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OUTLINE Introduction to Mass Spectrometry Ionization Methods Mass Analyzer Fragmentation and MS Interpretation Chemical Identification Comparison of Physical Properties Elemental Analysis Burn the compound and Boiling Point Melting Point Density Optical rotation Appearance Odor measure the amounts of CO2, H2O and other components that are produced to determine the empirical formula Spectroscopic Methods for Structure Determination Ultraviolet-Visible (UV/Vis) spectroscopy: determination of solutions of transition metal ions and highly conjugated organic compounds Infrared (IR) spectroscopy: Functional groups Mass spectrometry (MS): Molecular mass and formula and structure information Nuclear magnetic resonance (NMR) spectroscopy: Map of carbon-hydrogen framework Definition of Mass Spectrometry Mass spectrometry (MS) : An analytical technique by using mass spectrometry for the determination of the composition of a sample or molecule and elucidation of the chemical structures of molecules, such as peptides and other chemical compounds. Mass spectrometry has been described as the smallest scale in the world, not because of the mass spectrometer’s size but because of the size of what it weighs -- molecules. What information can be determined? • Molecular weight • Molecular formula (HRMS) • Structure (from fragmentation fingerprint) • Isotopic incorporation / distribution • Protein sequence (MS-MS) MS Principles • Find a way to “charge” an atom or molecule (ionization) • Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer) • Detect ions using microchannel plate or photomultiplier tube General principles on mass spectrometry Computer system Sample introduction Resulting chromatogram Ionization of sample in the ion source Separation of ions by mass analyzer Detector Analysis by GC-MS Mixture GC MS Separation Identification B m/z A m/z C m/z A+B+C B A C 8 Instrumentation Components of a mass spectrometry 1) Inlet system : introduce a very small amount of sample ( micromole or less) into the mass spectrometer, where its components are converted to gaseous ions. 2) Ion source : converts the components of a sample into ions by bombardment with electrons, ions, molecules, or photon. 3) Mass analyzer : separates the analyte ions according to their m/z ratios. 4) Detector : converts the beam of ions into an electrical signal(currents) ; the detector output can be displayed or stored, to yield the mass spectrum. 5) Electronics of power supply and control of the systems. 6) Vacuum systems : maintain low pressures ( 10–5 to 10–8 torr); rotary vacuum oil pump, diffusion pump, turbomolecular pump. Three Components of an MS • A typical mass spectrometer contains – Ionizer – Mass analyzer – Detector • Ion source charges the to-be-measured molecules. – Charge can be negative but often positive. – Two common types: MALDI and ESI. – John B. Fenn & Koichi Tanaka 2002 Nobel Prize in Chemistry for Electrospray and MALDI • Mass analyzer separates ions according to the mass to charge ratio (m/z) of the ions. – Iontrap, TOF, Quadrupole, FTICR. • Detector detects the ions. Ionization Methods • Electron bomb Ionization EI • Chemical Ionization • Matrix Assisted Laser Desorption Ionization • Fast atom bombardment • Electro Spray Ionization Sample introduction / ionization method: Ionization method Typical Analytes Sample Introduction Mass Range Electron Impact (EI) Relatively small volatile GC or liquid/solid probe to 1,000 Daltons Method Highlights Hard method versatile provides structure info Chemical Ionization (CI) Relatively small volatile GC or liquid/solid probe to 1,000 Daltons Soft method molecular ion peak [M+H]+ Liquid Electrospray (ESI) Peptides Proteins nonvolatile Chromatography or syringe to 200,000 Daltons Soft method ions often multiply charged Carbohydrates Organometallics Peptides nonvolatile Sample mixed in viscous matrix to 6,000 Daltons Soft method but harder than ESI or MALDI Peptides Proteins Nucleotides Sample mixed in solid matrix to 500,000 Daltons Soft method very high mass Fast Atom Bombardment (FAB) Matrix Assisted Laser Desorption (MALDI) Electron Ionization ( EI ) Sample is heated and energized by a beam of electrons, usually gives a molecular ion (M+) and a lot of fragments。 H H H C C H H H H H e- + H H H C C H H C C+ H H H H H (M-R2)+ Mass Spectrum (M-R )+ 1 + M+ (M-R3) H C+ H H C H H H Electron Impact Ionization (EI) • Most widely used method • Analytes are bombared with high-energy electrons (usually 70eV) • As a result of collision, an electron is removed from the analytes (M), generating a molecular ion M+ (radical cation) M + e- M+ + 2e- Electron Impact Ionization (EI) • Due to excess internal energy, fragmentation of the molecular ion will occur. • The fragmentation is reproducible and characteristic of the compund. • It is also possible to predict the fragmentation on the basis of chemical structures which is why MS is good tool for structure elucidation of unknown compounds Chemical Ionization (CI) • Softer ionization technique Less fragmentation Easier to find molecular ions. • Two different modes: Negative chemical ionization (NCI) and Positive Chemical Ionization (PCI). • NCI is used for analytes that are able to form stable negative ions, for example samples containing acidic groups or halogens. NCI is often used to analyze pesticides (contains Cl or Br). • PCI is used for samples that can form positive ions (most compounds). Chemical Ionization (CI) • The principle for NCI and PCI is similar: • Reagent gas (usually methane, isobutane or ammonia) is introduced into the source where it is ionized: PCI (simplified): CH4 + e- CH4+ +2eCH4 + CH4+ CH3 + C H5+ NCI: • CH4 + e- CH4- The ionized gas collide with the sample molecules generating a [M+H]+ or [M+H]- ion that is detected: PCI: CH5+ + M [M+H]+ + C H4 NCI: CH4- + M [M+H]- + C H4 Field ionization (FI) Field ionization (FI) is a method that uses very strong electric fields to produce ions from gas-phase molecules. 阳极 + + + + + + d<1mm + + + + + + + 阴极 Field ionization (FI) + + + - - + - + - + + - + + - ++ + + + - + + - + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Matrix Assisted Laser Desorption Ionization (MALDI) sample is co-crystallized with a matrix and then irradiated with laser. MALDI is achieved in two steps. In the first step, the compound to be analyzed is dissolved in a solvent containing in solution small organic molecules, called the matrix. The second step occurs under vacuum conditions inside the source of the mass spectrometer. Properties of MALDI Good solubility Vapour pressure must be sufficiently low to maintain vacuum conditions Viscosity must allow diffusion of the analyte from the bulk to the surface Polar : to solvate and separate preformed ion Less Sensitive to Salts Lower PRACTICAL detection limits Easier to interpret spectra (less multiple charges) Quick and easy Higher mass detection Higher Throughput (>1000 samples per hour) Principle of MALDI MALDI mass spectrometry has become a powerful analytical tool for both synthetic polymers and biopolymers. ElectroSpray Ionization (ESI) Electrospray is abbreviated to ESI ,ample is sprayed out of a narrow nozzle in a high potential field. Generates positive (M+nH)n+ and negative (M - nH)n- ions and almost no fragmentation. Generates multiple charged ions. Electrospray (Detail) Electrospray Ionization • Can be modified to “nanospray” system with flow < 1 mL/min • Very sensitive technique, requires less than a picomole of material • Strongly affected by salts & detergents • Positive ion mode measures (M + H)+ (add formic acid to solvent) • Negative ion mode measures (M - H)- (add ammonia to solvent) Fast atom bombardment ( FAB) Softer than EI and CI. Ions are produced by bombardment with heavy atoms. Gives (M+H)+ ions and litle fragmentation. Good for more polar compounds. Ar + e Ar+ + Ar fast slow Ar+ acceleration (5-15 KeV) Ar + Ar+ + 8 KeV fast slow Properties of FAB Advantages Parent Ion High Mass Compounds (10,000 amu) Thermally Labile Compounds (R.T.) Disadvantages No Fragment Library Solubility in Matrix (MNBA, Glycerol) Quantitation Difficult Needs Highly Skilled Operator Relatively Low Sensitivity Mass analyzers • After analytes have been ionized they are separated according to their mass-to-charge ratio (m/z) in a mass analyzer (mass filter). • Quadrupoles and ion traps are common mass filters in GC-MS systems. • Time of flight (TOF) mass filter is very much used nowadays in LCMS systems. Computer system Sample introduction Ionization of sample Separation of ions by mass analyzer Detector Transport of ions to the mass filter • The ionization takes place in the ion source. • Ions are then transported to the mass filter by focusing lenses. These have a voltage running through them and by either attracting or repelling the ions they guide them into the mass filter. Different Mass Analyzers • Magnetic Sector Analyzer (MSA) – High resolution, exact mass, original MA • Quadrupole Analyzer (Q) – Low (1 amu) resolution, fast, cheap • Time-of-Flight Analyzer (TOF) – No upper m/z limit, high throughput • Ion Trap Mass Analyzer (QSTAR) – Good resolution, all-in-one mass analyzer • Ion Cyclotron Resonance (FT-ICR) – Highest resolution, exact mass, costly Magnetic Sector Analyzer Magnetic sector analyzer – Uses electric and/or magnetic fields to separate ions Advantages Double focusing magnetic sector mass analyzers are the "classical" model against which other mass analyzers are compared. Classical mass spectra Very high reproducibility Best quantitative performance of all MS analyzers High resolution High sensitivity 10,000 Mass Range Linked scan MS/MS does not require another analyzer • Disadvantages Requires Skilled Operator Usually larger and higher cost than other mass analyzers Difficult to interface to ESI Low resolution MS/MS without multiple analyzers • Applications All organic MS analysis methods Accurate mass measurements Quantitation Isotope ratio measurements Time of Flight Analyzer TOF analyzer – ions are accelerated through a flight tube and the time of light to the detector is measured Ions are accelerated and their time of flight to the detector is measured. Principle of TOF Analyzer • Uses a pulse of ion mixtures, not steady stream • Ions accelerated into drift tube by a pulsed electric • field called the ion-extraction field • Drift Tube is usually 1-2 m long, under vacuum • Ions traverse the drift tube at different speeds • ( L / t ) = v = ( 2zV / m )½ Advantages of TOF Analyzer Good for kinetic studies of fast reactions and for use with gas chromatography to analyze peaks from chromatograph High ion transmission Can register molecular ions that decompose in the flight tube Extremely high mass range (>1MDa) Fastest scanning • Disadvantages Requires pulsed ionization method or ion beam switching (duty cycle is a factor) Low resolution (4000) Limited precursor-ion selectivity for most MS/MS experiments • Applications Almost all MALDI systems Very fast GC/MS systems Quadrupole Analyzers Quadrupole analyzers – ions are filtered or trapped in a device consisting of several metal rods using specifically tailored electromagnetic fields Quadrupole Analyzers • Electric/magnetic fields trap, store, eject ions • Requires an in-line quadrupole to act as mass pre-filter • Contains a single ring electrode and a top and bottom cap electrode • Varying RF frequency will vary the m/z ratios that are trapped • Additional fragmentation can be performed on ions stored in the ion trap • Advantages Easy to use ,simple construction,fast Good reproducibility Relatively small and low-cost systems Quadrupoles are now capable of routinely analyzing up to a m/q ratio of 3000,which is useful in electrospary ionization of biomolecules, which commonly produces a charge distribution below m/z 3000 MS detectors • Many different types available • Electron multipliers (EM) are often used • Continuous –Dynode Version mainly in GC-MS Computer system Sample introduction Ionization of sample Separation of ions by mass analyzer Detector Continious dynode EM • • The EM multiplies incident charges, thereby amplifying the signal. The current is measured that is proportional to the amount of analyte in the sample. EMs have limited lifetime which is dependent on the number of ions that hits the device, i.e., the amount of samples introduced and number of samples analyzed. +Fast response + High sensitivity Watson, Introduction to Mass Spectrometry, 4th ed Ions are detected with a microchannel plate primary ion -1000V + ee- L e- e -100V D L >> D What does a mass spectrometer do? 1. It measures mass better than any other technique. 2. It can give information about chemical structures. What are mass measurements good for? To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers Applications of Mass Spectrometry Pharmaceutical analysis Bioavailability studies Drug metabolism studies, pharmacokinetics Characterization of potential drugs Drug degradation product analysis Screening of drug candidates Identifying drug targets Biomolecule characterization Proteins and peptides Oligonucleotides Environmental analysis Pesticides on foods Soil and groundwater contamination Forensic analysis/clinical The mass spectrum shows the results MALDI TOF spectrum of IgG MH+ Relative Abundance 40000 30000 (M+2H)2+ 20000 10000 (M+3H)3+ 0 50000 100000 Mass (m/z) 150000 200000 ESI Spectrum of Trypsinogen (MW 23983) M + 15 H+ 1599.8 M + 16 H+ M + 14 H+ 1499.9 1714.1 M + 13 H+ 1845.9 1411.9 1999.6 2181.6 m/z Mass-to-charge ratio Protein Identification 1. Peptide Mass Finger Printing (PMF) from MS data 2. Database search using fragment ion masses from MS/MS data 3. Sequence Tags from MS/MS data PROBLEM Bank President Biologist Who robbed the bank? What protein was isolated? GATHER EVIDENCE Police Officer 1. Interview witnesses 2. Dust for fingerprints Mass Spectrometrist 1. Interview biologist who isolated the protein 2. Cleave protein to obtain peptide mixture enzyme 3. Analyze peptide mixture by MS to obtain peptide molecular masses! DATABASE SEARCH Police Officer Height: 5’7” Weight: 160 lbs Gender: male Age: 35-40 Fingerprints search DATABASE OF KNOWN FELONS Mass Spectrometrist Approx. molecular weight: 30,000 Origin: bovine liver Peptide mass list from MS analysis: 975.4832, 1112.5368, 632.3147, 803.4134, 764.3892 search PEPTIDE MASS DATABASE OF KNOWN PROTEINS DATABASE SEARCH RESULTS Police Officer Mass Spectrometrist Identifies the robber Identifies the protein Anthony J. Felon bovine carbonic anhydrase