Mass Spectrometry and Related Techniques 1

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Mass Spectrometry and Related Techniques 1
Lecture Date February 20th, 2012
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Mass Spectrometry

• Mass Spectrometry (a.k.a. MS or mass spec) a
  method of separating and analyzing ions by their
  mass-to-charge ratio
• MS does not involve a specific region of the
  electromagnetic spectrum (because it is not
  directly interested in the energies of emitted
  photons, electronic or vibrational transitions,
  nuclear spin transitions, etc)

Ion abundance
Ion
Ion
Ion
Up to m/z 100000!
m/z
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History of Mass Spectrometry

• J. J. Thomson at Cambridge reported the first MS
  experiment in 1913 and discovered isotopes.
• F. W. Aston built the first MS in 1919 and
  studied isotopes, winning the 1922 Nobel Prize in
  Chemistry.
• In the 1930s, Ernest Lawrence invented the
  calutrons used in WW2 to separate 235U.
• Nobel Prize in Physics (1989) to Wolfgang Paul
  for the ion trap.
• Nobel Prize in Chemistry (2002) to John Fenn
  (electrospray ionization) and Koichi Tanaka
  (MALDI).

J. J. Thomson
F, W, Aston
Calutron at the Y-12 Plant at Oak Ridge,
Tennessee, used during the Manhattan Project
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General Notes on Atomic and Molecular Mass

• Helpful units and conversions
• 1 amu 1 Da 1/12 the mass of a neutral 12C
  atom.
• 1 kDa 1000 amu
• Atomic weights of other elements are defined by
  comparison.
• Mass-to-charge ratio (m/z) the ratio of the
  mass of an ion (m) to its charge (z)
• Molecular ion an ion consisting of essentially
  the whole molecule

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Mass Spectrometers

• A block diagram of a generic mass spectrometer

Ionization Source
Mass Analyzer
Detector

• This lecture covers the ionization source the
  method of making the ions for MS analysis.

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Ionization Sources

• Electron Ionization (EI)
• Chemical Ionization (CI/APCI)
• Photo-ionization (APPI)
• Electrospray (ESI)
• Matrix-assisted Laser Desorption (MALDI)
• Field Desorption (FD)
• Plasma Desorption (PD)
• Fast atom bombardment (FAB)
• High-temperature Plasma (ICP)

Gas Phase
Desorption
Ionization Source
Mass Analyzer
Detector
See also Table 20-1 in Skoog, et al.
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EI Electron Ionization/Electron Impact

• The electron ionization (EI) source is designed
  to produce gaseous ions for analysis.
• EI, which was one of the earliest sources in wide
  use for MS, usually operates on vapors (such as
  those eluting from a GC)

Heated Incandescent Tungsten/Rhenium Filament
e-
Accelerate!
70 eV
Vaporized Molecules
To Mass Analyzer
Ions
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EI Electron Ionization/Electron Impact

• How EI works
• Electrons are emitted from a filament made of
  tungsten, rhenium, etc
• They are accelerated by a potential of 70 V
• The electrons and molecules cross (usually at a
  right angle) and collide
• The ions are primarly singly-charged, positive
  ions, that are extracted by a small potential
  (5V) through a slit

Diagram from F. W. McLafferty, Interpretation of
Mass Spectra, 3rd Ed., University Science Books,
Mill Valley, CA (1980).
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EI Electron Ionization/Electron Impact

• When electrons hit the molecules undergo
  rovibrational excitation (the mass of electrons
  is too small to really move the molecules)
• About one in a million molecules undergo the
  reaction

M e-
M 2e-
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EI Electron Ionization/Electron Impact

• Advantages
• Results in complex mass spectra with fragment
  ions, useful for structural identification
• Disadvantages
• Can produce too much fragmentation, leading to no
  molecular ion (makes structural identification
  difficult!)

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CI Chemical Ionization

• Chemical ionization (CI) is a form of gas-phase
  chemistry that is softer (less energetic) than
  EI
• In CI, ionization occurs via proton transfer
  reactions
• A gas (ex. methane, isobutane, ammonia) is
  introduced into the source at 1 torr.
• Example CH4 reagent gas

EI
CH4
CH4
CH4 CH4
CH5 CH3
AH CH5
AH2 CH4
Strong acid
See B. Munson, Anal. Chem., 49, 772A (1977).
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CI Hard and Soft Sources

• The energy difference between EI and CI is
  apparent from the spectra
• CI gases
• harshest (most fragments) methane
• softest ammonia

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APCI Atmospheric-Pressure Chemical Ionization

• APCI a form of chemical ionization using the
  liquid effluent in a spray chamber as the reagent
• APCI is a form of API (atmospheric pressure
  ionization or ambient ionization) - these are a
  range of ionization techniques that operate at
  higher pressures, outside the vacuum MS regions,
  and sometimes at normal pressures and
  temperatures
• Examples of ambient ionization methods to be
  discussed later in this lecture DESI, MALDI

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APCI Atmospheric-Pressure Chemical Ionization

• The APCI process
• The sample is in a flowing stream of a carrier
  liquid (or gas) and is nebulized at moderate
  temperatures.
• This stream is flowed past an ionizer which
  ionizes the carrier gas/liquid.
• 63Ni beta-emitters
• Corona (electric) discharge needle at several kV
• The ionized stream (which can be an LC solvent)
  acts as the primary reactant ions, forming
  secondary ions with the analytes.
• The ions are formed at AP in this process, and
  are sent into the vaccuum
• In the vaccuum, a free-jet expansion occurs to
  form a Mach disk and strong adiabatic cooling
  occurs.
• Cooling promotes the stability of analyte ions
  (soft ionization)

See A. P. Bruins, Mass Spec. Rev., 10, 53-77
(1991).
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APCI Chemical Ionization

• An APCI source

760 torr
10-6 torr
Diagram from Agilent Technologies
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APCI Chemical Ionization

• An APCI mass spectrum

Diagram from Agilent Technologies
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Electrospray Ionization (ESI)

• The ESI process
• Electrospray ionization (ESI) is accomplished by
  flowing a solution through an electrically-conduct
  ive capillary held at high voltage (several keV
  DC).
• The capillary faces a grid/plate held at 0 VDC.
• The solution flows out of the capillary and feels
  the voltage charges build up on nebulized
  droplets, which then begin to evaporate
• Coulombic explosions occur when the repulsion of
  the charges overcomes the surface tension of the
  solution (holding the drop together) known as
  the Rayleigh limit.
• Depending on whose theory you believe
• the analyte ion is eventually the only ion left
• orthe analyte ion is evaporated from a small
  enough droplet

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Electrospray Ionization (ESI)

• A picture of two ideas for the electrospray
  process

Note ions which are surface-active will be
preferentially ionized this can lead to ion
suppression!

• The Taylor cone the shape of the cone that
  shoots from the needle when surface tension is
  overcome by electrostatic forces, and forms a jet

El Aneed, et al. , Applied Spectroscopy Reviews,
44 210230, 2009. Jet image from
http//www.newobjective.com/electrospray/electrosp
ray.html
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Electrospray Ionization (ESI)

• An ESI source

Diagram from Agilent Technologies
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Electrospray Ionization (ESI)

• A selection of modern ESI and heated ESI designs

Stanke et al., J. Mass. Spectrom. 2012, 47,
875884.
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Typical ESI Spectra

• An ESI mass spectrum

Diagram from Agilent Technologies
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Typical ESI Spectra

• An ESI mass spectrum of a 14.4 kDa enzyme

Diagram from http//www.nd.edu/masspec/ions.html
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ESI and APCI

• ESI and APCI are complementary techniques for
  solution-phase analytes

Figure from Agilent Instruments
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ESI and APCI

• ESI and APCI complementary techniques

ESI APCI
Very soft ionization can ionize thermally labile samples Some sample volatility needed (nebulizer)
Ions formed in solution Ions formed in gas phase
Singly- and multiply-charged ions MH Singly-charged ions, MH and M-H-
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Atmospheric Phase Photo-ionization

• APPI ionizes using UV irradiation and (usually) a
  dopant

D. A. Robb and M. W Blades, Anal. Chim. Acta,
2008, 627, 34-49.
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Atmospheric Phase Photo-ionization

• APPI can ionize things that ESI and APCI cant

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Comparison of Ionization Methods

• How to choose an ionization technique

Figure from Agilent Instruments
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MALDI Matrix-Assisted Laser Desorption/Ionizatio
n

• A method for desorbing a sample with a laser,
  while preventing thermal degradation
• A sample is mixed with a radiation-absorbing
  matrix used to help it ionize
• MALDI is heavily used for large biomolecules and
  polymers.

Diagram from Koichi Tanaka (Nobel Lecture), 2002
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MALDI Matrix Effects

• The role of the matrix
• Must absorb strongly at the laser wavelength
• The analyte should preferably not absorb at this
  wavelength
• Common matrices include nicotinic acid and many
  other organic acids

Batoy et al., Applied Spectroscopy Reviews, 2008,
43, 485550.
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MALDI at Atmospheric Pressure

• Advantages fast, easy and sensitive
• Disadvantages no LC, matrix still needed

S. Moyer and R. Cotter, Atmospheric Pressure
MALDI, Anal. Chem., 74, 468A-476A (2002)
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FAB Fast Atom Bombardment

• A soft ionization technique
• Often used for polar, higher-mwt, thermally
  labile molecules (masses up to 10 kDa) that are
  thermally labile.
• Samples are atomized by bombardment with keV
  range Ar or Xe atoms.
• The atom beam is produced via an electron
  exchange process from an ion gun.

e-
Xe
Xe? 2e-
accel
Xe?
Xe? (high KE)
Xe? (high KE) Xe
Xe (high KE) Xe?

• Advantages
• Rapid sample heating reduced fragmentation
• A glycerol solution matrix is often used to make
  it easier to vaporize ions

K. L. Rinehart, Jr., Science, 218, 254 (1982) K.
Biemann, Anal. Chem., 58, 1288A, (1986).
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SIMS Secondary Ion MS

• Focused Ion Beam 3He, 16O, 40Ar
• Beam energy 5 to 20 keV
• Beam diameter 0.3 to 5 mm
• Beam Hits Target
• A small of the target material is sputtered
  off and enters the gas phase as ions (usually
  positive)
• Advantages
• Imaging of ions (characteristic masses) on a
  surface or in biological specimens
• Surface analysis using beam penetration
  depth/angle
• Can be used for both atomic and molecular
  analysis
• Sensitive to low levels, picogram, femtogram and
  lower
• Will discuss more in surface analysis/microscopy
  talk

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Desorption Electrospray DESI

• Desorption-electrospray ionization (DESI) is an
  ambient ionization technique
• A new technique for desorbing ions using
  supersonic jets of solvents (charged like in
  electrospray)

From Z. Takats et al., Science, 2004, vol 306,
p471.
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Inductively Coupled Plasma (ICP) as an MS Source

• The inductively-coupled plasma serves as an
  atomization and ionization source (two-in-one!)
  for elemental studies.

• See optical electronic lecture for more details
• Solution flow rates up to 50-100 mL/min

Photo by Steve Kvech, http//www.cee.vt.edu/progr
am_areas/environmental/teach/smprimer/icpms/icpms.
htmArgon20Plasma/Sample20Ionization
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Further Reading

• Required (please skim)
• J. Cazes, Ed. Ewings Analytical Instrumentation
  Handbook, 3rd Ed., Marcel Dekker, 2005, Chapter
  7.
• Optional
• http//www.spectroscopynow.com/raman/details/educa
  tion/sepspec13199education/Introduction-to-Raman-S
  pectroscopy-from-HORIBA-Jobin-Yvon.html
• D. A. Skoog, F. J. Holler and S. R. Crouch,
  Principles of Instrumental Analysis, 6th Edition,
  Brooks-Cole, 2006, Chapter 18.
• D. A. Long, The Raman Effect, Wiley, 2002.
• S. Hooker, C. Webb, Laser Physics, Oxford, 2010.
• P. W. Atkins and R. S. Friedman, Molecular
  Quantum Mechanics, 3rd. Ed., Oxford, 1997.
• http//www.rp-photonics.com/yag_lasers.html