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Title: Mass Spectrometry and Related Techniques 1


1
Mass Spectrometry and Related Techniques 1
Lecture Date February 20th, 2012
2
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
3
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
4
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

5
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.

6
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.
7
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
8
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).
9
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-
10
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!)

11
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).
12
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

13
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

14
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).
15
APCI Chemical Ionization
  • An APCI source

760 torr
10-6 torr
Diagram from Agilent Technologies
16
APCI Chemical Ionization
  • An APCI mass spectrum

Diagram from Agilent Technologies
17
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

18
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
19
Electrospray Ionization (ESI)
  • An ESI source

Diagram from Agilent Technologies
20
Electrospray Ionization (ESI)
  • A selection of modern ESI and heated ESI designs

Stanke et al., J. Mass. Spectrom. 2012, 47,
875884.
21
Typical ESI Spectra
  • An ESI mass spectrum

Diagram from Agilent Technologies
22
Typical ESI Spectra
  • An ESI mass spectrum of a 14.4 kDa enzyme

Diagram from http//www.nd.edu/masspec/ions.html
23
ESI and APCI
  • ESI and APCI are complementary techniques for
    solution-phase analytes

Figure from Agilent Instruments
24
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-
25
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.
26
Atmospheric Phase Photo-ionization
  • APPI can ionize things that ESI and APCI cant

27
Comparison of Ionization Methods
  • How to choose an ionization technique

Figure from Agilent Instruments
28
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
29
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.
30
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)
31
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).
32
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

33
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.
34
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
35
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
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