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Mass Spectrometry

Mass Spectrometry. Ionization Techniques. Mass Spectrometer. All Instruments Have: Sample Inlet Ion Source Mass Analyzer Detector Data System. http://www.asms.org. Ionization Techniques. Gas-Phase Methods Electron Ionization (EI) Chemical Ionization (CI). Desorption Methods

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Mass Spectrometry

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  1. Mass Spectrometry Ionization Techniques

  2. Mass Spectrometer • All Instruments Have: • Sample Inlet • Ion Source • Mass Analyzer • Detector • Data System http://www.asms.org

  3. Ionization Techniques Gas-Phase Methods • Electron Ionization (EI) • Chemical Ionization (CI) • Desorption Methods • Matrix-Assisted Laser Desorption Ionization (MALDI) • Fast Atom Bombardment (FAB) • Secondary Ion MS (SIMS) • Spray Methods • Electrospray (ESI) • Atmospheric Pressure Chemical Ionization (APCI)

  4. Electron Ionization http://www.noble.org/PlantBio/MS/ion_tech_main.html

  5. Electron Ionization • Samples must be vaporized in the ion source • Typically 1 of 1000 molecules entering the source is ionized • 10-20 eV of energy is imparted to the molecule • ~10eV is enough to ionize most molecules • Up to 230 kcal/mol is left to cause fragmentation

  6. Electron Ionizationb-lactam: Effects of ionization energy

  7. Electron Ionization (low picomole) Advantages • Well-Established • Fragmentation Libraries • No Supression • Insoluble Samples • Interface to GC • Non-Polar Samples • Disadvantages • Parent Identification • Need Volatile Sample • Need Thermal Stability • No Interface to LC • Low Mass Compounds (<1000 amu) • Solids Probe Requires Skilled Operator

  8. Chemical Ionization • Reagent gas is introduced into the source at ~0.5 torr • Reagent gas is preferentially ionized. Ions react mostly with neutral reagent gas • Reactions occurring depend on the nature of the reagent gas • Ions in the reagent gas plasma react with the analyte

  9. Chemical Ionization http://www.noble.org/PlantBio/MS/ion_tech_main.html

  10. Chemical Ionization: Methane • Methane primarily forms CH4+• with CH2+• and CH3+ • CH4+• + CH4→ CH5+ + CH3 (m/z 17) • CH2+• + CH4→ C2H3+ + H2 + H• • C2H3+ + CH4→ C3H5+ + H2 (m/z 41) • CH3+ + CH4→ C2H5+ + H2 (m/z 29)

  11. Chemical Ionization: Methane

  12. Chemical Ionization: Methane • Ions other than saturated hydrocarbons react via proton transfer • CH5+ + M → MH+ + CH4 (or via C2H5+ or C3H5+) • For saturated hydrocarbons, hydride abstractions is common • CH5+ + RH → R+ + CH4 + H2 • For polar molecules, adducts can form • CH3+ + M → (M+CH3)+ • MH+, R+, and adducts are pseudomolecular ions.

  13. Chemical Ionization: Isobutane

  14. Chemical Ionization: Isobutane

  15. Chemical Ionization: Isobutane • Reacts through Proton Transfer • C4H9+ + M → MH+ + C4H8 • For saturated hydrocarbons, no reaction • For polar molecules, adducts can form • C4H9+ + M → (M+C4H9)+ • Lack of reaction with hydrocarbons can be used for selective detection of compounds in mixtures containing hydrocarbons • Less fragmentation is observed with isobutane. (molecular species is more reliably formed)

  16. EI vs. Methane vs. Isobutane EI Methane CI Isobutane CI

  17. Chemical Ionization: Negative Ions • Low energy electrons are present in the CI plasma • These can attach to molecules with high electron affinities • There are two principal pathways: N2O/CH4 reagent gas • AB + e-→ AB-• (associative resonance capture) • AB + e-→ A• + B- (dissociative resonance capture) • Deprotonation can also occur if a basic ion is formed in the reagent gas plasma

  18. Chemical Ionization (low picomole) Advantages • Molecular 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

  19. Ionization Sources - II • EI and CI have limitations • Both require a volatile sample • Samples must be thermally stable • Neither lends itself to LC/MS analysis • Other techniques have been developed • FAB (Fast Atom Bombardment) • SIMS (Secondary Ion MS) • MALDI (Matrix Assisted Laser Desorption) • ESI (Electrospray)

  20. FAB • Sample is dissolved in a non-volatile liquid matrix • Glycerol and m-Nitrobenzyl alcohol are common matrices • A high energy (5kV) beam of neutral atoms (typically Ar or Xe) is focused onto the sample droplet • Dissolved Ions and Molecules are ejected into the gas phase for analysis

  21. FAB

  22. FAB • For Organic Molecules M+H and M+Na ions are typically observed • M+H ions typically fragment more than M+Na ions • Salts such as NaI can be added to the matrix to induce M+Na formation

  23. FAB (nanomole) Advantages • Stable Molecular 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 • Not amenable to automation • Relatively Low Sensitivity

  24. SIMS • Analysis of surfaces in situ • A high energy (15-40 keV) beam of primary ions (In+, Ga+) or clusters (SF6, Au3, C60) is focused onto the sample droplet • Surface and slightly sub-surface atoms or molecules are ejected and ionized • Clusters increase secondary ion yield dramatically

  25. SIMS Courtesy of Mike Kurczy, Winograd Group, Penn State University

  26. SIMSGa+ vs. C60+ J. Phys. Chem. B 2004, 108, 7831-7838 Videos

  27. SIMS Advantages • Surface analyses • Stable Molecular Ion • Mass Limit ~ 10000 amu • Small primary ion beam spots and beam rastering make imaging possible • Disadvantages • No Fragment Library • Quantitation Difficult • Needs Highly Skilled Operator • Not amenable to automation • Relatively Low Sensitivity

  28. MALDIMatrix Assisted Laser Desorption • Sample dissolved in a solid matrix • Typically mixed in solution • Small droplet applied to target and dried • A wide variety of matrices exist • Choose based on hydrophobic/hydrophilic character of sample • Also based on laser absorbance (usually UV) • An ionization agent is often added • Agent must bind to the sample • TFA and its Na+ Ag+ salts are common

  29. MALDI

  30. MALDI • Choice of matrix based on empirical evidence • http://polymers.msel.nist.gov/maldirecipes/maldi.html • Typically singly charged ions observed • Some matrix adducts/cluster ions • Difficult to analyze low MW compounds due to matrix background • Typically used for MW 500-500,000

  31. MALDI

  32. MALDI

  33. UV-MALDI Matrices

  34. MALDI (low femtomole) Advantages • Parent Ion • High Mass Compounds (>100,000 amu) • Thermally Labile Compounds (R.T.) • Easy to Operate • Easily Automated • Disadvantages • No Fragment Library • Wide variety of matrices • Quantitation Difficult • Matrix Background

  35. ESIElectrospray Ionization • Sample dissolved in a polar solvent • Solution flows into a strong electric field (3-6 kV potential) • Electric field induces a spray of highly charged droplets (charges at surface) • As droplets shrink, repulsion increases until they break into smaller droplets • In small enough droplets, surface charges can be desorbed into the gas phase.

  36. ESI

  37. ESI • Ions formed via charge-residue or ion-evaporation • Molecules form M+H+ or M-H- ions • Large molecules: 1 charge / 1000 amu • Small molecules: Usually singly charged • Molecules with no acid/base groups • Can form adduct ions with Na+ K+ NH4+ Cl- OAc-, etc. • Salts may be added or already present in sample.

  38. ESI • ESI ions formed at high pressure must be transferred into high vacuum • Differential pumping is needed to move ions through small openings while maintaining low pressures • Ions become super-cooled by expansion. Solvent can recondense • Two methods to reduce cluster formation • High temperature transfer tube • Heated counter-current flow of N2

  39. ESI

  40. ESI-Multiply Charged Ions • Large Molecules produce an envelope of charge states • Deconvolution must be done to determine the charge states if isotopic resolution is not possible • Typically, MS data systems use software to deconvolute automatically

  41. ESI-Multiply Charged Ions M=16953 Δm = 1 amu ; ∆(m/z) ≈ 0.055; z = 18 ; Δ(m/z) ≈ 0.10 Δm = 1 amu z = 10

  42. j(m2-mp) z1 = M = z1(m1-mp) (m2-m1) ESI-Multiply Charged Ions • Consider (M+zH)z+ • z1m1 = M + z1mp (m1 = measured m/z) • Consider a peak of m/z=m2 which is (j-1) charge states away from peak m1 • m2(z1-j) = M + (z1-j)mp

  43. 10(1621.3-1.0073) j(m2-mp) = 51.0 z1 = z1 = (m2-m1) (1621.3-1303.8) ESI-Multiply Charged Ions j=10 1303.8 1621.3 M = z1(m1-mp) M = 51.0(1303.8-1.0073) M = 66485

  44. ESI (low femtomole to zeptomole) Advantages • Parent Ion • High Mass Compounds (>100,000 amu) • Thermally Labile Compounds (<0º C) • Easy to Operate • Interface to HPLC • Zeptomole sensitivity with nanospray • Disadvantages • No Fragmentation • Need Polar Sample • Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best) • Sensitive to Salts

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