Mass Spectrometry(Mass Spec.) Prof. Yonghai Chai School of Chemistry & Materials Science For Bilingual Chemistry Education
OUTLINE Introduction to Mass Spectrometry Ionization Methods Mass Analyzer Fragmentation and MS Interpretation Hyphenated MS Techniques
By James Crawford How do two people with different languages communicate with each other? Then, how can I catch up, Ms.?
Chemical Identification • Comparison of • Physical Properties • Boiling Point • Melting Point • Density • Optical rotation • Appearance • Odor • Elemental Analysis • Burn the compound and 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.
Timeline for MS Development • 1897 Early Mass Spectrometry • 1919 The observation of isotopes using mass spectrometry • 1934 Double Focusing Analyzer • 1939 Accelerator Mass Spectrometry • 1946 Time-of-Flight Mass Spectrometry • 1947 Preparative Mass Spectrometry • 1949 Ion Cyclotron Resonance (ICR) • Reverse Geometry Double • focusing MS • 1953 Quadrupole Analyzers Joseph John Thomson "In recognition of the great merits of his theoretical and experimental investigations on the conduction of electricity by gases.“ 1906 Nobel Prize "At first there were very few who believed in the existence of these bodies smaller than atoms. I was even told long afterwards by a distinguished physicist who had been present at my  lecture at the Royal Institution that he thought I had been 'pulling their legs." Cited from: http://masspec.scripps.edu/mshistory/ Replica of J.J. Thomson's third mass spectrometer.
Continuation of Timeline • 1956 Gas Chromatography Mass Spectrometry (GC/MS) • Identifying Organic Compounds with Mass • Spectrometry • 1962 Mass Spectrometry Imaging • 1966 Chemical Ionization • 1966 Peptide Sequencing • 1966 Tandem Mass Spectrometry • 1966 Metabolomics • 1968 Electrospray Ionization • Collision Induced Dissociation • 1969 Field Desorption-MS of Organic Molecule Francis William Aston "For his discovery, by means of his mass spectrograph, of isotopes, in a large number of non-radioactive elements, and for his enunciation of the whole-number rule." Mass spectrometry of isotopes 1922 Nobel Prize Cited from: http://masspec.scripps.edu/mshistory/
Continuation of Timeline 1974 Fourier Transform Ion Cyclotron Resonance 1974 Extra-Terrestrial Mass Spectrometry 1975 Atmospheric Pressure Chemical Ionization (APCI) 1976 Californium-252 Plasma Desorption MS 1978 GC-C-IRMS 1978 Triple Quadrupole Mass Analyzer 1980 Inductively Coupled Plasma MS 1981 Matrix-Assisted Desorption Ionization 1984 Quadrupole/Time-Of-Flight Mass Analyzer 1985 Matrix-Assisted Laser Desorption Ionization (MALDI) Wolfgang Paul Hans Georg Dehmelt “For the development of the ion trap technique.” 1989Nobel prize Cited from: http://masspec.scripps.edu/mshistory/
Continuation of Timeline ESI 1987 Soft Laser Desorption of Proteins 1989 ESI on Biomolecules 1989 Monitoring Enzyme Reactions with ESI-MS 1990 Protein Conformational Changes with ESI-MS 1990 Clinical Mass Spectrometry 1991 MALDI Post-Source Decay 1991 Non-covalent Interactions with ESI 1992 Low Level Peptide Analysis 1993 Oligonucleotide Ladder Sequencing 1993 Protein Mass Mapping 1996 Intact Virus Analyses John B. Fenn MALDI Koichi Tanaka "For the development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules." Cited from: http://masspec.scripps.edu/mshistory/
Continuation of Timeline • 1998 Electron Capture Dissociation (ECD) • 1999 Nanostructure Desorption/Ionization • Quantitative Proteomics and Metabolomics with • Isotope Labels • 2000 Orbitrap • 2004 Desorption Electrospray Ionization (DESI) • 2004 Electron Transfer Dissociation (ETD) • 2005 Direct Analysis in Real Time (DART) Fred W. McLafferty Alfred O.C. Nier Alan G. Marshall Klaus Biemann R. Graham Cooks Donald F. Hunt Catherine Fenselau Franz Hillenkamp Carol V. Robinson Michael Karas Malcolm Dole Brian T. Chait Cited from: http://masspec.scripps.edu/mshistory/
What information can be determined? • Molecular weight • Molecular formula (HRMS) • Structure (from fragmentation fingerprint) • Isotopic incorporation / distribution • Protein sequence (MS-MS)
Schematic Mass Spectrometer 样品 检测器 离子源 质量分析器 数据分析处理器
What’s in a Mass Spectrum Mass-to-charge ratios of a molecule or its fragment are graphed or tabulated according to their relative abundance Fragment Ions Fragment Ions: derived from molecular ion or higher weight fragments
Applications Biomolecule characterization Pharmaceutical analysis • Proteins and peptides • Oligonucleotides Paleoclimatology and Archeology http://www.sciencemag.org/products/lst_20060901.dtl • Forensic analysis/clinical • Environmental analysis • Pesticides on foods • Soil and groundwater contamination Paleotemperature O16 and O18 foraminifera
Relative Abundance of Isotopes Atomic weight of an element is a weighted average of the naturally occurring isotopes.
Isotopic Ratio from the Spectra Mass spec. can be used to measure the isotopic ratios
Continuation of Isotopes • Chlorine (35Cl to 37Cl is 3:1, give M + 2)
Ionization Methods Electron bomb Ionization (电子轰击离子化) EI Chemical Ionization (化学电离) CI Field ionization (场电离) FI Matrix Assisted Laser Desorption Ionization (基质协助的激光解吸) MALDI Fast atom bombardment (快原子轰击) FAB Electro Spray Ionization(电喷雾) ESI
(M-R2)+ Mass Spectrum (M-R1)+ M+ (M-R3)+ Electron Bomb Ionization ( EI ) Sample is heated and energized by a beam of electrons, usually gives a molecular ion (M+) and a lot of fragments。
Properties of EI • Hard ionization • Gas-phase molecules enter source through heated probe or • GC column • 70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions • EI spectra can be matched to library stds CI (soft ionization) • Higher pressure of methane leaked into the source (mtorr) • Reagent ions transfer proton to analyte
ChemicalIonization (CI) Electron ionization leads to fragmentation of the molecular ion, which sometimes prevents its detection. Chemical ionization (CI): A technique that produces ions with little excess energy. Thus this technique presents the advantage of yielding a spectrum with less fragmentation in which the molecular species is easily recognized. Consequently, chemical ionization is complementary to electron ionization.
Advantages Parent Ion Interface to GC Insoluble Samples Disadvantages No Fragment Library Need Volatile Sample Need Thermal Stability Quantitation Difficult Low Mass Compounds (<1000 amu) Solids Probe Requires Skilled Operator Properties of CI
+ + + 阳极 + + + + + + + + + + d<1mm 阴极 Field ionization (FI) Field ionization (FI) is a method that uses very strong electric fields to produce ions from gas-phase molecules.
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)
MALDI mass spectrometry has become a powerful analytical tool for both synthetic polymers and biopolymers. Principle of MALDI
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+ acceleration (5-15 KeV) Ar+ + Ar Ar + Ar+ fast slow + 8 KeV fast slow
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 Properties of FAB
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.
Advantages Electrospray Ionization can be easily interfaced to LC. Absolute signals from Electrospray are more easily reproduced, therefore, better quantitation. Mass Accuracy is considered better. Multiple charging is more common then MALDI. Disadvantages No Fragmentation Need Polar Sample Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best) Sensitive to Salts Suppression Properties of ESI
Types of Mass Analyzers Magnetic sector analyzer （磁分析器） Time of Flight analyzer (TOF) (飞行时间分析器） Quadrupole analyzers (四极滤质器） Fourier Transform Ion-Cyclotron （傅立叶离子回旋共 振分析器）
Magnetic SectorAnalyzer Magnetic sector analyzer – Uses electric and/or magnetic fields to separate ions
Principle of Magnetic SectorAnalyzer The ion source accelerates ions to a kinetic energy given by : (1/2)m2= zV Where m is the mass of the ion ,v is its velocity, z is the charge on the ion ,and V is the applied voltage of the ion optics.
Principle of Magnetic SectorAnalyzer Only ions of mass-to-charge ratio that have equal centripetal and centrifugal forces pass through the flight tube: m v 2 / r = Bzv By rearranging the equation, m/z = B2r2/2V It shows that the m/q ratio of the ions that reach the detector can be varied by changing either the magnetic field or the applied voltage of the ion optics.
In summary ,by varying the voltage or magnetic field of the magnetic-sector analyzer ,the individual ion beams are separated spatially and each has a unique radius of curvature according to its mass/charge ratio.
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