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Mass Spectrometry

Mass Spectrometry. A.) Introduction : Mass Spectrometry (MS) measures the atomic or molecular weight of a ion from the separation based on its mass to charge ratio ( m/z ) - elemental composition of matter - structures of inorganic, organic and biological molecules

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Mass Spectrometry

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  1. Mass Spectrometry • A.) Introduction: • Mass Spectrometry (MS) measures the atomic or molecular weight of a ion from the separation based on its mass to charge ratio (m/z) • - elemental composition of matter • - structures of inorganic, organic and biological molecules • - qualitative and quantitative composition of complex mixtures • - isotopic ratios of atoms in the sample • One of the MOST Routinely used Analytical Techniques

  2. Mass Spectrometry Nobel Laureates: Joseph John Thomson Physics 1906 first mass spectrometer Francis William Aston Chemistry 1922 mass spectrometry of isotopes John B. Fenn Chemistry 2002 electrospray ionization of biomolecules Wolfgang Paul Physics 1989 quadrupole and quadrupole ion trap MS Koichi Tanaka Chemistry 2002 Matrix-assisted laser Desoprtion/ionization (MALDI) A Long and Continuing History of Achievements

  3. Mass Spectrometry Abundance Quantitative Information Qualitative Information Mass Spectrometry Data • Qualitative Analysis • Molecular Weight Determination • Structure Determination • Quantitative Analysis • Biotechnology • analysis of proteins & peptides • analysis of oligonucleotides • Pharmaceutical • drug discovery, combinatorial chemistry • pharmokinetics, drug metabolism • Clinical • neonatal screening, hemoglobin analysis • drug testing • Environmental • water, food, air quality (PCDs etcs) • Geological • oil composition • Toxicology • Forensics

  4. Mass Spectrometry • Advantages Over Atomic Optical Spectrometric • Detection limits three orders of magnitude better • Remarkably simple spectra that are unique and easily interpreted • Ability to measure isotopic ratios • Disadvantages • Instrument costs are two to three times higher • Instrument drift that can be as high as 5-10% per hour • Interference effects

  5. Atomic Weights in MS • Discriminates among the masses of isotopes • differs from other analytical techniques • Atomic mass units (amu) or daltons (Da) • Relative scale:  exactly 12 amu • amu or Da equals 1/12 mass of • 1.66054x10-24 g/atom • All measured masses relative to • has a mass 2.91407 times • 12.0000 Da x 2.91407 = 34.9688 Da  atomic weight = 34.9688 g/mol • Exact Mass (m) • Sum of specific set of isotopes within compounds • 12C1H4: m = 12.00000 x 1 + 1.007825 x 4 = 16.031 Da • 13C1H4: m = 13.00335 x 1 + 1.007825 x 4 = 17.035 Da • 12C1H32H1: m = 12.00000 x 1 + 1.007825 x 3 + 2.0140 x 1 = 17.037 Da • 3-4 significant figures to right of decimal • Nominal Mass (m) • Whole number precision in mass measurement • 12C1H4: m = 12 x 1 + 1 x 4 = 16 Da • 13C1H4: m = 13 x 1 + 1 x 4 = 17 Da • 12C1H32H1: m = 12 x 1 + 1 x 3 + 2 x 1 = 17 Da

  6. Atomic Weights in MS • Chemical atomic weight or average atomic weight • A1, A2, An :atomic masses in Da • p1, p2, pn :fractional abundance of each isotope • n :number of isotopes • Weight of interest for most purposes • Sum of chemical atomic weights for the atoms in the compound formula • CH4 (m) =12.01115 + 4 x 1.00797 = 16.0434 Da • typical atomic masses in periodic table Boron: 10B 23% 11B 100% B (m) = (23x10 + 100x11)/123 = 10.81 Zirconium: 90Zr 51.5% 91Zr 11.2% 92Zr 17.1 % 94Zr 17.4% 96Zr 2.8% Zr (m) = (51.5x90 + 11.2x91 + 17.1x92 + 2.8x96)/100 = 91.22

  7. Molecular Formulas from Exact Molecular Weights • Requires identification of molecular ion peak • Exact mass needs to be determined • High resolution instruments • detect mass differences of a few thousands of amu • Purine, C5H4N4 (m = 120.044) • Benzamidine, C7H8N2 (m = 120.069) • Ethyltolune, C9H12 (m = 120.096) • Acetophenone, C8H8O (m = 120.058) • Molecular ion peak is 120.070 ± 0.005  only C7H8N2 is possible formula • Precision of a few parts per million is routinely possible

  8. Molecular Formulas from Isotopic Ratios • Low-resolution instrument  only differentiate whole number masses • Requires sufficiently intense molecular ion peak • Requires accurate heights for (M+1)+ and (M+2)+

  9. Resolution in MS (R) • differentiate between masses • R = m/Dm • or • Higher the number the better the resolution • 500,000 is better than 500 • Resolution of 4000 would resolve peaks occurring at m/z values of: • 400.0 and 400.1 • 40.00 and 40.01

  10. Example 22: Calculate the resolution to differentiate (a) C2H4+ (m = 28.0313) and CH2N+ (m = 28.0187) and (b) N2+ (m = 28.0061) and CO+ (m = 27.9949).

  11. Identification of odd electron ions – The Nitrogen Rule • Based on an anomaly in the relationship between the atomic weights and valences of the common elements • An organic molecule containing the elements C, H, O, S, P or halogen • odd nominal mass if it contains an odd number of nitrogen atoms • even nominal mass if it contains an even number of nitrogen atoms (including 0) Even nominal mass  even valence Odd nominal mass  odd valence Nitrogen is the exception

  12. Double Bond Equivalent (D) • number of rings or double bonds that an ion contains • Calculated from the elemental formula as follows: • where: • D is unsaturation • imax is the total number of different elements • Ni the number of atoms of element i and • Vi the valence of atom i • valence of 1 (H, F, Cl), valence of 2 (O, S). • valence of 3 (N, P), valence of 4 (C, Si). Hexane: C6H14 Mass of molecular ion: 86 D = 1+1/2((6x(4-2) + 14x(1-2)) = 0 Hexene: C6H12 Mass of molecular ion: 84 D = 1+1/2((6x(4-2) + 12x(1-2)) = 1

  13. Basic MS Instrument Design • Atomic Mass Spectrometry involves the following steps: • atomization • conversion of atoms to ions • most ions are single charge • separation of ions based on mass-to-charge ratio (m/z) • Counting the number of ions of each type or measuring the ion current • Principal Components • Vacuum system  maintain low pressure(10-5 to 10-8torr) • Inlet: introduce m-amount of sample into ion source • Ion source: sample converted into gaseous ion by bombardment with: • Electrons • Photons • Ions • Molecules • Thermal/electric energy • Positive/negative ions accelerated into mass analyzer • Mass analyzer: disperse ions (m/z) • Transducer: convert beam of ions to electrical signal

  14. Types of Atomic and Molecular MS • Thermal ionization & Spark source  first MS • Inductively coupled plasma (ICP)  current common approach • Differ by types ion sources and mass analyzer

  15. MS Theory: • 1.) Mass analyzers use electric and magnetic fields to apply a force on charged particles • F= ma (Newton's second law) • F = e(E+ v x B) (Lorentz force law) • where: • F- force applied to the ion m - mass of the ion • a- acceleration e- ionic charge • v x B - vector cross product E- electric field • of the ion velocity and the applied • magnetic field • 2.)Force is therefore dependent on both mass and charge • - spectrometers separate ions according to their mass-to-charge ratio (m/z) • - not by mass alone

  16. Inlet Systems: • Introduce sample into ion source with minimum loss of vacuum • Spectrometer equipped with multiple inlets for different sample types • Batch Inlet • Direct Probe Inlet • Gas Chromatography • Liquid Chromatography

  17. Inlet Systems: • External (Batch) Inlet Systems: • Simplest • Gas & liquid samples (bp < 500oC) • Sample heated (<400 °C) in small external oven • Sample pressure 10-4 to 10-5torr • Vapor admitted to ionizer through valve • Gas stream added to analyte

  18. The direct probe is only ¼" in diameter. Probe moves through various lock system stages permits for a step-wise increase in the vacuum Inlet Systems: Direct probe inlet system for solids • Direct Probe • Solids and non-volatile samples • Less sample is required & wasted (few ng) • sample held on the surface of glass or aluminum capillary tube, fine wire or small cup • Sample vial inserted through air-lock into ionizer chamber • Lock system minimizes amount of air that must be pumped from system • Vial heated to vaporize sample • Vial can be reduced to capillary or surface plate for small quantities

  19. Inlet Systems: • LC & GC coupled to mass spectrometer • Permits separation and determination of components for complex mixtures • Requires specialized inlet systems • Major interface problem – carrier gas dilution • Jet separator (separates analyte from carrier gas) • Lighter carrier gas deflected by volume • Heavier sample travels in straight line

  20. Ion Sources: • Formation of gaseous analyte ions • Mass spectrometric methods are dictated by ionization techniques • Appearance of spectrum highly dependant on ionization technique • Gas-phase • Sample first vaporized then ionized • Thermally stable compounds boiling points < 500oC • MW < 100 amu • Desorption • Solid or liquid directly converted to gaseous ion • MW as large as 105daltons

  21. Ion Sources: • Hard sources • Sufficient energy so analyte are in highly excited energy state • Relaxation involves rupture of bonds • Produces fragment ions with m/z < molecular ion • Kinds of functional groups  structural information Hard Ionization

  22. Ion Sources: • Soft sources • Cause little fragmentation • Mass spectrum consists of molecular ion and only few, if any, other peaks • Accurate mass Soft Ionization

  23. Ion Sources: • Electron-Impact Source (EI) • Sample heated to produce molecular vapor • Bombard with a beam of electrons • Electrons emitted from heated tungsten or rhenium filament • Electrons accelerated by a potential of 70V • Path of electrons and molecular ion at right angles • Form positive ions  electron beam expels electron due to electrostatic repulsion M + e- M●+ + 2e- • Not very efficient  one molecule in a million ionized • Positive ions attracted to first slit by small potential 5V • High potential applied at accelerator plates 103 to 104 V • Generates molecular ion velocity

  24. Ion Sources: • Electron-Impact Source (EI) • Hard source 50V higher energy than chemical bond • Highly excited vibrational and rotational state • Electron beam does not increase translational energy • Relaxation results in extensive fragmentation • Large number of positive ions of various masses • Typically less mass than molecular ion • Lower mass ions called daughter ions • Sometimes molecular ion not present

  25. Ion Sources: • Electron-Impact Source (EI) • Base peak  most intense peak • Usually a daughter ion or fragment ion • Peaks at MW greater than molecular ion • Same chemical formula but different isotope composition • Size of peak depends on relative natural abundance of isotopes • Collision Product peak (M+1)+ • Collision transfers a hydrogen atom to the ion to generate a protonated molecule • Second order reaction  depends on concentration • Increases with increase in pressure

  26. Ion Sources: • Electron-Impact Source (EI) • Advantages • Good sensitivity • Fragmentation  unambiguous identification of analytes • Disadvantages • Need to volatize sample  thermal decomposition before ionization • Fragmentation  disappearance of molecular ion peak • MW not determined

  27. Ion Sources: • Chemical Ionization Source (CI) • Electron Impact and Chemical Ionization are Interchangeable in a Spectrometer • Chemical Ionization is the second most common procedure for generating ions • Gaseous atoms from the sample are: • Heated from a probe • Collide with ions produced reagent gas bombarded by electrons • Usually positive ions are used • Need to modify electron beam • Add vacuum pump capacity • Reduce width of slit for mass analyzer • Allow a reagent pressure of 1 torr in ionization area • Keep pressure below 10-5torr in analyzer • Concentration ratio of reagent to sample is 103 to 104 • Electron beam preferentially interacts with reagent instead of sample

  28. Ion Sources: • Chemical Ionization Source (CI) • Soft Source • Methane is common reagent • Also use propane, isobutane and ammonia • Reacts with high-energy electron beam to generate several ions • CH4+, CH3+ (~90% of product) and CH2+ • React with other methane molecules CH4+ + CH4 CH5+ + CH3 CH3+ + CH4  C2H5+ + H2 • Collisions Between sample molecule and CH5+ & C2H5+ are highly reactive • Involve proton or hydride transfer CH5+ + MH  MH2+ + CH4 proton transfer C2H5+ + MH  MH2+ + C2H4 proton transfer C2H5+ + MH  M+ +C2H6 hydride transfer • Proton transfer  (M + 1)+ • Hydride transfer  (M – 1)+ • C2H5+ transfer (M + 29)+ EI Hard Ionization Soft Ionization CI

  29. Ion Sources: • Matrix-Assisted Laser Desorption/Ionization (MALDI) • Accurate MW for polar biopolymers • DNA, RNA, Proteins • Few thousands to several hundred thousand Da • Sample is mixed with large excess of radiation-absorbing matrix material • Solution is evaporated onto solid surface • Sample exposed to pulsed laser beam • Sublimation of analyte ions • MS spectra recorded between laser beam pulses

  30. Ion Sources: • Matrix-Assisted Laser Desorption/Ionization (MALDI) • Low background noise • At high MW, matrix causes significant background at low MW • Absence of fragmentation • Multiple charged ions (+2, +3) • Observe dimerstrimers • Mechanism is not completely understood • Matrix compound must absorb the laser radiation • Soluble enough in sample solvent to be present in large excess • Analyte should not absorb laser radiation • Fragmentation will occur Simulation of MALDI

  31. Ion Sources: • Matrix-Assisted Laser Desorption/Ionization (MALDI) • MALDI spectra are greatly influenced by type of matrix, solvent and additive • At high MW, matrix causes significant background at low MW Dariusz Janecki et al. (2002) ASMS

  32. Ion Sources: • Electrospray Ionization (ESI) • One of the most important techniques for analyzing biomolecules • Polypeptides, proteins and oligonucleotides • Inorganic species synthetic polymers • MW >100,000 Da • Uses atmospheric pressure and temperature • Sample pumped through a stainless steel capillary • Rate of a few mls per minute • Needle at several KVs potential • Creates charged spray of fine droplets

  33. Ion Sources: • Electrospray Ionization (ESI) • Passes through desolvating capillary • Evaporation of solvent • Attachment of charge to analyte molecule • Molecules become smaller  charge density becomes greater  desorption of ions into ambient gas

  34. Ion Sources: • Electrospray Ionization (ESI) • Little fragmentation of thermally fragile biomolecules • Ions are multiply charged • m/z values are small • Detectable with quadrupole with mass range of 1500 or less • Average charge state increases ~linearly with MW • MW determined from peak distribution

  35. Ion Sources: • Fast Atom Bombardment Sources (FAB) • Major role for MS studies of polar high molecular-weight species • Soft Ionization technique • MW > 10,000 • Structural information for MW ~3,000 • Samples are in a condensed state • Glycerol solution matrix • Liquid matrix helps reduce lattice energy • Ionized by bombardment with energetic (several keV) xenon or argon atoms • Very rapid sample heating • Reduces sample fragmentation • Positive & negative analyte ions are sputtered from the surface • Desorption process • Must overcome lattice energy to desorb an ion and condense a phase • “healing” the damage induced by bombardment

  36. Ion Sources: • Fast Atom Bombardment Sources (FAB) • Beam of fast energetic atoms are generated by: • Passing accelerated argon or xenon ions from an ion source through a chamber • Chamber contains argon or xenon atoms at 10-5 torr • High-velocity ions undergo a resonant electron-exchange reaction without substantial loss of translational energy Focusing Atom beam Extraction plate Analyte ion beam (secondary ions) Probe tip Analyte metrix Production of “fast atoms” Charge transfer Ar+* + Ar0 ----------------> Ar+ + Ar0* Accelerated argon ion from “ion gun” Ground state argon atom “slow ion” “fast atom”

  37. Example 23: Identify the ions responsible for the peaks in the following mass spectrum for 1,1,1,2-tetrachloroethane (C2H2Cl4): MW = 167.85

  38. Mass analyzers: • Double-Focusing analyzer • Two devices for focusing an ion beam • Electrostatic analyzer • Magnetic sector analyzer • Ions accelerated through slit into curved electrostatic field • Focus beam of ions with narrow band of kinetic energies into slit • Ions enter curved magnetic field • Lighter ions deflected more than heavier ions

  39. Mass analyzers: • Double-Focusing analyzer • Curved magnetic field of 180, 90 or 60 degrees • Vary magnetic field strength or accelerating potential to select for ions of different mass • Kinetic energy of accelerated ion (before magnetic field) where: V = voltage v = ion velocity e = electronic charge (1.60x10-19C) z = ion charge • Path through magnet depends on the balance of two forces: • Magnetic force (FM) and centripetal force (Fc) where: B = magnetic field strength r = radius of curvature of magnetic sector • Substitute velocity into kinetic energy equation • Select masses by varying B by changing current in magnet

  40. Mass analyzers: • Double-Focusing analyzer

  41. Mass analyzers: • Quadrupole mass analyzer • More compact, less expensive, rugged • High scan rate  spectrum in < 100ms • Four parallel cylindrical rods serve as electrodes • Opposite rods are connected electrically • One pair attached to positive side of variable dc source • One pair attached to negative side of variable dc source • Variable radio-frequency ac potential (180o out of phase) applied to each pair of rods • Ions accelerated through space between rods • Potential of 5 to 10 V • ac and dc voltages increased simultaneously with ratio being constant • All ions without specific m/z strike rods and become neutral • only ions having a limited range of m/z reach transducer (detector)

  42. Mass analyzers: • Quadrupole mass analyzer • Positive rods • Alternating ac causes ions to converge during positive arc and diverge during negative arc • If ions strike rod during negative arc  neutralized and removed by vacuum • Striking a rod depends on: • rate of movement through rod • Mass to charge ratio • Frequency and magnitude of the ac signal • dc current effects momentum of ions • Momentum directly related to square-root of mass • More difficult to deflect heavy ions than lighter ions • Prevents heavier atoms from striking rods • High-pass mass filter

  43. Mass analyzers: • Quadrupole mass analyzer • Negative rods • In the absence of ac, all positive ions drawn to rods  annihilated • Offset for lighter ions by ac • Low-pass mass filter • For ions to pass through the rods (band of ions): • Significantly heavy to pass positive rods • Significantly light to pass negative rods • One pair attached to positive side of variable dc source • One pair attached to negative side of variable dc source • Adjusting ac & dc moves the center of band of ions which pass the rods

  44. Mass analyzers: • Time of Flight (TOF) Mass Analyzers • Ions generated by bombardment of the sample with a brief pulse of: • Electrons, secondary ions, laser-generated photons • Frequency of pulse 10 to 50 kHz, duration of pulse 0.25 ms • Ions accelerated by electric field pulse 103 to 104 V • Same frequency of ionization pulse, but lags behind • Accelerated particle enter field-free drift tube • Ions enter tube with same kinetic energy • Ion velocity vary inversely with mass • Lighter particles arrive at detector before heavier particles • Flight times are 1 to 30 ms • Requires fast electronics • Peak broadening due to variability in ion energies and initial position • Limits resolution compared to magnets and quadrupole • Less widely used than quadrupole • Advantages: unlimited mass range, rapid data acquisition, simplicity, ruggedness, ease of access to ion source

  45. Transducers (detectors) for Mass Spectrometry: • Electron Multipliers • Detects positive ions • Similar to photomultiplier used in UV/vis • Each dynode held at successfully higher voltage • Dynodes have Cu/Be surfaces • Burst of electrons emitted when struck with energetic ions or electrons • Electron multiplies contain upwards of 20 dynodes • Typical current gain of 107 • Continuous-Dynode electric multiplier • Glass that is heavily doped with lead • Potential of 1.8 to 2 V across length of transducer • Trumpet shaped • Ions strike surface near entrance that ejects electrons • Electrons then skip along surface ejecting more electrons with each impact • Typical current gain of 105 to 108 • Rugged, reliable with high current gains • Positioned directly at the exit slit of magnet • Ions have enough energy to eject electrons • Requires accelerator with quadrupole MS

  46. Transducers (detectors) for Mass Spectrometry: • Faraday Cup • hollow collector, open at one end and closed at the other, used to collect beams of ions • incident ion strikes the dynode surface which emits electrons • induces a current which is amplified and recorded • Surrounded by cage • Prevents the escape of reflected ions and ejected secondary electrons • Inclined with respect to ion beam • Particles striking or leaving the electrode are reflected away from entrance • Connected to ground potential through a large resistor • Ions striking plate are neutralized by flow of electrons from resistor • Causes a potential drop across resistor • Independent of the energy, mass or chemical nature of ion • Inexpensive and simple mechanical and electronic device • Disadvantages: • Need for a high-impedance amplifier • Limits speed at which spectrum can be scanned • Less sensitive than electron multipliers

  47. Atomic Mass Spectra and Interferences: • Spectroscopic interference • An ionic species in the plasma has the same m/z values as an analyte • Isobaric interference • Two elements that have isotopes with nearly the same mass • Differ by less than 1 amu • 113In+ overlaps with 113Cd+ and 115In+ overlaps with 115Sn+ • Isobaric interference occurs with the most abundant and most sensitive isotope • 40Ar+ overlaps with 40Ca+ (97%) need to use 44Ca+ (2.1%) • 58Ni+ overlaps with 56Fe+ need to use 56Fe+ • Exactly predictable from abundance tables • Polyatomic ion interference • More serious than isobaric • Polyatomic species form from interactions with species in plasma, matrix or atmosphere • Several molecular ions can form and interfere • Typically observed for m/z < 82 amu • Potential interference: 40Ar2+, 40ArH+, 16O2+, H216O,16OH+, 14N+ • Serious interference: 14N2+ with 28Si+, NOH+ with 31P+, 16O2+ with 32S+, 40ArO+ with 56Fe+, 40Ar2+ with 80Se+ • Correct with blank

  48. Atomic Mass Spectra and Interferences: • Spectroscopic interference • Oxide and Hydroxide species interference • Most serious • Oxides an hydroxides of analyte, matrix solvent plasma gases • MO+ and MOH+ ions • Formation depends on injector flow rate, RF power, sampler skimmer spacing, sample orifice size, plasma gas composition, oxygen elimination solvent removal efficiencies • Matrix Effects • Occur at concentrations > 500 to 1000 mg/ml • Reduction in analyte signal, sometimes signal enhancement • General effect • Minimized by using more dilute solutions, altering sample introduction procedure or by separating out offending species • Use internal standard to correct effect Affect of pesticide response to matrix effects Add protectant to remove matrix effect Analytical Sciences 2005, 21, 1291

  49. Fourier Transform (FT) MS: • Improves signal-to-noise ratios • Greater Speed • Higher sensitivity and resolution • Requires an Ion Trap • Ions are circulated in well-defined orbits for extended periods • Ion cyclotron resonance (ICR) • Ions in a magnetic field circulate in a plane perpendicular to the direction of the field • Angular frequency of this motion  cyclotron frequency (wc) • Ion trapped in a magnetic field can absorb energy from an ac electricfield • Frequency of field must match cyclotron frequency • Absorbed energy increases velocity of ion and radius without affecting wc

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