Introduction to Mass Spectrometry March 2008 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 ! 12 units 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 ! 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 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 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, neutrons, and electrons – Many atoms together make up molecules ATOM U Carbon Nitrogen Oxygen Hydrogen Carbon Atom 6 protons (+) 6 neutrons 6 electrons(-) Carbon 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 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 ! O Caffeine CH3 H3C N N H O N CH3 N C8H10N4O2 Total Mass 194 Daltons 3.22x10-22 grams O Caffeine CH3 H3C N N H O N CH3 N C8H10N4O2 Total Mass 194 Daltons 3.22x10-22 grams So we must devise a machine which can measure ~ 10-22 grams. Analytical Chemistry Instrumental Methods Mass Spectrometry Spectroscopy Chemical Methods Titration Gravimetric Analysis Solution Chemistry Optical Emission NMR Microwave Optical Absorption FT-ICR TOF Quadrupole Ion Trap Linear Trap Magnetic Sector 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 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 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 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 Separate Based on Mass/Charge Sample So, we could come up with 6x5x4 = 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 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 Caffeine 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) + 2eThis reaction creates the molecular ion so is very useful. However, the excess energy from the electron can cause the molecular ion to fall apart: Neutral Molecule IP2 Excess Energy get redistributed throughout ion to cause fragmentation. s1 IP s0 s1 s0 Ionized Molecule Electron Impact e- e- e- M A+ B M(g) + (g) + M+(g) A+Fragment 1 (g) + BFragment 2 (g) e- M+ 2e- •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 eV “Classical Spectra” to be used for comparisons O MW 194 CH3 H3C N N H O N CH3 N O CH3 H3C N N H O N CH3 N 109 m/z O CH3 H3C N N H O N CH3 N 55 m/z 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 of CH4: • CH4 + e- CH4+ + 2e– Fragmentation forms CH3+, CH2+, CH+ 2. ion-molecule reactions create stable reagent ions: • • CH4+ + CH4 CH3 + CH5+ CH3+ + CH4 H2 + C2H5+ – CH5+ and C2H5+ are the dominant methane CI reagent ions Methane CI Reagent Ions – Ions at m/z 17, 29, and 41 are from methane; • H3O+ is also formed from water vapor in the vacuum system Reagent Ions React with Analytes • Several Types of Reactions May Occur – Form Pseudomolecular Ions (M+1) – CH5+ + M CH4 + MH+ – M+1 Ions Can Fragment Further to Produce a Complex CI Mass Spectrum – Form Adduct Ions – C2H5+ + M [M + C2H5]+ – C3H5+ + M [M + C3H5]+ M+29 Adduct M+41 Adduct – Molecular Ion by Charge Transfer – CH4+ + M M+ + CH4 – Hydride Abstraction (M-1) – C3H5+ + M C3H6 + [M-H]+ » Common for saturated hydrocarbons EI Spectrum of Cocaine • Extensive Fragmentation • Molecular Ion is Weak at m/z 303 Methane CI of Cocaine Pseudo molecular Ion and Fragment Ions Isobutane CI of Cocaine • Soft Reagent - Less Fragmentation Polarity, MW and Volatility Liquid Techniques • Depending on solvent composition and molecular properties either – APPI – ESI – APPI APPI 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 discharge • CI process ionizes sample molecules • Positive mode: proton transfer or charge exchange • Negative mode: proton abstraction or electron capture APCI – Cut Away View 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 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 ! 2)Droplet Shrinkage 3)Gaseous Ion Formation •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 (5 kv) MS LC Column Flow Drying Gas Nebulizing Gas 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 • Scanning (Filter) – Linear Quadrupole – Sector ( Separation in Space) • Pulsed (Batch) – Ion Trap – FT-ICR – Time-of-Flight ( Separation in Time) Basics of Ion Physics F ma F qE F qvB 1 2 K .E. mv qV 2 m – mass a – acceleration B – Magnetic Field q – charge E - electric field F – Force K.E. – kinetic energy V – electric potential v - velocity Combine 1st two equations ma qE qE a m We can measure this. We can control this. (volts/meter) qE a m We can measure this. We can control this. (volts/meter) qE a m We can deduce This ! -40V 0V + 1 meter 0V + qE a m -40V Time of Flight MS 1 2 K .E. mv qV 2 m 2Vt 2 q l 2 Time of Flight (TOF) m/z t2 + 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 SECTOR MS 2 mv F qvB r 2 2 m Br q 2v MStation™ Double Focusing Magnetic Sector Mass Spectrometer FROM JEOL High resolution (60,000 at 10% valley). 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 2 mv F qvB r v qB r m 1347.734 1348.736 m Resolution m 1349.741 PROFILE Scan 35 from ...ta sept 24.04\0.01+0.036 extrste m ode 1 609.xm s Spectrum 1A BP 609.50 (1384=100% ) 0.01+0.036 extrste mode 1 609.xms PROFILE 0.472 min. Scans: 3-67 Channel: 1 Ion: 2000 us RIC: 21543 kCounts Reserpine 1.5 Resolution ~ 1200 609 609.50 0.3541 1384 1.0 610 610.45 0.4533 471 0.5 607 611 611.45 0.3952 114 612 0.0 605.0 607.5 610.0 612.5 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 615.0 617.5 m/z 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 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 of Bonn in 1953 – Nobel Prize 1989) • 3D and 2D 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 rely time varying electric fields (RF) + + - - + + + - - + + Splat + - - + - + + - + - + + - + - + + + - + - + + Ion Trap + Quadrupole Theory • Forces on ion are simple to understand • As always Fz eEz ma Where Fz = the force in the z direction e = charge on the particle m = mass of the particle a = acceleration Ez= electric field Fz ma eEz d z FZ maz m 2 e dt z 2 2 d z e2 z FZ ma m 2 2 (U V cos t ) dt ro Ion Motion in an Ion Trap • After several pages of math, we can derive an equation for ion motion as a function of time: d 2 z 2e 2 (U V cos t ) z 0 2 dt ro m d 2r e (U V cos t )r 0 2 2 dt ro m • 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 - 8eU az m 2 ro2 - 4eV qz m 2 ro2 Mathieu Equation for an ion trap 15 10 z stable au 5 z stable 0 5 10 15 20 25 -5 r stable -10 r stable -15 - 16eU az m 2 ro2 - 8eV qz m 2 ro2 qu Stability Diagram ( Area 1) az z 0.1 1.0 0.8 0.6 0.0 0.2 0.4 qz 0 0.4 - 0.1 r - 0.2 0.6 - 0.3 - 0.4 0.8 - 0.5 1.0 - 0.6 0.2 az 0.4 0.6 - 16eU m 2 ro2 0.8 1.0 1.2 1.4 qz - 8eV m 2 ro2 • 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 - 8eU az m 2 ro2 - 4eV qz m 2 ro2 Stability Diagram for a Quad V=200V U=0V 200 - 8eU az m 2 ro2 100 50 - 4eV qz m 2 ro2 Stability Diagram for a Quad V=200V U=50V 50 100 200 - 8eU az m 2 ro2 - 4eV qz m 2 ro2 Stability Diagram for a Quad 50 100 V=200V U=100V 200 - 8eU az m 2 ro2 - 4eV qz m 2 ro2 Stability Diagram for a Quad 50 150 200 - 8eU az m 2 ro2 V=400V U=200V - 4eV qz m 2 ro2 Stability Diagram for a TRAP az z 0.1 1.0 0.8 0.6 0.0 0.2 0.4 qz 0 0.4 - 0.1 Quad operates by selectively passing one m/z at a time. r - 0.2 0.6 - 0.3 - 0.4 0.8 - 0.5 1.0 - 0.6 0.2 - 16eU az m 2 ro2 0.4 0.6 0.8 1.0 1.2 1.4 - 8eV qz m 2 ro2 Trap operates by collecting all ions simultaneously and then ramping them out one at a time. Stability Diagram for a Trap V=200V U=0V Eject when q=0.908 200 - 16eU az m 2 ro2 100 50 - 8eV qz m 2 ro2 Stability Diagram for a Trap V=300V U=0V Eject when q=0.908 200 - 16eU az m 2 ro2 100 - 8eV qz m 2 ro2 50 Stability Diagram for a Trap V=400V U=0V Eject when q=0.908 200 - 16eU az m 2 ro2 - 8eV qz m 2 ro2 100 50 Mass Spectrum on a Quad or Trap RF Spectrum Ramp RF (in trap) or ramp RF/DC in Quad Stability Diagram for a Trap m ? - 16eU az 2 2 m ro - 8eV qz 2 2 m ro Potential Well Model eV 2 D z 2 Dr 4mzo2 2 Need for helium buffer gas Secular Frequency • Ion Motion in Trap contains many frequency components • These depend on a and q parameters – (When q < 0.40) 1 2 q u u au 2 2 2 2 u m/z= 1500 q = 0.0605 ω = 16.7 kHz 0 Low Mass Cut Off 100 m/z m/z= 500 q = 0.1816 ω = 50.5 kHz 0.2 m/z= 1000 q = 0.0908 ω = 25.1 kHz 0.4 0.6 0.8 m/z= 106 q = 0.850 ω = 301.9 kHz 1 Varian Eject m/z= 1500 q = 0.0605 ω = 16.7 kHz 0 Low Mass Cut Off 100 m/z m/z= 500 q = 0.1816 ω = 50.5 kHz 0.2 m/z= 1000 q = 0.0908 ω = 25.1 kHz 0.4 0.6 0.8 m/z= 106 q = 0.850 ω = 301.9 kHz 1 Notched Broad Band Waveform Fourier Transform Amplitude frequency Frequency Notch 180kHz m/z= 1500 q = 0.0605 ω = 16.7 kHz 0 240kHz Low Mass Cut Off 100 m/z m/z= 500 q = 0.1816 ω = 50.5 kHz 0.2 m/z= 1000 q = 0.0908 ω = 25.1 kHz 0.4 0.6 0.8 m/z= 106 q = 0.850 ω = 301.9 kHz 1 Practical Mass Spectrometer Load Time Notched Waveform Ion Ejection Dipole Ejection Mass Spectrum Ion trap Benefits High sensitivity Multi-stage mass spectrometry (analogous to FTICR experiments) Compact mass analyzer Cheap 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 Classical mass spectra Good reproducibility Relatively small and low-cost systems Potentially 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, 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 quads ( Triple Quad) • In an Ion trap MS/MS is done in time. Q1 Pass only 195 Q2 RF ONLY -Pass Everything -Collisions with Ar cause fragmentation Q3 Scan from 100-195 Look at daughter ions Triple Quad vs. Ion Trap 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 ppb in the processed grapefruit • Major matrix interferent: Similar retention time Similar spectrum Concentration much greater than TBZ Interferent Interferent MS, MS/MS, and MS/MS/MS of TBZ Matrix peak MS (500 pg) MS/MS (10 pg) 20180-220 Matrix peak MS/MS/MS (10 pg) 20117465-220 No matrix peak Quadrupole 1 MS 1 Select Scanning Scanning Quadrupole 2 Collision Cell Quadrupole 3 MS 1 Scanning Product Ion Scan Select Precursor ion Scan Scanning Neutral Loss Scan Real Life System PROFILE Scan 35 from ...ta sept 24.04\0.01+0.036 extrste m ode 1 609.xm s Spectrum 1A BP 609.50 (1384=100% ) 0.01+0.036 extrste mode 1 609.xms PROFILE 0.472 min. Scans: 3-67 Channel: 1 Ion: 2000 us RIC: 21543 kCounts Reserpine 1.5 609 609.50 0.3541 1384 1.0 610 610.45 0.4533 471 0.5 607 611 611.45 0.3952 114 612 0.0 605.0 607.5 610.0 612.5 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 615.0 617.5 m/z PROFILE Scan 85 from ...ultip charge ions\4500-cytochrome c-6-17-04.xms Spectrum 1A BP 816.43 (2316=100%) 4500-cytochrome c-6-17-04.xms PROFILE 4.898 min. Scans: 10-160 Channel: 1 Ion: 500 us RIC: 159835 kCounts Cytochrome C – MW 12220 +15 2.5 816.43 0.3790 2316 m/z = mass/charge MW nH nH 2.0 +14 874.45 0.4061 1507 1.5 +16 765.65 0.6099 1186 1.0 +13 941.55 0.4655 693 +8 +12 0.5 +17 1020.13 0.4678 413 +11 +10 1112.72 0.4881 247 721.00 1.3347 238 +9 1223.87 0.6065 183 1529.57 0.5139 456 1359.76 0.5506 236 +7 1747.75 0.7672 180 0.0 500 750 1000 1250 1500 1750 2000 m/z Market Segments and Where Varian Sits GC/MS mass analyzer type GC/MS Initial Sales $280M Sector $5 M TOF $7 M Ion Trap $50 M 2% 3% 18% Triple Quadrupole 4% $ 14 M Single Quadrupole $204 M 73% LC/MS mass analyzer type 2004 LC/MS Initial Sales $698M Q-TOF $128 M Sector/FTICR $30 M 13% Single Quadrupole $114 M 18% 3% API TOF $65 M 8% 33% Ion Trap $140 M 25% Triple Quadrupole $221 M Agilent Bruker Single Quad JEOL Micro Mass 1 Triple Quad Sector 3 FT-ICR 3D Trap 3 2 1 1 1 2 4 4 1 1 1 3 2 1 1 1 TOF/TOF 2 Q-TOF 2 TOTAL (LC/MS) 1 2 Linear Trap TOF Sciex Thermo Varian 5 10 1 4 1 1 2 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, 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 $330M Academic, 40M (5%) Food/AG, 14M (15%) Indep.Test 16M (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 ! (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