UVVIS Spectroscopy

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Chapter 5Mass Spectrometry
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Mass Spectrometry Basics

• What information can be determined?
• Molecular weight
• Molecular formula (HRMS)
• Structure (from fragmentation fingerprint)
• Isotopic incorporation / distribution
• Protein sequence (MS-MS)

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Mass Spectrometry Basics

• Mass spectrometry has 4 basic operations
• Sample introduction (analyte must be in vapor
  phase)
• Ionization
• Mass analysis ( separating ions by mass/charge
  ratio)
• Detection and quantitation

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Sample Introduction

• Method Applications
• Batch (reservoir) gases, volatile liquids
• Direct insertion probe very low vapor
  pressure solids and liquids
• Membrane aqueous solutions, air samples
• Chromatography eluent LC-MS, GC-MS, etc.

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Ionization Methods

• 1. Electron Ionization (EI)
• most common ionization technique, limited to
  relatively low MW compounds (lt600 amu)
• 2. Chemical Ionization (CI)
• ionization with very little fragmentation, still
  for low MW compounds (lt800 amu)
• 3. Desorption Ionization (DI)
• for higher MW or very labile compounds
• 4. Spray ionization (SI)
• for LC-MS, biomolecules, etc.

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Electron Ionization (EI)

• vaporized sample is bombarded with high energy
  electrons (typically 70 eV)
• hard ionization method leads to significant
  fragmentation
• ionization is efficient but non-selective
• PLK Chapter 8 is written from an EIMS perspective

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Electron Ionization

• Advantages
• inexpensive, versatile and reproducible
• fragmentation gives structural information
• large databases if EI spectra exist and are
  searchable
• Disadvantages
• fragmentation at expense of molecular ion
• sample must be relatively volatile

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Chemical Ionization (CI)

• Vaporized sample reacts with pre-ionized reagent
  gas via proton transfer, charge exchange,
  electron capture, adduct formation, etc.
• common CI reagents
• methane, ammonia, isobutane, hydrogen, methanol
• soft ionization gives little fragmentation
• selective ionization--only exothermic or
  thermoneutral ion-molecule reactions will occur
• choice of reagent allows tuning of ionization

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Desorption Ionization (DI)

• Allows analysis of non-volatile or thermally
  labile materials
• Sample hit with large amount of energy in short
  time
• Secondary ion mass spectrometry (SIMS)
• Fast atom bombardment (FAB)
• Plasma desorption (PD)
• Laser desorption (LD)
• Matrix-assisted laser desorption (MALDI)

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SIMS and FAB

• Sample in solid or liquid matrix in bombarded
  with a stream of high energy ions (SIMS) or
  neutral atoms (FAB)
• The matrix absorbs most of the energy and has a
  dramatic effect on ionization
• Matrix contributes large amount of background
  ions
• Common matrices
• glycerol, dithiothreitol, diethanolamine,
  m-nitrobenzyl alcohol
• Additives can dictate ions formed

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MALDI

• Sample/matrix hit by short laser pulse
• Matrix usually organic acid with strong
  electronic absorption at lasers wavelength
• Efficiently produces intact molecular ions (
  usually MH and MNa )
• Useful for large biomolecules (up to 100,000 MW)
• Requires only pico- or femtomoles of material
• Matrix chosen for specific applications

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Spray Ionization (SI)

• Sample solution shot through capillary to create
  microdroplets, and ultimately intact molecular
  ions
• Good for high MW, thermally labile compounds
• Thermospray (TS)
• first spray method developed, no longer very
  common
• solution of sample sprayed through hot nozzle,
  creating charged mini-droplets
• Electrospray (ES)
• solution sprayed through nozzle with 2-5 kV
  potential

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Mass Analysis

• Five main methods of separating ions based on
  mass/charge ratio
• Magnetic sector analyzer
• Time of Flight (TOF) analyzer
• Quadrupole analyzer
• Ion trap (Varian Saturn MS)
• Ion cyclotron resonance

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Fragmentation

• Governed by product ion stability
• consideration
• octet rule
• resonance delocalization
• polarizability and hyperconjugation
• electronegativity
• Stevensons Rule
• For simple bond cleavage, the fragment with
  lowest ionization potential takes the charge
• (in other words, the most stable ion is formed)

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General Fragmentation Pathways

• One-bond cleavages (a-cleavages)
• Two-bond cleavages
• eliminate H-X
• retro Diels-Alder
• McLafferty rearrangement

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Alkane Fragmentation

• Long chains give homologous series of m/z 14
  units
• Long chains rarely lose methyl radical
• Straight chain alkanes give primary carbocation
• branched alkanes have small or absent M
• enhanced fragmentation at branch points
• Cycloalkanes
• loss of side chain
• loss of ethylene fragments

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Alkene Fragmentation

• Fairly prominent M
• fragment ions of CnH2n and CnH2n-1
• terminal alkenes lose allyl cation if possible
• Cycloalkenes
• prominent molecular ion
• retro Diels-Alder cleavage

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Alkyne Fragmentation

• Molecular ion readily visible
• terminal alkynes readily lose hydrogen atom
• terminal alkynes lose propargyl cation if
  possible

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Aromatic Hydrocarbon Fragmentation

• Molecular ion usually strong
• alkylbenzenes cleave at benzylic carbon
• tropylium ion formation
• McLafferty rearrangement of aromatics
• need g-hydrogens

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Alcohol Fragmentation

• Molecular ion strength depends on substitution
• primary alcohol weak M
• secondary alcohol VERY weak M
• tertiary alcohol M usually absent
• Dehydration fragmentation
• thermal vs. 1,4-dehydration of M
• Loss of alkyl group
• largest R group lost as radical

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Ether Fragmentation

• Alpha-cleavage
• C-O cleavage
• requires stable cation to lose
• ion rearrangement

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Carbonyl Compounds

• Dominant fragmentation pathways
• a-cleavage
• b-cleavage
• McLafferty rearrangement

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Halide Fragmentation

• Loss of halogen atom
• Elimination of HX
• alpha-cleavage
• 1,4-rearrangement

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MS analysis examples
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C5H12O MW 88.15
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C7H12Br MW 171.04
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C9H10O MW 134.18
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C11H12O 3 MW 192.21
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C5H8O2 MW 100.12