for Q1 and two for Q3. Each feedthrough carries the combined RF and DC voltages for one pair of opposing quadrupole rods.

The feedthroughs are installed through punch-outs in the tops of the Q1 and Q3 coil boxes into designated holes in the bottom of the vacuum chamber. One end of each feedthrough lead is connected to the respective interconnect circuit board inside the vacuum chamber; the other end is connected to a sleeve in the respective coil box.

Collision CellThe collision cell is a ceramic housing pressurized with CAD gas. The housing contains Q2 and is closed at both ends by interquad lenses IQ2 and IQ3.

Ions enter Q2 through IQ2 and collide with the CAD gas molecules in the cell. The collisions provide the energy needed to dissociate precursor ions into fragment ions. All ions in the collision cell are transferred to Q3 where the precursor or fragment ions can be selectively filtered and transferred to the ion detector for counting.

CAD gas is fed through a vacuum fitting on the end of the flange of the vacuum chamber. The CAD gas line is then routed along the mass filter rail and fed through a hollow locating pin in the top of the collision cell. The user controls the gas flow from the applications computer.

Since the degree of fragmentation is a function of collision gas thickness (CGT), the CGT must be controlled. This is accomplished by controlling the flow of gas that has been redirected from the differentially pumped interface and fed through a gas flow controller to the collision cell. The gas flow is set by the operator from the applications computer.

Ion OpticsThe ion optics are designed to help guide and focus the sample ions through the mass filters and deliver these selected ions to the ion detector. Voltage potentials are applied to the ion optics by the operator at the applications computer and can be varied for different sample and application requirements. The figure Q Trap LC/MS/MS ion optics path on page 38 shows the ion optics path.

37


Q Trap LC/MS/MS ion optics path

The ion optics consist of the following:

• Curtain plate

• Orifice plate (OR)

• Stubbies (ST)

• Interquad lenses (IQ1, IQ2, IQ3)

• Exit lens (EX)

• Deflector (DF)

The curtain plate and the orifice plate are part of the vacuum interface.

The stubbies, high vacuum region, and the exit lens are mounted on the mass filter rail. The stubbies help transfer the ions from the Q0 region to the Q1 mass filter in the high vacuum region. This lens is actually a shortened version of an RF-only quadrupole. The interquad lenses help the transmission of ions into the respective quadrupoles, while the deflector helps to improve the collection efficiency of the ion detector.

The deflector, ion detector, and support electronics are contained in a separate module that attaches to the front of the vacuum chamber at the detector end of the instrument.

38


Ion Detector and Signal Handling

Ion detector

Deflector VoltageThe deflector voltage is supplied by the lens power supply board. It varies from –400 to +400 volts and can be set by the operator at the acquisitions computer. The gas resistor (DS1) acts as a surge voltage protector for this circuit.

Power Distribution ModuleThe power distribution module is the interface between the external 230 VAC power supply and the instrument’s electronics. The module supplies all required power for operating the mass spectrometer.

The instrument requires two separate 230 VAC power sources operated at 50 or 60 Hz. One single phase 230 VAC power source is required for the instrument’s main console. The second power source is required for the roughing pump. The applications computer, printer, and other accessories (including LC equipment) are powered separately from standard wall outlets. An optional line adjustment transformer (LAT) can be purchased to provide accurate, consistent power for the instrument and the roughing pump.

The instrument operates within design specification with line voltage variations of 230 VAC +5%. The OEM equipment including the roughing pump specify that line variations also be minimized to +5%.

A DC power supply converts the AC input power into DC voltages.

39


AC Power DistributionThe 230 VAC power enters the main console by a detachable cable and is fed through a filter to the main power switch on the power distribution panel. The switch doubles as a circuit breaker that trips and disables power to the instrument (if there is a power surge). When the power switch is on, power is directed straight to the power distribution panel where it is divided and connected by detachable cables to the following equipment on the main console:

• Main DC power supply

• Card cage blower

• Heated Nebulizer inlet (optional)

DC Power DistributionThe DC power supply converts the 230 VAC input power into four DC voltages:+5.0 V, -18 V, +24 V A and +24 V B. The DC voltages are supplied to the motherboard where they are available to the system electronics box and the main module equipment. The DC power supply has a feedback sensing circuit that ensures the consistency of the DC voltages across the motherboard.

ARF ModuleThe ARF power supply module supplies Q3 with an RF voltage allowing the mass spectrometer to operate as a linear ion trap. The ARF module is located adjacent to the vacuum chamber.

The System Electronics Box The system electronics box houses the following seven printed circuit boards:

• System controller

• Ion path DACs and vacuum gauge controller

• Lens power supply

• HV power supply

• QPS exciter

• Q1 amplifier board

• Q3 amplifier board

These boards are contained in individual modules. Each module plugs into the common motherboard that forms the back of the system electronics box. Together, the modules control the instrument and convert the input power into the precise RF and DC voltages that drive the mass filters and supporting ion optics.

The QPS exciter board and the Q1 and Q3 amplifiers form part of the quadrupole power supply, providing the precise AC and DC voltages to the Q1 and Q3 mass filters.

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4
Operating the Q Trap LC/MS/MS System
Work Process FlowPerforming qualitative and quantitative analysis, from start to finish, involves various modes and steps which you set in the Analyst software. The following figure Work process flow illustrates the process flow from beginning to end at a very basic level.

Work process flow

The top half of the figure indicates the instrument parameters. These parameters can be divided into three types:

• Instrument-specific (set up on initial installation and on mass calibration)

• Compound-specific (set up for each analysis)

• Source-specific (set up for each analysis)

41

Operating the Q Trap LC/MS/MS System Q Trap LC/MS/MS Hardware Manual

Once you assign values to instrument parameters for a particular analysis, they become the “working parameters,” describing the control parameters for the instrument.

The bottom half of the figure describes the processing of the sample data according to the instrument parameters; you introduce samples and quantitate them.

To process another batch, no further parameter setup is required; you simply submit another batch. To process another compound, you should redefine the compound- and source-specific parameters.

Setting Up Instrument-Specific ParametersYou need to set up instrument parameters on a periodic basis, either on initial installation or if you need to recalibrate the instrument. This process is not required for each analysis. For more information, refer to the Analyst 1.3 Operator’s Guide or the Analyst online Help.

To set up instrument-specific parameters1. Create a hardware profile in the Analyst Hardware Configuration Editor.

2. Introduce a sample containing the compound of interest (usually a reference compound, such as PPG).

3. Tune the mass spectrometer:

a) Define the acquisition method (scan type and masses).

b) Examine the shape for sensitivity, peak width, resolution, and mass assignment. (The last two verify the mass spectrometer’s performance.)

c) Adjust your method as necessary to obtain the maximum sensitivity for your analyte(s) or mass(es) of interest.

Setting Up Compound-Specific ParametersEach time you want to quantitate a new compound, you need to define the analysis conditions for the compound. Depending upon your level of expertise, you set compound-specific parameters in two ways:

• Automatically (for novice users)

• Manually (for experienced users)

To set up compound-specific parameters automatically1. Introduce the compound into the mass spectrometer.

2. Start the Quantitation Optimization wizard. Analyst produces an acquisition method for the mass spectrometer. Refer to the online Help for detailed instructions about using the Quantitation Optimization wizard.

42

Q Trap LC/MS/MS Hardware Manual Operating the Q Trap LC/MS/MS System

To set up compound-specific parameters manually1. Introduce the compound into the mass spectrometer.

2. Start Manual Tune. Refer to the Analyst online Help for detailed instructions about using Manual Tune.

3. Optimize individual instrument parameters as needed. The application software produces an acquisition method for the mass spectrometer.

Setting Up Source-Specific ParametersSource parameters can be optimized for the LC conditions used during analysis. These parameters are accessed either by clicking the Source/Gas tab in the Tune Method Editor, or by clicking Edit Parameters in the Acquisition Method Editor while in Acquire mode.

Once you have created an acquisition method for the mass spectrometer, you need to define or modify the acquisition methods for the peripheral devices (such as LC pumps and autosamplers) so that they synchronize with the instrument.

For more information, refer to the Analyst 1.3 Operator’s Guide or the Analyst online Help.

Shutting Down and Powering Up theQ Trap LC/MS/MS System

The power to the Q Trap LC/MS/MS system should be left on to maintain the high vacuum conditions required for operation. The instrument remains in a warmed-up state so that operation can begin immediately. If the instrument is already on and warmed up, the green status light on the front panel will be on and will not be flashing. If the instrument is not on, you should follow the procedure “Powering Up the Q Trap LC/MS/MS System” on page 44.

The instrument power is only turned off when service on the vacuum or electrical components is required. When first powered up, the instrument, under control of the system firmware, goes into Pump-Down mode and attempts to start the turbo pump and bring the vacuum chamber to operating pressure. The process is transparent and does not require operator intervention. While the system is pumping down, the ion optics, detector, and ion source voltages are disabled. When the necessary vacuum conditions are reached and the safety interlocks are satisfied, the instrument switches to Analysis mode and the operating voltages are enabled.

Certain conditions outside the direct control of the instrument firmware must be satisfied before the turbo pump will be initiated. The curtain gas must be turned on at the source, and the roughing pump must be turned on manually. Interlocks monitored by the firmware detect if the curtain gas and the roughing pump are switched on. If the interlocks are not satisfied, the turbo pump is not initiated.

43


Powering Up the Q Trap LC/MS/MS SystemIf the instrument was vented during the shut-down procedure, the covers must be replaced before the mass spectrometer can be powered up. For more information, see “Removing the Instrument Covers” on page 48.

To power up the Q Trap LC/MS/MS system1. Replace the venting screw on the front of the vacuum chamber, if the instrument was

vented during the shut-down procedure.

2. Replace the instrument covers.

3. Turn on the roughing pump, if it was turned off.

4. Ensure that the curtain gas supply is flowing to the instrument. The pressure should be regulated to 60 psig.

5. Ensure that the 207 V to 242 V main power supply is plugged into the electrical connections panel.

6. Turn on the main power switch.

7. Ensure that the General Purpose Interface Bus (GPIB) cable is connected to both the Q Trap LC/MS/MS system and the applications computer.

8. Turn on the applications computer.

Note: Should the ion source be removed, the system electronics will be disabled interrupting any data acquisition tasks. The turbo pump and the vacuum system will not be affected.

Instrument WarmupThe mass filters must be adequately warmed up before proper performance can be achieved.

If the power has been off for more than five minutes, the instrument should warm up for approximately one hour after the operating vacuum conditions are established. If the instrument has been off for an extended period of time (for example, overnight), a warm up period of four hours is recommended.

Shutting Down the Q Trap LC/MS/MS SystemThe power to the Q Trap LC/MS/MS system is typically always left on, thereby maintaining the high vacuum conditions required for operation. The instrument also remains in a warmed up state so that operation can begin immediately. The instrument power is usually only turned off when service to the vacuum or electrical components is required.

When shutting down the instrument, care must be taken to prevent the roughing pump’s exhaust from being drawn into the vacuum chamber through the turbo pump’s exhaust ports. The likelihood of this occurring is reduced because the roughing pump has a valve that isolates the pump exhaust from the pump intake when the pump is shut off or fails. Shutting down the instrument properly will greatly reduce the possibility of pump exhaust being drawn into the vacuum chamber.

44


To shut down the Q Trap LC/MS/MS system1. Complete any ongoing scans, or select the Abort Sample from the Acquire menu.

2. Shut off the sample flow to the instrument.

CAUTION! The sample flow must be shut off before shutting down the instrument.

3. Close the application software.

4. Shut off the main power switch to the instrument. The switch is located on the bulkhead at the back right corner of the chassis.

Note: When the main power switch is turned off, the turbo pump continues to rotate without power for a few minutes and continues to provide vacuum to the vacuum chamber. If, during this time, the roughing pump is powered down, the pressure in the vacuum line between the roughing pump and the turbo pump increases. The increase in back pressure can create an incorrect load on the turbo pump bearings and cause a catastrophic failure of the turbo pump.

CAUTION! To prevent damage to the turbo pump, leave the roughing pump running for a minimum of five minutes after shutting off the instrument’s main power switch. This prevents the buildup of pressure in the vacuum lines from the turbo pump to the roughing pump.

CAUTION! If the instrument is to be shut down for any length of time, we recommend that the vacuum chamber be vented to prevent the roughing pump exhaust from being sucked back into the vacuum chamber. (To vent the vacuum chamber, follow steps 5 to 7.)

CAUTION! If the vacuum chamber is not going to be vented while the instrument is shut down, we recommend the roughing pump remain turned on to prevent the pump exhaust from being sucked back into the vacuum chamber. (If you do not want to vent the vacuum chamber, skip steps 5 to 7.)

5. Shut off the roughing pump. The power switch is located beside the power supply input attachment on the roughing pump.

6. Unplug the main power source to the instrument from the rear panel at the back right corner of the chassis.

7. Vent the vacuum chamber for twenty minutes. To do so, remove the venting screw (with a 5 mm socket wrench) from the front of the chamber.

Note: To vent the vacuum chamber, you must first remove the instrument covers. For more information, see “Removing the Instrument Covers” on page 48.

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Pumping DownThe following chart shows the Pump-Down sequence.

Vacuum Pump-Down sequence

Pump-Down SequenceThe Pump-Down sequence is initiated when the instrument’s power is switched on. Before attempting to initiate the turbo pump, the status of the system interlocks and fault conditions, including the vacuum gauge and the turbo pump status, are evaluated. The proper setting of the curtain gas is verified and, when the initial conditions are correct, the control sequence initiates the turbo pump.

46


The status of the turbo pump is monitored by the firmware control circuitry. If the turbo pump does not reach the normal operating mode within a specified time-out period, the sequence triggers a Turbo Transition fault. The system attempts to start the pump three times. If the pump fails to stabilize after the three attempts, the firmware controller registers a hard fault and aborts the Pump-Down sequence.

The turbo pump switches to normal operating mode when its turbo blades reach their rated rotational speeds. Power to the vacuum gauge filament is enabled 55 seconds after the turbo pump has reached its normal status. The vacuum gauge output is not monitored until ten seconds after the gauge has been enabled. This delay allows the gauge output to stabilize before it is used as a variable in the Pump-Down sequence.

There is a two-stage Pump-Down sequence. When the vacuum chamber pressure reaches 10-4 torr, the gases are put under the control of the Analyst software. Before the electronics are enabled, the vacuum must reach 2 X 10-5 torr.

To initiate the Pump-Down sequence1. Replace the venting screw on the front of the vacuum chamber, if the instrument was

vented.

2. Replace the instrument covers. See “Removing the Instrument Covers” on page 48.

3. Turn on the roughing pump, if it was turned off.

4. Ensure that the curtain gas supply is flowing to the instrument. The pressure should be regulated to 60 psig.

5. Ensure that the 207 V to 242 V main power supply is plugged into the electrical connections panel.

6. Turn on the main power switch.

7. Ensure that the General Purpose Interface Bus (GPIB) cable is connected to both the Q Trap LC/MS/MS system and the applications computer.

8. Turn on the applications computer.

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Removing the Instrument CoversThere are four covers that enclose the operating modules of the Q Trap LC/MS/MS system. The covers can be opened to allow access to the instrument's component modules, the system electronics box, and the operational parameter checkpoints. The covers are designed to prevent access to the instrument when high operating voltages are engaged.

Removing the Front CoverYou must remove the front cover before you can open the remaining covers.

Q Trap LC/MS/MS system—front cover

Opening the front cover exposes the main components of the Q Trap LC/MS/MS system, including many of the system test points. The cover is secured at the top by three quarter-turn screws that are mounted on top of the card cage and coil box. The cover is not hinged to the chassis but is anchored at the bottom by the three tabs. You must lift the cover off the tabs and away from the chassis to access the front of the Q Trap LC/MS/MS system.

To remove the front cover1. Shut down the instrument. See “To shut down the Q Trap LC/MS/MS system” on page

45.

2. Remove the ion source. See “Removing the TurboIonSpray Ion Source” on page 51.

WARNING! Do not remove the Q Trap LC/MS/MS system covers unless the instrument has been shut down properly and the main power disconnected (see “Shutting Down the Q Trap LC/MS/MS System” on page 44). Failure to follow this sequence will expose the operator to hazardous voltages.

3. Unscrew the three captured bolts that secure the front cover to the instrument.

4. Grasp the top corners of the front cover and gently pull and lift the cover to remove it.

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Removing the Top Cover

Q Trap LC/MS/MS system—top cover


1. Remove the front cover.

2. Unscrew the two captured bolts that secure the top cover to the back of the instrument.

3. Tilt the front end of the cover up and gently pull towards the rear. Lift the cover to remove it.

49


Removing the Back CoverThe back cover encloses most of the systems cabling. It is not hinged and must be removed to access the back of the Q Trap LC/MS/MS system.

Q Trap LC/MS/MS system—back cover


1. Remove the front cover.

2. Remove the top cover.

3. Unscrew the two captured bolts that secure the back cover to the back of the instrument.

4. Gently lift the cover to remove it.

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Power Distribution Cover

Q Trap LC/MS/MS system—power distribution cover

WARNING! There are no operator serviceable items located behind the power distribution cover.

Removing the TurboIonSpray Ion SourceIf you intend to vent the vacuum chamber during the shut-down procedure, the ion source must be removed.

Note: This procedure instructs you on how to remove the TurboIonSpray source. For instructions on removing the Heated Nebulizer or the Flow Nanospray ion sources, refer to either the Q Trap APCI Heated Nebulizer Ion Source Manual or the Q Trap Flow Nanospray Ion Source Manual.

To remove the TurboIonSpray ion source1. Finish or abort any ongoing scans.

2. Shut down the sample flow to the ion source.

3. Turn the latches on the front plate of the source. Loosen the latches completely, but be aware that they cannot be removed. Remove the TurboIonSpray source from the front of the Q Trap LC/MS/MS system. Pull the ion source away from the vacuum chamber so that the latches clear the connections in the vacuum interface housing. For more information, refer to the Q Trap LC/MS/MS TurboIonSpray Ion Source Manual.

51


Replacing the TurboIonSpray Ion Source1. Install the ion source against the vacuum chamber so that the latches slide over the

connections in the vacuum interface housing.2. Turn the latches on the front plate of the ion source until the ion source is locked in

position against the vacuum chamber.

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Q Trap LC/MS/MS Hardware Manual Appendix A: Maintenance Checklist

Appendix A: Maintenance Checklist

Q Trap LC/MS/MS System Periodic Maintenance

The Q Trap LC/MS/MS system requires regular maintenance to ensure optimum performance. You should ensure that the proper maintenance procedures are performed at regular intervals. These procedures can only be performed by a Qualified Maintenance Person or a Service Engineer. You should consult the appropriate person before performing any of the maintenance procedures listed below.

A Qualified Maintenance Person can perform the following procedures:

• Cleaning the curtain plate

• Cleaning the orifice and skimmer

• Cleaning Q0

• Replacing the card cage blower filter

A Service Engineer can perform all of the above procedures as well as the additional procedures below:

• Replacing the roughing pump oil

• Replacing the roughing pump filter trap

• Replacing the roughing pump mist eliminator filter

Serial No._________ Year_________

Procedure Performed by Interval Date Date Date Date Date

Clean the curtain plate

Qualified Maintenance Person

When it appears dirty or performance suffers

Clean the orifice and skimmer



Clean Q0 Qualified Maintenance Person


Replace the card cage blower filter


Maximum every three months, when it appears dirty, or performance suffers

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Appendix A: Maintenance Checklist Q Trap LC/MS/MS Hardware Manual

Replace the roughing pump oil

Service Engineer Maximum every six months, when it appears dirty, or if the instrument has been stored for an extended period.

Replace the roughing pump filter trap

Service Engineer When it becomes clogged or perfor-mance suffers

Replace the roughing pump mist eliminator filter

Service Engineer When it appears dirty or performance suffers

Serial No._________ Year_________

Procedure Performed by Interval Date Date Date Date Date

54

Q Trap LC/MS/MS Hardware Manual Appendix B: PPG Exact Mass Table

Appendix B: PPG Exact Mass Table

The following table contains the exact monoisotopic masses and charged species (positive and negative) observed with the polypropylene glycol (PPG) calibration solutions. The masses and ions were calculated using the formula M = H[OC3H6]nOH, while the positive ion MS/MS fragments used the formula [OC3H6]n(H+). In all calculations, H = 1.007825, O = 15.99491, C = 12.00000, and N = 14.00307.

Note: When performing calibration with the PPG solutions, ensure that the correct isotope peak is used.

n Exact Mass (M) (M = NH4)+ MS/MS

Fragments (M = 2NH4)2+ (M = COOH)-

1 76.052 94.087 59.0 56.061 121.0502 134.094 152.129 117.1 85.082 179.0923 192.136 210.171 175.1 114.102 237.1344 250.178 268.212 233.2 143.123 295.1765 308.220 326.254 291.2 172.144 353.2186 366.262 384.296 349.2 201.165 411.2597 424.304 442.338 407.3 230.186 469.3018 482.346 500.380 465.3 259.207 527.3439 540.388 558.422 523.4 288.228 585.38510 598.430 616.464 581.4 317.249 643.42711 656.471 674.506 639.4 346.270 701.46912 714.513 732.548 697.5 375.291 759.51113 772.555 790.590 755.5 404.312 817.55214 830.597 848.631 813.6 433.333 875.59415 888.639 906.673 871.6 462.354 933.63616 946.681 964.715 929.7 491.373 991.67817 1004.723 1022.757 987.7 520.396 1049.72018 1062.765 1080.799 1045.7 549.417 1107.76219 1120.807 1138.841 1103.8 578.438 1165.80420 1178.849 1196.883 1161.8 607.459 1223.84521 1236.890 1254.925 1219.9 636.480 1281.88722 1294.932 1312.967 1277.9 665.501 1339.92923 1352.974 1371.009 1335.9 694.521 1397.97124 1411.016 1429.050 1394.0 723.542 1456.01325 1469.058 1487.092 1452.0 752.563 1514.05526 1527.100 1545.134 1510.1 781.584 1572.097

55

Appendix B: PPG Exact Mass Table Q Trap LC/MS/MS Hardware Manual

27 1585.142 1603.176 1568.1 810.605 1630.13828 1643.184 1661.218 1626.2 839.626 1688.18029 1701.226 1719.260 1684.2 868.647 1746.22230 1759.268 1777.302 1742.2 897.668 1804.26431 1817.309 1835.344 1800.3 926.689 1862.30632 1875.351 1893.386 1858.3 955.710 1920.34833 1933.393 1951.428 1916.4 984.731 1978.39034 1991.435 2009.469 1974.4 1013.752 2036.43135 2049.477 2067.511 2032.5 1042.773 2094.47336 2107.519 2125.553 2090.5 1071.794 2152.51537 2165.561 2183.595 2148.5 1100.815 2210.55738 2223.603 2241.637 2206.6 1129.836 2268.59939 2281.645 2299.679 2264.6 1158.857 2326.64140 2339.687 2357.721 2322.7 1187.878 2384.68341 2397.728 2415.783 2380.7 1216.899 2442.72442 2455.770 2473.805 2438.7 1245.920 2500.76643 2513.812 2531.847 2496.8 1274.940 2558.80844 2571.854 2589.888 2554.8 1303.961 2616.85045 2629.896 2647.930 2612.9 1332.982 2674.89246 2687.938 2705.972 2670.9 1362.003 2732.93447 2745.980 2764.014 2729.0 1391.024 2790.97648 2804.022 2822.056 2787.0 1420.045 2849.01749 2862.064 2880.098 2845.0 1449.066 2907.05950 2920.106 2938.140 2903.1 1478.087 2965.10151 2978.147 2996.182 2961.1 1507.108 3023.14352 3036.189 3054.224 3019.2 1536.129 3081.18553 3094.231 3112.266 3077.2 1565.150 3139.22754 3152.273 3170.307 3135.2 1594.171 3197.26955 3210.315 3228.349 3193.3 1623.192 3255.31156 3268.357 3286.391 3251.3 1652.213 3313.35257 3326.399 3344.433 3309.4 1681.234 3371.39458 3384.441 3402.475 3367.4 1710.255 3429.43659 3442.483 3460.517 3425.5 1739.276 3487.47860 3500.525 3518.559 3483.5 1768.297 3545.520261 3558.566 3576.601 3541.5 1797.318 3603.56262 3616.608 3634.643 3599.6 1826.339 3661.60463 3674.650 3692.685 3657.6 1855.359 3719.64564 3732.692 3750.726 3715.7 1884.380 3777.687



56

Q Trap LC/MS/MS Hardware Manual Appendix B: PPG Exact Mass Table

65 3790.734 3808.768 3773.7 1913.401 3835.72966 3848.776 3866.810 3831.7 1942.422 3893.77167 3906.818 3924.852 3889.8 1971.443 3951.81368 3964.860 3982.894 3947.8 2000464 4009.85569 4022.902 4040.936 4005.9 2029.485 4067.89770 4080.944 4098.978 4063.9 2058.506 4125.93871 4138.985 4157.020 4122.0 2087.527 4183.98072 4197.027 4215.062 4180.0 2116.548 4242.02273 4255.069 4273.104 4238.0 2145.569 4300.06474 4313.111 4331.145 4296.1 2174.590 4358.10675 4371.153 4389.187 4354.1 2203.611 4416.14876 4429.195 4447.229 4412.2 2232.632 4474.19077 4487.237 4505.271 4470.2 2261.653 4532.23178 4545.279 4563.313 4528.3 2290.674 4590.27379 4603.321 4621.355 4586.3 2319.695 4648.31580 4661.363 4679.397 4644.3 2348.716 4706.35781 4719.404 4737.439 4702.4 2377.737 4764.39982 4777.446 4795.481 4760.4 2406.758 4822.441



57

Appendix B: PPG Exact Mass Table Q Trap LC/MS/MS Hardware Manual

58

Q Trap LC/MS/MS Hardware Manual Appendix C: Scan Parameter Settings

Appendix C: Scan Parameter SettingsQ1 Scan Parameter Settings

Parameter ID

Parameter Name

Parameter Group

Ion Sources

Access Rule

Access ID

Access Name Equation Default (Offset)

Access Min.

Access Max

IS IonSpray Voltage

Source / Gas FNS Operator IS IonSpray Voltage

700 0 3000

IS IonSpray Voltage

Source / Gas IS, TIS Operator IS IonSpray Voltage

5000 (- 4200)

0 5000

NC Nebulizer Current

Source / Gas HN Operator NC Nebulizer Current

2 0 6

TEM Temperature Source / Gas TIS, HN

Operator TEM Temperature 0 0 550

GS1 Gas 1 Source / Gas ALL Operator GS1 Gas 1 20 0 90


CUR Curtain Gas Source / Gas ALL Operator CUR Curtain Gas 20 10 55

CAD Collision Gas Source / Gas ALL Fixed CAD Collision Gas 0

OR Orifice Plate Compound ALL Potential Diff.

DP Declustering Potential

DP = OR 20 0 200

Q0 Focusing Rod Offset

Compound ALL Potential Diff.

EP Entrance Potential

EP = -Q0 * 10 1 12

IQ1 Focusing Lens 1

Compound ALL Param. Dep.

IQ1 = Q0 + offset

–1

ST Prefilter Compound ALL Param. Dep.

ST = Q0 + offset

–5

RO1 Q1 Rod Offset

Resolution ALL Potential Diff.

IE1 Ion Energy 1 IE1 = Q0 – RO1

1 0.5

FI2 Focusing Interface 2

Compound ALL Hidden

RO2 Collision Cell Rod Offset

Compound ALL Fixed RO2 Collision Cell Rod Offset

–30

AF2 Excitation RF Amplitude

Compound ALL Hidden

IQ3 Focusing Lens 3

Compound ALL Fixed IQ3 Focusing Lens 3

–150

RO3 Q3 Rod Offset

Compound ALL Fixed RO3 Q3 Rod Offset

–150

AF3 QTrap RF Amplitude

Compound ALL Hidden

EX Exit Lens Detector ALL Fixed EX Exit Lens –100

EXB Exit Barrier Compound ALL Hidden

DF Deflector Detector ALL Fixed DF Deflector –200

59

Appendix C: Scan Parameter Settings Q Trap LC/MS/MS Hardware Manual

CEM CEM Detector ALL Operator CEM CEM 1800 500 3000

ihe Interface Heater

Source / Gas ALL Operator ihe Interface Heater

1 0 1

C2 Collar 2 Compound ALL Fixed C2 Collar 2 0

C2B Collar 2 Barrier

Compound ALL Hidden

XA3 Trap RF Amplitude (original)

Compound ALL Fixed XA3 Trap RF Amplitude (original)

0

XA2 Excitation Energy (original)

Compound ALL Fixed XA2 Excitation Energy (original)

0

Q1 Scan Parameter Settings (Continued)Parameter ID

Parameter Name

Parameter Group

Ion Sources

Access Rule

Access ID


Access Min.

Access Max

60


.

Q3 Scan Parameter SettingsParameter ID

Parameter Name

Parameter. Group

Ion Source

Access Rule

Access ID Access Name Equation Default (Offset)

Access Min.

Access Max

IS IonSpray

Voltage


700 0 3000

IS IonSpray

Voltage


5000 (-4200)

0 5500

NC Nebulizer

Current

Source / Gas IS, TIS Operator NC Nebulizer Current

2 0 6

TEM Temperature Source / Gas HN Operator TEM Temperature 0 0 550

GS1 Gas 1 Source / Gas TIS, HN

Operator GS1 Gas 1 20 0 90



CAD Collision Gas

Source / Gas ALL Fixed CAD Collision Gas 1



DP = OR 20 0 200




EP = –Q0 10 1 12

IQ1 Focusing Lens 1


IQ1 = Q0 + offset

–1


ST = Q0 + offset

–5

RO1 Q1 Rod Offset


RO1 = Q0 – 1

IQ2 Focusing Lens 2


IQ2 = RO2 + 2

FI2 Focusing

Interface 2

Compound ALL Hidden


Compound ALL Operator RO2 Collision Cell Rod Offset

–20 –142 –1


Compound ALL Hidden

IQ3 Focusing Lens 3


CXP Collision Cell Exit Potential

CXP = RO2 – IQ3

Q3mass 0 58

RO3 Q3 Rod Offset


IE3 Ion Energy 3 IE3 = RO2 – RO3

4 0.5 8


Compound ALL Hidden

EX Exit Lens Detector ALL Fixed EX Exit Lens –100



61





1 0 1



Compound ALL Hidden



0



0

Q3 Scan Parameter Settings (Continued)Parameter ID

Parameter Name

Parameter. Group

Ion Source

Access Rule

Access ID Access Name Equation Default (Offset)

Access Min.

Access Max

62


.

MS/MS Scan Parameter SettingsParameter ID

Parameter Name

Parameter Group

Ion Source

Access Rule

Access ID


Access Min.

Access Max

IS IonSpray Voltage


700 0 3000

IS IonSpray Voltage


5000 (-4200)

0 5500


Source / Gas FNS Operator NC Nebulizer Current

2 0 6

TEM Temperature Source / Gas IS, TIS Operator TEM Temperature 0 0 550

GS1 Gas 1 Source / Gas HN Operator GS1 Gas 1 20 0 90

GS2 Gas 2 Source / Gas TIS, HN

Operator GS2 Gas 2 0 0 90


CAD Collision Gas Source / Gas ALL Simplified CAD Collision Gas medium low high



DP = OR 20 0 200




EP = – Q0 10 1 12

IQ1 Focusing Lens 1


IQ1 = Q0 + offset

–1


ST = Q0 + offset

–5

RO1 Q1 Rod Offset


IE1 Ion Energy 1 IE1 = Q0– RO1

1 0.5 2

IQ2 Focusing Lens 2


CEP Collision Cell Ent. Potential

CEP = Q0 – IQ2

Q1 mass 0 188


Compound ALL Hidden



CE Collision Energy

CE = Q0 – RO2

30 5 130


Compound ALL Hidden

IQ3 Focusing Lens 3


CXP Collision Cell Exit Potential

CXP = RO2 – IQ3

15 0 58

RO3 Q3 Rod Offset


IE3 Ion Energy 3 IE3 = RO2 – RO3

4 0.5 8


Compound ALL Hidden

EX Exit Lens Detector ALL Fixed EX –100




63




1 0 1



Compound ALL Hidden



0



0

MS/MS Scan Parameter Settings (Continued)Parameter ID

Parameter Name

Parameter Group

Ion Source

Access Rule

Access ID


Access Min.

Access Max

64

Q Trap LC/MS/MS Hardware Manual .

.LIT Scan Parameter Settings

Parameter ID

Parameter Name

Parameter Group

Ion Source

Access Rule

Access ID


Access Min.

Access Max

IS IonSpray Voltage

Source / Gas Operator IS IonSpray Voltage

5000 (-4200)

0 5500


Source / Gas Operator NC Nebulizer Current

2 0 6

TEM Temperature Source / Gas Operator TEM Temperature 0 0 550

GS1 Gas 1 Source / Gas Operator GS1 Gas 1 20 0 90

GS2 Gas 2 Source / Gas Operator GS2 Gas 2 0 0 90

CUR Curtain Gas Source / Gas Operator CUR Curtain Gas 20 10 55

CAD Collision Gas Source / Gas Simplified CAD Collision Gas medium

low high

OR Orifice Plate Compound Potential Diff.


DP = OR 20 0 200


Compound Potential Diff.

Q0 Entrance Potential

EP = -Q0* 10 1 12

IQ1 Focusing Lens 1

Compound Hidden

ST Prefilter Compound Parameter Dep.

ST = Q0 + offset

–5

RO1 Q1 Rod Offset

Resolution Potential Diff.

IE1 Ion Energy 1 IE1 = Q0 – RO1

1 0.5 2

IQ2 Focusing Lens 2

Compound Hidden



CEP Collision Cell Ent. Potential

CEP = Q0 – FI2

Q1 mass

0 188



CE Collision Energy

CE = Q0 – RO2

30 5 130


Compound Operator AF2 Q2 Auxiliary RF Amplitude

0 0 20

IQ3 Focusing Lens 3

Compound Hidden

RO3 Q3 Rod Offset

Compound Hidden


Compound Operator AF3 Q3 Auxiliary RF Amplitude

Q3 mass and scan speed

0 5

EX Exit Lens Compound Hidden Q3 mass and scan speed

0 5

EXB Exit Barrier Compound Operator EXB Exit Barrier 4 2 15

65

. Q Trap LC/MS/MS Hardware Manual

DF Deflector Detector Fixed DF Deflector –400

CEM CEM Detector Operator CEM CEM 1800 500 3000


Source / Gas Operator ihe Interface Heater

1 0 1

C2 Collar 2 Compound ALL Hidden 0


Compound ALL Operator C2B Collar 2 Barrier


Compound ALL Hidden Q3 mass and scan

-500 500


Compound ALL Hidden 0

LIT Scan Parameter Settings (Continued)Parameter ID

Parameter Name

Parameter Group

Ion Source

Access Rule

Access ID


Access Min.

Access Max

66

Q Trap LC/MS/MS Hardware Manual Appendix D: Consumables

Appendix D: Consumables

The following is a list of consumables for the Q Trap LC/MS/MS system:

Item Part No. Description 1 011556 Tube, Teflon 1/32"ID × 1/16"OD 2 011555 Tube, Teflon 1/16" × .007"ID 3 016316 Tube 1/16 OD × .005 BORE 4 015968 Fitting, Union 1/16, .010 ORIF 5 012627 Fitting, Rheflex 1/16 6 012637 Ferrule, Rheflex 1/16 7 014629 Tool, Swab, Anti-Static Foam 8 020783 Fuse, 2.5A 250V 5 × 2 OMM SB9 017363 Filter, Intake Air

10 010615 Syringe, Gas Tight, 1.0 mL11 024676 Syringe, Gas Tight, 10 uL12 010613 Needle, Syringe, Removable13 010616 Needle, Syringe, Luer Hub, 2"14 001935 Tube, TFL 1/8 OD × 0.15 WALL15 019670 Tool, Swab Anti-Static Foam16 016492 Cap, Tubing Fused SIL 0.16 OD17 010352 Fuse, 4A 250V 5x20 SLO-BLO18 019667 Tube, Loop Diverter Valve 10UL19 019668 Fitting, Adapter Syringe 1/16"20 011281 Tool, Tube Cutter, Peek

67

Appendix D: Consumables Q Trap LC/MS/MS Hardware Manual

68

Q Trap LC/MS/MS Hardware Manual Appendix E: Sample Experiments

Appendix E: Sample Experiments

The examples below are sample experiments you can set up to familiarize yourself with the function of the Q Trap LC/MS/MS system. The examples indicate the procedures you should follow for creating small and large molecule experiments.

Small Molecules: EPI Use the example experiment below as a guide when creating EPI experiments.

To create an EPI scan1. On the Analyst Navigation bar, click Acquire, and then double-click Build

Acquisition Method.

The Acquisition Method Editor appears.

2. In the Acquisition method pane, highlight Period, right-click, and then select Add Experiment

3. Click the MS tab.

4. In the Scan Type list, select Enhanced Product Ion (EPI).

5. Specify parameters as shown in the example below.

6. Click Edit Parameters.

The Period/Experiment/Parameter Table dialog box appears.

7. Click the Source/Gas tab.

The dialog box below shows typical values for the parameters. You may have to alter these parameters for optimal performance based on the LC flow rate. For example, if

69

Appendix E: Sample Experiments Q Trap LC/MS/MS Hardware Manual

you are not in Simplified mode, you will need to set the CAD gas high enough to achieve a base pressure of 4e-5 torr.

8. Click the Compound tab.

The dialog box below shows typical values for the parameters.

9. Click OK.

10. Click the Advanced MS tab.

Specify parameters as shown in the following figure. You may have to alter these parameters for optimal performance. For example, a resolution of 1000 amu/s will give you high sensitivity, however, if you want to see more points across a chromatographic peak, set the resolution to 4000 amu/s.

70


To increase sensitivity you can select Q0 Trapping or increase the Trap fill time. Be aware that too much ion current can saturate the ion trap, cause space charge, and result in poor resolution of peaks.

Small Molecules: MS3 Use the MS/MS/MS (MS3) scan to gather further information about a fragment ion. This is useful in determining the structure of metabolites, for example, where the modification occurred in the molecule. You can use the example experiment below as a guide when creating MS3 experiments.

To create an MS3 scan1. On the Navigation bar, click Acquire, and then double-click Build Acquisition

Method.




4. In the Scan Type list, select MS/MS/MS (MS3).

5. Specify parameters as shown in the example below. You may want to alter these parameters for optimal performance. For example, if you select No Fragmentation, the second precursor ion is isolated but not fragmented. Therefore the spectrum will display only the second precursor ion.

71







The following dialog box shows typical values for the parameters. You may want to alter these parameters for optimal performance. For example, you should set CE to give the best results for the second precursor ion. You can also optimize AF2. A good starting point for AF2 is 60.

72


9. Click OK.


Specify parameters as shown in the example below. You may have to alter these parameters for optimal performance. For example, you can increase the Excitation Time to increase the energy applied to the second precursor ion.

To increase the sensitivity of the scan, set the Q1 Resolution to Low or Open. The Q3 Resolution determines the isolation window of the second precursor ion. Set Q3 Resolution to Open to see the entire isotope pattern in the spectrum.

73


Large Molecules: Protein Identification—Tryptic Digest

The QTrap LC/MS/MS system provides better sensitivity, higher resolution and faster scan speeds than a conventional triple quadrupole mass spectrometer when analyzing proteolytic digests. This new technology, integrated with information dependent data acquisition (IDA), provides a powerful tool to design specific experiments based on each protein applications. You can use the example experiment below as a guide when creating your own experiments.

This example requires you to create multiple scans. You must perform the following steps in order:

1. Create an Enhanced MS scan.

2. Create an Enhanced Resolution scan.

3. Create an IDA experiment.

4. Create an Enhanced Product Ion scan.

5. Submit the samples.

6. View the results in Explore mode.

Step 1: Create an EMS scan1. On the Analyst Navigation bar, click Acquire, and then double-click Build

Acquisition Method.




4. In the Scan Type list, select Enhanced MS (EMS).

5. Specify parameters as shown in the following example.


74







9. Click OK.


Specify parameters as shown in the dialog box below.

75


Step 2: Create an ER scan1. In the Acquisition method pane, highlight Period, right-click, and then select Add

Experiment


3. In the Scan Type list, select Enhanced Resolution (ER).



Specify parameters as shown in the figure below.

76


Step 3: Create an IDA experiment1. In the Acquisition method pane, highlight Period, right-click, and then select Add

IDA Criteria Level.

2. Click the IDA First Criteria Level tab.

3. Set a range for the most intense peaks.


Step 4: Create an EPI scan1. In the Acquisition method pane, highlight Period, right-click, and then select Add

Experiment


77


3. In the Scan Type list, select Enhanced Product Ion (EPI).



Specify parameters as shown in the figure below.

6. On the toolbar, click the Save button to save the acquisition method.

Step 5: Submit the samples1. On the Navigation bar, click Acquire, and then double-click Build Acquisition

Batch.

2. Click the Sample tab.

3. Click Add Set.

The Add Samples dialog box appears.

78


4. Select an Acquisition method from the list.

5. Click the Submit tab.

6. Click Submit.

7. From the Acquire menu, click Start Sample.

Step 6: View the results in Explore mode1. On the Navigation bar, click Explore, and then double click Open Data File.

The Select Sample dialog box appears.

2. Click the data file and sample you just created, and then click OK.

The data is displayed in four spectra: TIC, EMS, ER, EPI.

79


80

Q Trap LC/MS/MS Hardware Manual Index

IndexA

anti-suckback valve 28

CCAD process 8collision cell 9, 36, 37collisionally activated dissociation 8components of the system electronics

box 40compound-specific parameters,

setting instrument parameters 42Curtain gas 23–24, 37

turning on the Curtain gas 43Curtain gas interlocks 33

DDC distribution board 39deflector voltage 39differentially pumped interface 23–24

Eelucidation, structural 9enhanced modes 11enhanced product ions scans 6entrance optics 24

Ffragmenting

precursor ions 7, 37product ions 8

Ggas ballast valve 29gas control assembly 25–26gas control sequence 33gas control system 31gas curtain interface 22gas flow controllers 31

HHeated Nebulizer inlet 40

Heated Nebulizer ion source 6, 17Heater gas flow 32

Iinstrument

warming up 44instrument parameters 41instrument-specific parameters,

setting 42interface vacuum 28interquad lenses 10ion count 9ion optics path 37ion path chamber 35ion source

Heated Nebulizer 17ions

fragmentation 9precursor 8, 9, 10product 8, 9

Llenses, interquad 10Linear Ion Trap 36linear ion trap mode 5

Mmaintenance checklist 53mass filter rail 35mass filters 35mass spectrometry

principles 7principles of MS 7–8principles of MS/MS 8–10principles of MS/MS/MS 10–11single quadrupole 7triple quadrupole 8, 9

modesenhanced 11

NNebulizer gas flow 32

Pparameters

compound-specific 41instrument-specific 41source-specific 41

power distribution module, AC, DC distribution boards 39

powering up the mass spectrometer 43

PPG Exact Mass Table 55precursor 6precursor ions 8, 10precursor ions, fragmenting 7principles, mass spectrometry 7product ions 8product ions, fragmenting 8pump-down sequence 46pumping down 46pumping system 25

QQ Trap

enhanced scans 6quadrupole 36quadrupole, single 7quadrupoles, RF-only 9, 35–36

Rremoving

instrument covers 48ion source 51

RF-onlyquadrupoles 9, 35–36

roughing pump 28roughing pump interlock 34

Ssafe disposal, source exhaust gases 19safety interlocks, vacuum control

system 33sample introduction system 14setting parameters

compound-specific 42instrument-specific 42

81

Index Q Trap LC/MS/MS Hardware Manual

source-specific 43shutting down the mass spectrometer

44source exhaust gases

safe disposal 19toxic gases 19

source exhaust system, overview 19structural elucidation 9support, technical 3system electronics 48systems electronic box, components

of 40

Ttables, PPG Exact Mass 55technical support 3toxic gases

source exhuast gases 19turbo pump 24–27, 33, 43, 46turbo pump controllers 25–26TurboIonSpray ion source 15

Vvacuum control system 24vacuum control system, safety

interlocks 33vacuum feedthroughs 37vacuum gauge 24, 29–30, 46, 47vacuum gauge controller 30–31vacuum interface 22, 24, 28vacuum off sequence 25, 31–33vacuum system 21, 44

Wwarming up instrument 44work flow process 41

82

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