Ionisation techniques

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Lecture 3

• Ionisation techniques
• Gas Phase Ionisation Techniques
• Chemical Ionisation

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At the end of this lecture you should be able

• To explain how chemical ionisation works
• To instruct a MS operator about the type of
  chemical ionisation reagent gas required for your
  experiment

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Ionisation Techniques Overview

• Gas-Phase Methods
• Electron Impact (EI)
• Chemical Ionization (CI)

• Desorption Methods
• Secondary Ion MS (SIMS) and Liquid SIMS
• Fast Atom Bombardment (FAB)
• Laser Desorption/Ionization (LDI)
• Matrix-Assisted Laser Desorption/Ionization
  (MALDI)

• Spray Methods
• Atmospheric Pressure Chemical Ionization (APCI)
• Electrospray (ESI)

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EI electron ionisation recap

• 1st step sample must be in gas phase
• 2nd step bombarded by electron beam
• Generates high-energy analyte ions, which can
  fragment
• Analyte ions are always odd-electron
• Advantages Simple to use, provides
  library-searchable fingerprint data
• Disadvantages
• Applicable only to volatile (i.e. small) and
  thermally stable compounds
• Extensive fragmentation, can be difficult to
  detect molecular ion

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Chemical ionisation

• Introduced by Munson and Field 1966
• Ion source similar to that for EI
• Suitable for small, volatile molecules
• Higher pressures ca. 1 Torr for ionisation, 10-4
  Torr for injection into mass analyser
• Generates less energetic, more stable ions
• CI yields even-electron ions more stable
• Mainly molecular ion
• Simple spectra But fragmentation not
  straightforward
• Good for mixtures and quantitation
• Routinely used in gas chromatography (GC-MS)

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Chemical ionisation - details

• Step 1 Reagent gas R, present in large excess
  (10 to 100 fold higher partial pressure) over
  analyte, is ionised (leading to R?) at 0.1-1
  Torr by electron beam of 200-500 eV e.g. CH4 ?
  CH4? ? CH3, CH2?
• Step 2 Stable reagent ions are generated via
  ion-molecule interaction e.g. CH4? CH4 ?
  CH5 CH3? CH3 CH4 ? C2H5 H2
  CH2? CH4 ? C2H3 H2 H? C2H3
  CH4 ? C3H5 H2
• Step 3 Ion-molecule interactions generate MH
  of analyte (see next slide)

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Mechanisms of chemical ionisationIon-molecule
interactions between reagent gas and analyte

• Most important Proton transfer
• Reagent gases generate Brønsted acids, e.g. CH5,
  C2H5, H3
• Gas-phase acid-base reactions, e.g.M C2H5 ?
  MH C2H4
• Other mechanisms
• Adduct formation M C2H5 ? MC2H5
• Anion abstraction M C2H5 ? M-H C2H6

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Selective fragmentation after proton transfer

• Parent ion, e.g. MH, can fragment
• Extent of fragmentation is proportional to
  transferred energy during ion-molecule
  interaction
• Transferred energy depends on exothermicity of
  reaction
• Exothermicity is function of proton affinities
  (PA) of reagent gas (R) and analyte (M)
• R H ? RH PA(R) -DH (of this
  reaction)
• M H ? MH PA(M) -DH (of this
  reaction)
• M RH ? MH R
• DH0 PA(M) PA(R)
• Exothermic (DH0lt0) if PA(M)gtPA(R)

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Proton affinities of common reagent gases
(kJ/mole)

• Methane, CH4 423
• Ammonia, NH3 854
• Iso-butane, (CH3)3CH 819
• Ethane 601
• Water 697
• Methanol 761
• Hydrogen 423
• Acetone 823
• Methylamine 882

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Example selective fragmentation
137
H
O

O
CH4
PA423 kJ/mole
Lavanduyl acetate (MW 196) PA 840 kJ/mole
Iso-butane
137
197 MH
PA819 kJ/mole
95
123
81
109
NH3
214 MNH4
197
PA854 kJ/mole
137
100
200
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Other modes of CI

• Charge-Exchange Chemical Ionisation with
  toluene, benzene, NO, CS2, COS, Xe, CO2, CO, N2,
  Ar, He as reagent gas
• M X? ? M? X (creates radical cations)
• Can use mixtures to generate both kinds of ions
  (conventional CI and CE-CI)
• Negative CI electron capture

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Self-assessment questions

• Q1 Describe chemical ionisation mass
  spectrometry. How does it work, what is the
  nature of the reagent gas, what function(s) does
  the gas serve, and what type of mass spectra are
  generated from the analyte species ?
• Q2 Compare and contrast EI and CI
• Q3 Explain why EI and CI are not applicable to
  large non-volatile samples.
• Q4 Explain how the choice of reagent gas (eg NH3
  or CH4) affects the appearance of the mass
  spectra in chemical ionisation with respect to
  ionisation by proton transfer.

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Lecture 4

• Condensed phase ionisation techniques (1)
• Desorption methods

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At the end of this lecture you should be able to

• describe the differences and similarities of
  SIMS, LSIMS and FAB
• explain how laser desorption works
• describe MALDI and preparation of samples

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Condensed phase ionisation techniques (1) solid
state samples

• Field desorption (FD)
• Plasma desorption (PD)
• Secondary-ion Mass Spectrometry (SIMS)
• Fast Atom Bombardment (FAB)
• Laser Desorption/Ionisation (LDI)
• MALDI

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Field ionisation/field desorption

• Developed in 1969 by Beckey
• No primary beam to bombard sample
• Field ionisation Volatile samples brought into
  gas phase e.g. by heating
• Field desorption Non-volatile sample is applied
  to whiskers which are grown on thin metallic
  wire filament (emitter)
• FD Suitable for non-volatile and thermally
  labile samples, e.g. peptides, sugars, polymers,
  organometallics, carbohydrates

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Field ionisation/ field desorption

• Ionisation is induced by high electric field
  gradient (108 V/cm)
• Distorts electron cloud around atoms and
  facilitates electron tunnelling from sample
  molecules to emitter electrode
• Yields M? , then MH
• Hardly any fragmentation

emitter
to cathode
M adsorbed on emitter
electron tunnels
M? is desorbed
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Secondary Ion Mass Spectrometry (SIMS)

• Mainly for surface analysis
• Beam of Ar (or Xe) ions with energy of 5-15 keV
  bombards solid surface
• Secondary ions from surface are sputtered
• Used for
• Mass analysis
• Chemical composition of material
• Drawback Rapid damage to surface rapid decrease
  in signal

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rch/hurricanes_slide9.html
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Variations of SIMSFast atom bombardment (FAB)
and Liquid SIMS

• FAB Developed in 1980 by Barber et al.
• Improved version of SIMS
• Sample is dissolved in inert liquid matrix
• Common Matrix Glycerol (amongst others).
  Protects sample from destruction and helps
  ionisation and desorption
• FAB Bombardment with high-energy ATOMS (e.g. Xe)
• LSIMS Similar, but bombardment with IONS (e.g.
  Cs at 25-40 keV) instead of ATOMS
• Mass limits 7 kDa standard, 24 kDa possible
• Often used in conjunction with magnetic sector
  mass analysers

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FAB schematic
Slow Xe0
1. Ionisation ? slow Xe 2. Acceleration of Xe
ions 3. Neutralisation by collision and charge
exchange with slow atoms Xe(fast)
Xe(slow)? Xe(fast) Xe(slow)
Atom gun
Primary beam
Fast Xeo
MH
Sample ion beam
probe
Sample
Extraction and focusing
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Laser Desorption/Ionisation (LDI)

• Solid sample
• Laser beam with UV, Vis, or IR wavelength
• Sample required to absorb at laser wavelength
• Applied in surface and cluster analysis
• Drawbacks
• Difficult to control
• Thermal degradation
• No or low molecular ion
• Only useful for lt 1kDa

Laser beam
Desorbed ions and neutral species
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Matrix-assisted Laser Desorption/Ionisation
(MALDI)

• Nobel Prize in 2002
• Soft ionisation technique
• Generates low-energy ions
• Lasers UV or IR
• Most frequently combined withTOF mass analyser
• Can work forup to 1 MDa

Laser beam
Analyte molecule/ion
Matrix molecules
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Analyte ionisation in MALDI

• Step 1 Laser beam generates reactive/ excited
  matrix ionic species
• Matrix ions can be protonated, deprotonated,
  sodiated, or radical cations
• Step 2 In-plume ion-molecule charge transfer
  reactions between matrix ions and neutral analyte
  molecules
• Reactions Proton transfer,cation transfer,
  electrontransfer, electron capture

Plume Ions and molecule in gas phase
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MALDI sample preparation

• Sample/matrix mix (110,000 molar excess) in
  volatile solvent
• Requires only pico- to femtomoles of analyte
• Matrices Solid organic, liquid organic, ionic
  liquids, inorganic materials

Drying
80x magnification of dried sample/matrix drop on
target
Sample target
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Instrumentation
Insertion of target into instrument
Most common combination MALDI-TOF Instrument
MALDI generates pulses of ions, TOF works with
pulses of ions
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MALDI matrices
Most common Organic solids, e.g.
2,5-Dihydroxybenzoic acid (gentisic acid C7H6O4)
3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic
acid C11H12O5)
a-Cyano-4-hydroxycinnamic acid (4-HCCA C10H7O3N)
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MALDI matrix

• absorbs photon energy and transfers it to analyte
• minimises aggregation between analyte molecules
• Matrix must
• Absorb strongly at Laser wavelength
• Have low sublimation temperature
• Have good mixing and solvent compatibility with
  analyte
• Have ability to participate in photochemical
  reaction

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Matrices and analytes desired photochemical
characteristics
Absorbance
Laser
matrix
analyte
200
500
Wavelength (nm)
Common lasers N2 (337 nm), ArF excimer (193),
Nd-YAG frequency tripled (355 nm) and quadrupled
(266 nm)
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ApplicationsMass determination of intact
proteins
www.membrane.unsw.edu.au/alumni/robert.htm

• MALDI-TOF spectrum of a protein mixture
• Predominantly M ions (singly charged)

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Applications Molecular weight distribution of
polymers
www.arkat-usa.org/?VIEWMANUSCRIPTMSID869
poly(dimethyl)siloxane 2.25 kD
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Summary - MALDI

• Disadvantages
• MALDI matrix cluster ions obscure low m/z (lt600)
  range
• Analyte must have very low vapor pressure
• Pulsed nature of source limits compatibility with
  many mass analyzers
• Coupling MALDI with chromatography is very
  difficult
• Analytes that absorb laser light can be
  problematic

• Advantages
• Relatively gentle ionization technique
• Very high MW species can be ionized
• Molecule need not be volatile
• Very easy to get femtomole sensitivity
• Usually 1-3 charge states, even for very high MW
  species
• Positive or negative ions from same spot

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Self-assessment questions

• Q1 Describe SIMS, LSIMS and FAB
• Q2 In FAB, how is the fast atom beam produced and
  why is a fast atom beam used instead of the ion
  beam for the production of the secondary ions?
• Q3 How does Laser Desorption/Ionisation work?
• Q4 Why is LDI not being used with high molecular
  weight molecules?
• Q5 Describe MALDI and sample preparation for
  MALDI
• Q6 Explain why time-of-flight is suitable for
  mass detection in MALDI. Given the choice
  between a sector instrument and a TOF instrument,
  which one would you use to detect MALDI produced
  ions of 100 kDa and why ?