Mass Spectrometry Lecture 1

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Mass SpectrometryLecture 1

• Dr Kevin Welham
• F229

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Mass Spectrometry

• Mass- quantity of matter in a body.
• Weight force exerted by a body due to its
  position in a gravitational field.
• Atomic Mass e.g. AKZ where z mass number
  (protons neutrons) and A atomic number
  (protons).
• Relative Atomic Mass ratio of the atomic masses
  to an integer reference C1212.0000. These are
  non-integer,
• H11.00783 N1414.00307 O1615.9949

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Mass Spectrometry

• Molecular Mass the sum of the atomic masses
  making up the molecule.
• CH4 16 (low res) 16.0313 (high res)
• units are Daltons (Da)
• Absolute atomic mass 1 da 1.66. 10-24g
• 931 meV
• Isotopes elements containing the same number of
  protons but different numbers of neutrons in the
  nucleus
• 1H1 1H2 1H3 17Cl35 17Cl37
• Chemical atomic masses Include the contribution
  from isotopes giving non-integer mean atomic
  masses.
• Cl35Cl37 exhibits 31 natural abundance,
  therefore mean atomic mass is (35x3)37 35.5
• 4
• Spectrum A collection of similar but
  significantly different objects e.g.
• people, wavelengths, ionic masses.

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Mass Spectrometry

• Main Components of a modern MS
• Vacuum System
• Sample Introduction
• Ionisation Source
• Mass Analyser
• Ion Detection System
• Computer for data recording and processing

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Mass Spectrometry

• Vacuum system required for the instrument to
  operate.
• Ions once formed have to travel through the
  instrument to reach a detector, this can mean
  travelling distances in excess of 1 metre.
• At atmospheric pressure this would not be
  possible as ions would be scattered by collisions
  with gas molecules.
• High vacuum is required and is provided by rotary
  rough vacuum pumps (10-3 torr) in combination
  with turbo-molecular pumps (10-8 torr). On older
  systems the high vacuum may be provided by oil
  vapour diffusion pumps.
• High vacuum is required particular in the mass
  analyser region of the instrument for correct
  operation.

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Mass Spectrometry

• Sample introduction
• Solids or liquids on probe - direct insertion
  probe (DIP) can be heated to vapourise sample.
• Volatile liquids and gasses can be sampled
  through a heated inlet with a molecular leak
  directly into the ion source. Provides a
  continuous steady flow of sample into the ion
  source. Often used to introduce mass calibration
  compounds for tuning and calibration of the
  instrument.
• Gas Chromatograph
• Liquid Chromatograph

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Mass Spectrometry

• Ionisation sources and methods
• Electron Ionisation
• Utilises a heated filament (usually Re or W) to
  produce electrons. These are accelerated through
  a small potential difference (typically 70V to
  give electrons with 70eV energy)
• Small quantity of sample is then introduced into
  the source.
• Electrons collide with the sample molecules and
  produce ions e- M ? M. 2e-
• Ions formed are radical cations which
  subsequently undergo a series of fragmentation
  reactions to yield the fingerprint mass
  spectrum.

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Mass Spectrometry
Ion Repeller
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Mass Spectrometry

• Electron Ionisation cont.
• Ions formed are at very low pressure (5x10-6
  torr).
• Fragmentation is a result of UNIMOLECULAR
  reactions and is solely due to the internal
  energy of the ions.
• Internal energy is imparted during the ionisation
  step.
• Ionisation energies of most organic molecules are
  in range 7-10eV (1eV 96.5 kJ mol-1).
• Ionising electrons have 70eV and usually impart
  5-8eV of internal energy to the molecular ion as
  it is formed.
• The internal energy imparted to the ions during
  formation is more than enough to break bonds
  (2-4eV) and cause fragmentation.
• Fragmentation is through real chemical reactions
  with defined mechanisms and IS NOT random.
• Ions are present in the ion source region for a
  few microseconds so fragmentation reactions have
  to be fairly fast.
• Ion source is maintained at around 200 C to
  prevent contamination.

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Mass Spectrometry

• 70eV chosen as it is where the graph is flat so
  any variation in energy will not affect the
  spectra.
• Lower eV is sometimes used to boost the relative
  amount of the molecular ion, however it always
  results in many fewer ions being produced (see
  next slide).

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Mass Spectrometry
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Mass Spectrometry

• Electron Ionisation cont.
• The spectra feature extensive fragmentation which
  is related to the molecular structure.
• The spectra are fingerprints of the molecules
  and as such can be used to search spectral
  databases for identification of unknown samples.
• Unknown spectra can be interpreted from a
  knowledge of chemistry and mass spectrometry.
• Disadvantages of the technique The molecules
  have to be heated to vapourise them which can
  cause thermal decomposition is some cases,
  particularly more polar substances.
• Occasionally the fragmentation is too extensive
  and the molecular ion is not seen. This
  represents a loss of valuable information.

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Mass Spectrometry

• Ionisation Sources and methods
• Chemical Ionisation
• Alternative to electron ionisation which utilises
  a reagent gas to produce reagent ions which will
  undergo ion-molecule interactions with the
  sample.
• Suitable reagent gases are many and varied but
  include methane, iso-butane and ammonia.
• Basic source design is similar to EI but is made
  more gas tight so that a local high pressure of
  the reagent gas is created inside the ion source
  volume (1 -10 torr)

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Mass Spectrometry

• Chemical Ionisation cont.
• Reagent ion formation for methane
• CH4 e- ? CH4. CH3. CH2.
• CH4. CH4 ? CH5 CH3. (reagent ion)
• also
• CH3. CH4 ? C2H5 H2
• Main reaction is proton transfer to sample
• M CH5 ? MH CH4

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Mass Spectrometry

• Chemical Ionisation cont.
• Ammonia is another popular reagent gas
• see if you can write down equivalent reactions
  for ammonia below

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Mass Spectrometry

• Chemical Ionisation cont.
• Other reactions are possible including
• Charge exchange (electron transfer)
• Electrophilic addition
• Anion abstraction
• Some fragmentation can occur and the degree of
  fragmentation can be somewhat controlled by
  choice of reagent gas. The degree of
  fragmentation is related to the internal energy
  of the protonated species formed and this is
  dependant on the difference in proton affinity
  (P.A.) between the sample and the reagent.
• RH M ? R MH Enthalpy (H) P.A.(R)
  P.A.(M)
• P.A. (Organic Molecules) 600-900 kJ mol-1
• P.A. (CH4) 536 kJ mol-1
• P.A. (NH3) 847 kJ mol-1

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Mass Spectrometry
Methyl Stearate EI and CI spectra
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Mass Spectrometry

• Chemical Ionisation cont.
• Advantages low energy soft ionisation
  technique. This gives good molecular mass
  information but limited, if any, fragmentation.
  Technique is good for quantitative analysis or
  for identifying molecular mass when EI fails.
  However it is not good for qualitative work as
  the structural information from fragmentation is
  very limited.
• Disadvantages still relies on heating to
  vapourise the sample. Therefore the same issues
  with thermal decomposition of more polar analytes
  still arise.

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Mass Spectrometry
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Mass Spectrometry
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Mass Spectrometry

• Ionisation Sources and methods
• Desorption techniques
• Desorption Chemical Ionisation (DCI)
• Experimentally simple fits with existing CI
  system
• Molecular Ion and fragment information but not
  to very high mass.
• Thermal degradation can occur, therefore need to
  use rapid scanning with resultant loss in
  sensitivity.
• Negative ion spectra readily obtained.
• Compound application lowest of these three
  techniques.
• Inexpensive.

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Mass Spectrometry

• Desorption techniques cont.
• Field Desorption/ Field Ionisation (FD/FI)
• Considerable expertise required in preparing and
  handling emitters.
• Molecular ion data up to 10,000 Da.
• Low sensitivity.
• Polar compounds easily analysed.
• Negative ions possible but difficult.
• Extremely expensive.

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Mass Spectrometry

• Desorption techniques cont.
• Fast Atom Bombardment (FAB)
• Less complex than FD/FI, requires less operator
  technique.
• Excellent results with bio-molecules but less
  successful with polar compounds.
• Negative ion spectra easily obtained.
• High background ions particularly at low mass
  may obscure sample peaks.
• Intermediate in price.

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Mass Spectrometry

• Interfacing to GC systems.
• Interfacing to GC relatively easy due to all gas
  phase technique.
• Packed column GC requires interface and sample
  enrichment due to high gas flow rates e.g. 30-40
  ml min-1.
• Typical interfaces for packed column are
• membrane separator and jet separator.
• Both designed to enrich sample vapour at the
  expense of carrier gas (mobile phase) and allow
  the MS to retain the required high vacuum
  conditions.
• Can work with EI and CI ionisation techniques.
• Capillary column GC uses flow rates of 1-2ml
  min-1 of carrier gas. These flow rates are
  compatible with modern MS vacuum systems so no
  interface is required and the GC column exits
  directly into the MS ion source.
• Can work with both EI and CI ionisation methods.

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Mass Spectrometry

• Interfacing to LC systems
• Causes an immediate problem since analytical HPLC
  carried out with solvent flows of 1-2 ml min-1.
  This amount of solvent on expansion into the MS
  vacuum system overwhelms the pumps resulting in
  loss of vacuum and failure of the instrument.
• Therefore need to remove mobile phase solvent and
  enrich sample without losses.
• Early attempts used direct liquid introduction
  with flow splitters but this resulted in loss of
  sensitivity.
• A number of interfaces have been developed and
  fallen away including moving belt and
  thermospray.
• Currently Atmospheric Pressure Chemical
  Ionisation (APCI) and Electrospray (ESP) are the
  interfaces and ionisation methods of choice for
  combining LC with MS. Both operate at atmospheric
  pressure in the ion source region.

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Mass Spectrometry
Thermospray Ion Source
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Mass Spectrometry

• Electrospray Principles
• Electrospray requires three consecutive steps.
• NEBULISATION Production of small highly charged
  droplets at near atmospheric pressure conditions.
• Formation of multiply charged ions by ion
  desorption.
• Measurement of the m/z ratio of the ions produced.

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Mass Spectrometry
Electrospray Ion Source
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Mass Spectrometry

• Electrospray ionisation mechanism
• Critical field value is called Rayleigh Limit
• Occurs when electrostatic repulsion between ions
  is sufficient to overcome the surface tension
  holding the drop together.
• Emitted ions are highly charged, multiple
  protonation.

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Mass Spectrometry
Typical electrospray mass spectrum of a protein
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Mass Spectrometry

• If a pair of neighbouring peaks are selected then
  the only difference is in the measured m/z value
  and the number of protons attached.
• A pair of simultaneous equations can be set up
  and solved for n (the number of protons)
• The molecular mass can then easily be calculated.
• The data system will do this for each pair of
  peaks to give the molecular mass and the standard
  deviation of the measurements.

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Mass Spectrometry

• Electrospray summary
• Works best with aqueous solvents or aqueous
  /organic mixtures e.g. reverse phase.
• Soft ionisation technique- produces
  predominantly molecular ion species, MH,
  MNa, MNH4 and occasionally Msolvent
• Clusters are sometimes observed, 2MH,
  3MH, 4MH etc.
• Structural information from fragmentation is only
  available from tandem mass spectrometry
  techniques.
• Very good for large molecules up to approx.
  200kDa.
• Less useful for small molecules due to
  interferences from solvent ions at low mass.

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Mass Spectrometry

• Ionisation Sources and methods
• Desorption techniques cont.
• Matrix Assisted Laser Desorption / Ionisation
  (MALDI)
• Sample is admixed with a large excess of matrix
  (1500).
• Sample matrix mixture (1µL) deposited on sample
  target and dried.
• Target placed inside ion source of instrument.
• Sample irradiated with laser light typically from
  N2 laser at 337 nm (UV).

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Mass Spectrometry

• MALDI cont.
• Laser irradiation causes ions and neutrals to be
  desorbed from the solid surface.
• Ions are then accelerated into the mass analyser
  by an applied voltage (up to 30kV).
• Positive and negative ions are formed but
  neutrals are most abundant.
• Ions formed by mixed and complex reactions
  including some or all of the following
• Solution phase acid/base equilibria.
• Gas phase ion/molecule reactions.
• Photoionization.

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Mass Spectrometry
Common matrices for MALDI with N2 laser at 337nm
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Mass Spectrometry
MALDI MS of some salivary proteins in the 3,000
to 17,000 Da range
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Mass Spectrometry
MALDI MS of DNA oligomers (11,15 and 20 mer)
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Mass Spectrometry
MALDI MS of Glycoprotein, note the multiple
charged ions
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Mass Spectrometry

• MALDI summary.
• Despite the laser this technique is regarded as a
  soft ionisation method.
• Produces protonated or cation adduct ions,
  MH, MNa and MK and occasionally
  Mmatrix
• Sometimes ion clusters are observed, 2MH,
  3MH etc.
• Little fragmentation is seen.
• Generally singly charged ions dominate the
  spectrum, but multiple charges are seen.
• Structural information from fragmentation is
  available from tandem mass spectrometric methods.
• MALDI is fairly tolerant of interferences from
  sample buffers etc.
• although suppression effects are sometimes noted.

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Mass Spectrometry

• Maximum tolerated contaminant concentrations for
  peptide analysis using sinapinic acid matrix.
• Phosphate buffer 20mM
• Tris buffer 50nM
• Detergents 0.1
• SDS 0.01
• Alkali metal salts 1M
• Glycerol 2
• NH4 bicarbonate 30mM
• Guanidine 1
• Sodium azide 1