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Introduction to Mass Spectrometry March 2008 Introduction to Mass Spectrometry March 2008

What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! 0 units

What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! 12 units

What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! 12 units 8 9 10 11 12 13 14 15 16

What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! 14 units 8 9 10 11 12 13 14 15 16

What is a Mass Spectrometer? 12 units Number of counts A Mass Spectrometer is What is a Mass Spectrometer? 12 units Number of counts A Mass Spectrometer is a machine that weighs molecules ! 8 9 10 11 12 13 14 15 16 mass

Outline • Basic Chemistry • Analytical Chemistry • Mass Spectrometry – Types of Ion Outline • Basic Chemistry • Analytical Chemistry • Mass Spectrometry – Types of Ion Sources • EI, CI, ESI, APCI, APPI, MALDI – Types of MS • Ion Traps, Quads, FT-ICR, TOF, Sector • MS/MS • Performance Comparisons – Market Segments

Basic Chemistry • Everything is made of Atoms – Atoms are made of protons, Basic Chemistry • Everything is made of Atoms – Atoms are made of protons, neutrons, and electrons – Many atoms together make up molecules ATOM U

Carbon Nitrogen Oxygen Hydrogen Carbon Nitrogen Oxygen Hydrogen

Carbon Atom 6 protons (+) 6 neutrons 6 electrons(-) Carbon Atom 6 protons (+) 6 neutrons 6 electrons(-)

Carbon Carbon

More Carbon • 6 protons (1 mass unit each) + 6 neutrons (1 mass More Carbon • 6 protons (1 mass unit each) + 6 neutrons (1 mass unit each) = 12 mass units – Electrons are negligible ( 1/3600 of mass unit) • Some carbon (about 1%) has 7 neutrons so weigh 13 units

12. 00 x 99%+13. 00 x 1% = 12. 01 amu 12. 00 x 99%+13. 00 x 1% = 12. 01 amu

But how much does an atom weigh ? • It was found that 12 But how much does an atom weigh ? • It was found that 12 grams of carbon contains 6. 02 x 1023 atoms of carbon. ( 1023 seconds have not elapsed since the beginning of time !) • So one atom of carbon weighs 1. 99 x 10 -23 grams !

Caffeine C 8 H 10 N 4 O 2 Total Mass 194 Daltons 3. Caffeine C 8 H 10 N 4 O 2 Total Mass 194 Daltons 3. 22 x 10 -22 grams

Caffeine C 8 H 10 N 4 O 2 Total Mass 194 Daltons 3. Caffeine C 8 H 10 N 4 O 2 Total Mass 194 Daltons 3. 22 x 10 -22 grams So we must devise a machine which can measure ~ 10 -22 grams.

Analytical Chemistry Instrumental Methods Mass Spectrometry Spectroscopy Optical Emission NMR Microwave Optical Absorption Chemical Analytical Chemistry Instrumental Methods Mass Spectrometry Spectroscopy Optical Emission NMR Microwave Optical Absorption Chemical Methods ØTitration ØGravimetric Analysis ØSolution Chemistry ØFT-ICR ØTOF ØQuadrupole ØIon Trap ØLinear Trap ØMagnetic Sector

3 Elements to Mass Spectrometry (J. J. Thomson ~ 1910) Gas Phase/ Ionize Separate 3 Elements to Mass Spectrometry (J. J. Thomson ~ 1910) Gas Phase/ Ionize Separate Based on Mass/Charge Sample Why Ionize ? Difficult to manipulate neutral particles on molecular scale. If they are charged, then we can use electric fields to move them around. Detector

3 Elements to Mass Spectrometry (J. J. Thomson ~ 1910) Gas Phase/ Ionize Separate 3 Elements to Mass Spectrometry (J. J. Thomson ~ 1910) Gas Phase/ Ionize Separate Based on Mass/Charge Sample • • • Electron Impact (EI) Chemical Ionization (CI) Electrospray (ESI) Atmospheric Pressure Chemical Ionization (APCI) Photo-ionization (APPI) Matrix Assisted Laser Desorption and Ionization (MALDI) Detector

3 Elements to Mass Spectrometry (J. J. Thomson ~ 1910) Gas Phase/ Ionize Separate 3 Elements to Mass Spectrometry (J. J. Thomson ~ 1910) Gas Phase/ Ionize Separate Based on Mass/Charge Sample • Scanning (Filter) – Linear Quadrupole – Sector • Pulsed (Batch) – Ion Trap – FT-ICR – Time-of-Flight Detector

3 Elements to Mass Spectrometry (J. J. Thomson ~ 1910) Gas Phase/ Ionize Separate 3 Elements to Mass Spectrometry (J. J. Thomson ~ 1910) Gas Phase/ Ionize Separate Based on Mass/Charge Sample • • Faraday Cup Discrete Dynode Continuous Dynode Multi-channel Plate Detector

3 Elements to Mass Spectrometry (J. J. Thomson ~ 1856 -1940) Gas Phase/ Ionize 3 Elements to Mass Spectrometry (J. J. Thomson ~ 1856 -1940) Gas Phase/ Ionize Separate Based on Mass/Charge Sample So, we could come up with 6 x 5 x 4 = 120 Unique Mass Spectrometers. In reality, not all combinations make sense, but many extra “hybrid” MS systems have value. For example Q-TOF’s and LT-FT-ICR Detector

6 Types of Ion Sources 6 Types of Ion Sources

Ion Source Depends on Sample Solid Sample Liquid Sample Make into Solid ? Make Ion Source Depends on Sample Solid Sample Liquid Sample Make into Solid ? Make into Solution ? APCI APPI Turn into Gas? Chemical Properties of analyte in gas phase ? Chemical Properties of analyte in solution phase ? MALDI Gas Sample ESI CI EI

Polarity, MW and Volatility Polarity, MW and Volatility

Polarity, MW and Volatility Caffeine Polarity, MW and Volatility Caffeine

Gas Phase Ionization • EI and CI are gas phase ionization techniques • Sample Gas Phase Ionization • EI and CI are gas phase ionization techniques • Sample is heated to cause volatilization • The molecule must have a low enough MW and polarity so that: TBoil< TDecomposition

Electron Impact e- e- e- M M(g) + e- M+(g) + 2 e. This Electron Impact e- e- e- M M(g) + e- M+(g) + 2 e. This reaction creates the molecular ion so is very useful. However, the excess energy from the electron cause the molecular ion to fall apart:

Neutral Molecule IP 2 Excess Energy get redistributed throughout ion to cause fragmentation. s Neutral Molecule IP 2 Excess Energy get redistributed throughout ion to cause fragmentation. s 1 IP s 0 s 1 s 0 Ionized Molecule

Electron Impact e- e- e- M M(g) + e- M+(g) + 2 e. M+(g) Electron Impact e- e- e- M M(g) + e- M+(g) + 2 e. M+(g) A+Fragment 1 (g) + BFragment 2 (g) • Electron energy is chosen by compromise. • Fragment Information is useful. It can help structural determination. However, many ions produce only fragments with no molecular ion remaining. Molecular ion often very unstable. • 70 e. V “Classical Spectra” to be used for comparisons

MW 194 MW 194

109 m/z 109 m/z

55 m/z 55 m/z

Chemical Ionization • EI is not appropriate for some molecules (it causes too much Chemical Ionization • EI is not appropriate for some molecules (it causes too much fragmentation) • Instead, ionize a reagent gas (by EI) then react it with a analyte molecules • Typically use methane or ammonia for reagent gas

CI: Form Reagent Ions First • For Example - Methane CI 1. electron ionization CI: Form Reagent Ions First • For Example - Methane CI 1. electron ionization of CH 4: • CH 4 + e- CH 4+ + 2 e– Fragmentation forms CH 3+, CH 2+, CH+ 2. ion-molecule reactions create stable reagent ions: • • CH 4+ + CH 4 CH 3 + CH 5+ CH 3+ + CH 4 H 2 + C 2 H 5+ – CH 5+ and C 2 H 5+ are the dominant methane CI reagent ions

Methane CI Reagent Ions – Ions at m/z 17, 29, and 41 are from Methane CI Reagent Ions – Ions at m/z 17, 29, and 41 are from methane; • H 3 O+ is also formed from water vapor in the vacuum system

Reagent Ions React with Analytes • Several Types of Reactions May Occur – Form Reagent Ions React with Analytes • Several Types of Reactions May Occur – Form Pseudomolecular Ions (M+1) – CH 5+ + M CH 4 + MH+ – M+1 Ions Can Fragment Further to Produce a Complex CI Mass Spectrum – Form Adduct Ions – C 2 H 5+ + M [M + C 2 H 5]+ – C 3 H 5+ + M [M + C 3 H 5]+ M+29 Adduct M+41 Adduct – Molecular Ion by Charge Transfer – CH 4+ + M M+ + CH 4 – Hydride Abstraction (M-1) – C 3 H 5+ + M C 3 H 6 + [M-H]+ » Common for saturated hydrocarbons

EI Spectrum of Cocaine • Extensive Fragmentation • Molecular Ion is Weak at m/z EI Spectrum of Cocaine • Extensive Fragmentation • Molecular Ion is Weak at m/z 303

Methane CI of Cocaine Pseudo molecular Ion and Fragment Ions Methane CI of Cocaine Pseudo molecular Ion and Fragment Ions

Isobutane CI of Cocaine • Soft Reagent - Less Fragmentation Isobutane CI of Cocaine • Soft Reagent - Less Fragmentation

Polarity, MW and Volatility Polarity, MW and Volatility

Liquid Techniques • Depending on solvent composition and molecular properties either – APPI – Liquid Techniques • Depending on solvent composition and molecular properties either – APPI – ESI – APPI

APPI APPI

APPI • Lamp Wavelength chosen to only excite analytes not solvent/background – Low amount APPI • Lamp Wavelength chosen to only excite analytes not solvent/background – Low amount of photo dissociation results • New technique with few novel applications • Less universal than electrospray

APCI Principles • Rapidly vaporize entire liquid flow • Ionize solvent molecules in corona APCI Principles • Rapidly vaporize entire liquid flow • Ionize solvent molecules in corona discharge • CI process ionizes sample molecules • Positive mode: proton transfer or charge exchange • Negative mode: proton abstraction or electron capture

APCI – Cut Away View APCI – Cut Away View

What applications need APCI? • APCI works well for small molecules that are moderately What applications need APCI? • APCI works well for small molecules that are moderately polar to non-polar • APCI works well for samples that contain heteroatoms • Avoid samples that typically are charged in solution • Avoid samples that are very thermally unstable or photosensitive

Why Electrospray ? • Most Samples are delivered as liquids. – GC analysis requires Why Electrospray ? • Most Samples are delivered as liquids. – GC analysis requires heating sample to cause evaporation – Ionization occurs through electron impact or chemical reaction – Not all analytes are thermally stable • Electrospray was designed to provide a gentle method of creating gas phase ions

Taylor Cone Three Step Process 1) Droplet formation Electrospray process does not ionize samples Taylor Cone Three Step Process 1) Droplet formation Electrospray process does not ionize samples ! 2) Droplet Shrinkage 3) Gaseous Ion Formation

 • Solutions delivered to the tip of the electrospray capillary experience the electric • Solutions delivered to the tip of the electrospray capillary experience the electric field associated with the maintenance of a high potential. • Assuming a potential gradient, positive ions will accumulate at the surface. • Positively Charged Ions will “bud” off the surface when the applied electrostatic force is bigger than the surface tension.

Assisted Electrospray Low Voltage (0. 1 kv) Low Voltage (0. 5 kv) High Voltage Assisted Electrospray Low Voltage (0. 1 kv) Low Voltage (0. 5 kv) High Voltage (5 kv) MS LC Column Flow Drying Gas Nebulizing Gas

MALDI • • • Matrix Assisted Laser Desorption Ionization Analyte co-deposited with Matrix Laser MALDI • • • Matrix Assisted Laser Desorption Ionization Analyte co-deposited with Matrix Laser excites matrix which transfers energy to analyte Produces singly charged species Typically used for large biomolecules / polymers MALDI is a high mass/pulsed source so usually combined with TOF

5 Types of Mass Spectrometers 5 Types of Mass Spectrometers

5 Types of Mass Spectrometers • Scanning (Filter) – Linear Quadrupole – Sector ( 5 Types of Mass Spectrometers • Scanning (Filter) – Linear Quadrupole – Sector ( Separation in Space) • Pulsed (Batch) – Ion Trap – FT-ICR – Time-of-Flight ( Separation in Time)

Basics of Ion Physics m – mass a – acceleration B – Magnetic Field Basics of Ion Physics m – mass a – acceleration B – Magnetic Field q – charge E - electric field F – Force K. E. – kinetic energy V – electric potential v - velocity

Combine 1 st two equations Combine 1 st two equations

We can measure this. We can control this. (volts/meter) We can measure this. We can control this. (volts/meter)

We can measure this. We can deduce This ! We can control this. (volts/meter) We can measure this. We can deduce This ! We can control this. (volts/meter)

-40 V 0 V + 1 meter 0 V + -40 V -40 V 0 V + 1 meter 0 V + -40 V

Time of Flight MS Time of Flight MS

Time of Flight (TOF) m/z t 2 + Very high mass range + Both Time of Flight (TOF) m/z t 2 + Very high mass range + Both high resolution and high sensitivity + Mass accuracy + High scan speed + Mechanically simple - High vacuum critical - Demanding high voltage/ pulsed/ high precision electronics - Expensive Bruker, Waters-Micromass, JEOL, Analytica

Time of Flight Time of Flight

SECTOR MS SECTOR MS

MStation™ Double Focusing Magnetic Sector Mass Spectrometer FROM JEOL High resolution (60, 000 at MStation™ Double Focusing Magnetic Sector Mass Spectrometer FROM JEOL High resolution (60, 000 at 10% valley).

ü ü ü Very high reproducibility Best quantitative performance of all mass spectrometer analyzers ü ü ü Very high reproducibility Best quantitative performance of all mass spectrometer analyzers High resolution High sensitivity High dynamic range - Large Expensive Not suited for pulsed sources

FT-ICR FT-ICR

1347. 734 1348. 736 1349. 741 1347. 734 1348. 736 1349. 741

Reserpine Resolution ~ 1200 609 610 607 611 612 Reserpine is used to treat Reserpine Resolution ~ 1200 609 610 607 611 612 Reserpine is used to treat high blood pressure. It works by decreasing your heart rate and relaxing the blood vessels so that blood can flow more easily through the body. It also is used to treat severe agitation in patients with mental disorders

LC/MS/MS with data dependent acquisition using Bruker’s simple, unified Compass software package Exact mass LC/MS/MS with data dependent acquisition using Bruker’s simple, unified Compass software package Exact mass MS analysis to sub-ppm levels for unambiguous determination of elemental chemical composition. Automated software to confirm composition with m/z and isotopic pattern information Exact mass MS(n) capability for detailed structural analysis and peptide sequencing Qh-hybrid along with CID and ECD for “top-down” proteomics (Top↓Pro™) facilitates selected gas phase ion enrichment Extreme resolution capability for direct analysis of complex mixtures (> 600, 000 FWHM) Wide m/z range simultaneous detection of ions (e. g. 100 - 7, 000 m/z) Sub fmol sensitivity

FT-ICR ü ü ü The highest recorded mass resolution of all mass spectrometers Powerful FT-ICR ü ü ü The highest recorded mass resolution of all mass spectrometers Powerful capabilities for ion chemistry and MS/MS experiments Well-suited for use with pulsed ionization methods such as MALDI Non-destructive ion detection; ion remeasurement Stable mass calibration in superconducting magnet FTICR systems • • • Limited dynamic range High Vacuum Demands Subject to space charge effects and ion molecule reactions Many parameters (excitation, trapping, detection conditions) comprise the experiment sequence that defines the quality of the mass spectrum Generally low-energy CID, spectrum depends on collision energy, collision gas, and other parameters

Ion Traps, Transmission Quadrupoles and Linear Traps • Electrodynamic quadrupole fields – Paul (University Ion Traps, Transmission Quadrupoles and Linear Traps • Electrodynamic quadrupole fields – Paul (University of Bonn in 1953 – Nobel Prize 1989) • 3 D and 2 D traps • Created a “high resolution quad” that was 5. 82 m long ! • A quadrupole field is linearly dependant on the coordinate axis • Ions are confined or rejected based on Voltage, Frequency, Dimension, Mass and Charge

Ion Traps and Quads • Traps are Pulsed • Quads are Continuous • Both Ion Traps and Quads • Traps are Pulsed • Quads are Continuous • Both rely time varying electric fields (RF)

+ + - - + + + - - +

+ + - - + + + - - +

+ Splat - - + + Splat - - +

- + + - + - + + - +

- + + - + - + + - +

- + + + - - + + + -

+ - + + + - + +

Ion Trap + Quadrupole Theory • Forces on ion are simple to understand • Ion Trap + Quadrupole Theory • Forces on ion are simple to understand • As always Where Fz = the force in the z direction e = charge on the particle m = mass of the particle a = acceleration Ez= electric field

Ion Motion in an Ion Trap • After several pages of math, we can Ion Motion in an Ion Trap • After several pages of math, we can derive an equation for ion motion as a function of time: • These second order differentials are not trivial to solve. • Mathieu Equation ! ( solved in 1868 , sub type of Hill’s equations) • Graphical Solution – (Slightly different for Traps and Quads because of symmetry. ) NEED SOLUTIONS WHICH ARE BOUND AND STABLE IN TIME

Stable Solutions to the Mathieu Equation For a Quadrupole Stable Solutions to the Mathieu Equation For a Quadrupole

Mathieu Equation for an ion trap Mathieu Equation for an ion trap

Stability Diagram ( Area 1) • Operated in RF only mode • Light ions Stability Diagram ( Area 1) • Operated in RF only mode • Light ions have a higher qz than heavier ions • Ions stable in z axis when qz < 0. 908 • Ions selectively ejected when RF amplitude is raised • Light ions leave first, heavier ions later

Stability Diagram for a Quad Stability Diagram for a Quad

Stability Diagram for a Quad V=200 V U=0 V 200 100 50 Stability Diagram for a Quad V=200 V U=0 V 200 100 50

Stability Diagram for a Quad V=200 V U=50 V 50 100 200 Stability Diagram for a Quad V=200 V U=50 V 50 100 200

Stability Diagram for a Quad 50 100 200 V=200 V U=100 V Stability Diagram for a Quad 50 100 200 V=200 V U=100 V

Stability Diagram for a Quad 50 150 200 V=400 V U=200 V Stability Diagram for a Quad 50 150 200 V=400 V U=200 V

Stability Diagram for a TRAP Quad operates by selectively passing one m/z at a Stability Diagram for a TRAP Quad operates by selectively passing one m/z at a time. Trap operates by collecting all ions simultaneously and then ramping them out one at a time.

Stability Diagram for a Trap V=200 V U=0 V Eject when q=0. 908 200 Stability Diagram for a Trap V=200 V U=0 V Eject when q=0. 908 200 100 50

Stability Diagram for a Trap V=300 V U=0 V Eject when q=0. 908 200 Stability Diagram for a Trap V=300 V U=0 V Eject when q=0. 908 200 100 50

Stability Diagram for a Trap V=400 V U=0 V Eject when q=0. 908 200 Stability Diagram for a Trap V=400 V U=0 V Eject when q=0. 908 200 100 50

Mass Spectrum on a Quad or Trap RF Spectrum Ramp RF (in trap) or Mass Spectrum on a Quad or Trap RF Spectrum Ramp RF (in trap) or ramp RF/DC in Quad

Stability Diagram for a Trap Stability Diagram for a Trap

Potential Well Model Need for helium buffer gas Potential Well Model Need for helium buffer gas

Secular Frequency • Ion Motion in Trap contains many frequency components • These depend Secular Frequency • Ion Motion in Trap contains many frequency components • These depend on a and q parameters – (When q < 0. 40)

m/z= 1500 q = 0. 0605 ω = 16. 7 k. Hz m/z= 500 m/z= 1500 q = 0. 0605 ω = 16. 7 k. Hz m/z= 500 q = 0. 1816 ω = 50. 5 k. Hz m/z= 1000 q = 0. 0908 ω = 25. 1 k. Hz Low Mass Cut Off 100 m/z= 106 q = 0. 850 ω = 301. 9 k. Hz

Varian Eject m/z= 1500 q = 0. 0605 ω = 16. 7 k. Hz Varian Eject m/z= 1500 q = 0. 0605 ω = 16. 7 k. Hz m/z= 500 q = 0. 1816 ω = 50. 5 k. Hz m/z= 1000 q = 0. 0908 ω = 25. 1 k. Hz Low Mass Cut Off 100 m/z= 106 q = 0. 850 ω = 301. 9 k. Hz

Notched Broad Band Waveform Fourier Transform Amplitude frequency Notched Broad Band Waveform Fourier Transform Amplitude frequency

Frequency Notch 180 k. Hz m/z= 1500 q = 0. 0605 ω = 16. Frequency Notch 180 k. Hz m/z= 1500 q = 0. 0605 ω = 16. 7 k. Hz m/z= 500 q = 0. 1816 ω = 50. 5 k. Hz m/z= 1000 q = 0. 0908 ω = 25. 1 k. Hz 240 k. Hz Low Mass Cut Off 100 m/z= 106 q = 0. 850 ω = 301. 9 k. Hz

Practical Mass Spectrometer Load Time Notched Waveform Ion Ejection Dipole Ejection Mass Spectrum Practical Mass Spectrometer Load Time Notched Waveform Ion Ejection Dipole Ejection Mass Spectrum

Ion trap Benefits üHigh sensitivity üMulti-stage mass spectrometry (analogous to FTICR experiments) üCompact mass Ion trap Benefits üHigh sensitivity üMulti-stage mass spectrometry (analogous to FTICR experiments) üCompact mass analyzer üCheap and Easy to build Limitations • Poor quantitation • Poor inherent dynamic range • Subject to space charge effects and ion molecule reactions • Collision energy not well-defined in CID MS/MS • Many parameters (excitation, trapping, detection conditions) comprise the experiment sequence that defines the quality of the mass spectrum

Transmission Quadrupole Mass Spectrometer Benefits üClassical mass spectra üGood reproducibility üRelatively small and low-cost Transmission Quadrupole Mass Spectrometer Benefits üClassical mass spectra üGood reproducibility üRelatively small and low-cost systems üPotentially good conversion efficiency for MS/MS Limitations • Limited resolution • Peak heights variable as a function of mass (mass discrimination). Peak height vs. mass response must be 'tuned'. • Not well suited for pulsed ionization methods • Low-energy collision-induced dissociation (CID) MS/MS spectra in triple quadrupole and hybrid mass spectrometers depend strongly on energy, collision gas, pressure, and other factors.

Linear Trap + Newest Generation MS • Many of the advantages of ion traps, Linear Trap + Newest Generation MS • Many of the advantages of ion traps, without normal trap limitations. • Less Space Charge Problems • MSN • Great loading Efficiency =

MS/MS • In a transmission Quadrupole, MS/MS is done in Space – need three MS/MS • In a transmission Quadrupole, MS/MS is done in Space – need three quads ( Triple Quad) • In an Ion trap MS/MS is done in time. Q 1 Pass only 195 Q 2 RF ONLY -Pass Everything -Collisions with Ar cause fragmentation Q 3 Scan from 100 -195 Look at daughter ions

Triple Quad vs. Ion Trap Triple Quad vs. Ion Trap

Why MS/MS • Unknown Identification • Potentially two compounds of interest have the same Why MS/MS • Unknown Identification • Potentially two compounds of interest have the same mass ( and same retention time) • Quantitation improvements ( background signal reduced)

Problem: Thiabendazole in Grapefruit Extract • Antifungal agent, thiabendazole (TBZ) must be below 10 Problem: Thiabendazole in Grapefruit Extract • Antifungal agent, thiabendazole (TBZ) must be below 10 ppb in the processed grapefruit • Major matrix interferent: Similar retention time Similar spectrum Concentration much greater than TBZ Interferent

Interferent Interferent

MS, MS/MS, and MS/MS/MS of TBZ Matrix peak MS (500 pg) MS/MS (10 pg) MS, MS/MS, and MS/MS/MS of TBZ Matrix peak MS (500 pg) MS/MS (10 pg) 201 80 -220 Matrix peak MS/MS/MS (10 pg) 201 174 65 -220 No matrix peak

Quadrupole 1 MS 1 Select Scanning Quadrupole 2 Collision Cell Quadrupole 3 MS 1 Quadrupole 1 MS 1 Select Scanning Quadrupole 2 Collision Cell Quadrupole 3 MS 1 Scanning Product Ion Scan Select Precursor ion Scanning Neutral Loss Scan

Real Life System Real Life System

Reserpine 609 610 607 611 612 Reserpine is used to treat high blood pressure. Reserpine 609 610 607 611 612 Reserpine is used to treat high blood pressure. It works by decreasing your heart rate and relaxing the blood vessels so that blood can flow more easily through the body. It also is used to treat severe agitation in patients with mental disorders

+15 Cytochrome C – MW 12220 m/z = mass/charge +14 +16 +13 +8 +12 +15 Cytochrome C – MW 12220 m/z = mass/charge +14 +16 +13 +8 +12 +17 +11 +10 +9 +7

Market Segments and Where Varian Sits Market Segments and Where Varian Sits

GC/MS mass analyzer type GC/MS Initial Sales $280 M TOF $7 M Sector $5 GC/MS mass analyzer type GC/MS Initial Sales $280 M TOF $7 M Sector $5 M Ion Trap $50 M Triple Quadrupole $ 14 M Single Quadrupole $204 M

LC/MS mass analyzer type 2004 LC/MS Initial Sales $698 M Q-TOF $128 M Sector/FTICR LC/MS mass analyzer type 2004 LC/MS Initial Sales $698 M Q-TOF $128 M Sector/FTICR $30 M Single Quadrupole $114 M API TOF $65 M Ion Trap $140 M Triple Quadrupole $221 M

Agilent Bruker Single Quad JEOL Micro Mass 1 2 Sector 3 FT-ICR 1 1 Agilent Bruker Single Quad JEOL Micro Mass 1 2 Sector 3 FT-ICR 1 1 1 2 Triple Quad 3 D Trap 4 4 1 1 1 2 3 1 3 2 Linear Trap TOF Sciex Thermo Varian 1 1 1 TOF/TOF 2 1 2 2 Q-TOF 1 1 TOTAL (LC/MS) 5 10 4 4 1 11 6 10 3

The High-end LC/MS Vendors High-end LC/MS Vendor Market Share High-end TQ (55%) Waters, Thermo, The High-end LC/MS Vendors High-end LC/MS Vendor Market Share High-end TQ (55%) Waters, Thermo, ABI High-end Ion Traps (23%) Bruker/Agilent, Thermo LTQ -Thermo LC-TOF, TOF-TOF, QTOF (13%) Q-Trap (5%) Magnetic sector (4%)

Markets served by the high-end LC/MS Total Market $330 M Academic, 40 M (5%) Markets served by the high-end LC/MS Total Market $330 M Academic, 40 M (5%) Food/AG, 14 M (15%) Indep. Test 16 M (6%) Varian participates in less than 25% of the market, with a 1% overall market share

What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules What is a Mass Spectrometer? A Mass Spectrometer is a machine that weighs molecules ! (by measuring the mass to charge ratio of ions) Source Dispersion Detector EI CI ESI APCI APPI MALDI TOF FT-ICR Sector Quad Trap Faraday Cup Channeltron MCP