Mass Spectrometry and Related
Techniques 1
Lecture Date: February 20th, 2012
Mass Spectrometry
 Mass Spectrometry (a.k.a. MS or “mass spec”) – a
method of separating and analyzing ions by their mass-tocharge 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
m/z
Up to m/z = 100000!
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 1930’s, Ernest Lawrence
invented the calutrons used in
WW2 to separate 235U.
Nobel Prize in Physics (1989) to
Wolfgang Paul for the ion trap.
J. J. Thomson
F, W, Aston
 Nobel Prize in Chemistry (2002) to
John Fenn (electrospray ionization)
and Koichi Tanaka (MALDI).
Calutron at the Y-12 Plant at Oak Ridge,
Tennessee, used during the Manhattan Project
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:
molecule
an ion consisting of essentially the whole
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.
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)
Ionization
Source
Mass
Analyzer
See also Table 20-1 in Skoog, et al.
Gas Phase
Desorption
Detector
EI: Electron Ionization/Electron Impact
 The electron ionization
(EI) source is designed
to produce gaseous ions
for analysis.
Heated Incandescent
Tungsten/Rhenium Filament
eAccelerate!
 EI, which was one of the
earliest sources in wide
use for MS, usually
operates on vapors
(such as those eluting
from a GC)
70 eV
Vaporized
Molecules
Ions
To
Mass
Analyzer
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).
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-
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!)
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
CH4
EI
CH4+
CH4+ + CH4
CH5+ + CH3
AH + CH5+
AH2+ + CH4
Strong acid
See B. Munson, Anal. Chem., 49, 772A (1977).
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
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
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).
APCI: Chemical Ionization
 An APCI source:
760 torr
10-6 torr
Diagram from Agilent Technologies
APCI: Chemical Ionization
 An APCI mass spectrum:
Diagram from Agilent Technologies
Electrospray Ionization (ESI)
 The ESI process:
– Electrospray ionization (ESI) is accomplished by flowing a
solution through an electrically-conductive 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
 or…the analyte ion is evaporated from a small enough droplet
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: 210–230, 2009.
Jet image from http://www.newobjective.com/electrospray/electrospray.html
Electrospray Ionization (ESI)
 An ESI source:
Diagram from Agilent Technologies
Electrospray Ionization (ESI)
 A selection of modern ESI and heated ESI designs:
Stanke et al., J. Mass. Spectrom. 2012, 47, 875–884.
Typical ESI Spectra
 An ESI mass spectrum:
Diagram from Agilent Technologies
Typical ESI Spectra
 An ESI mass spectrum of a 14.4 kDa enzyme:
Diagram from http://www.nd.edu/~masspec/ions.html
ESI and APCI
 ESI and APCI are complementary techniques for solutionphase analytes:
Figure from Agilent Instruments
ESI and APCI
 ESI and APCI –complementary techniques:
ESI
APCI
Very “soft” ionization –
can ionize thermally
labile samples
Ions formed in solution
Some sample volatility
needed (nebulizer)
Singly- and multiplycharged ions [M+H]+
Singly-charged ions,
[M+H]+ and [M-H]-
Ions formed in gas
phase
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.
Atmospheric Phase Photo-ionization
 APPI can ionize things that ESI and APCI can’t:
Comparison of Ionization Methods
 How to choose an ionization technique:
Figure from Agilent Instruments
MALDI: Matrix-Assisted Laser
Desorption/Ionization
 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
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, 485–550.
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)
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.
Xe
Xe+
Xe+ (high KE) + Xe
eaccel
Xe+ + 2eXe+ (high KE)
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).
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…
Desorption Electrospray: DESI
 Desorptionelectrospray
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.
Inductively Coupled Plasma (ICP) as an MS Source
 The inductively-coupled
plasma serves as an
atomization and
ionization source (two-inone!) for elemental
studies.
 See optical electronic lecture

for more details
Solution flow rates up to: 50100 mL/min
Photo by Steve Kvech, http://www.cee.vt.edu/program_areas/environmental/teach/smprimer/icpms/icpms.htm#Argon%20Plasma/Sample%20Ionization
Further Reading
Required (please skim):
J. Cazes, Ed. Ewing’s Analytical Instrumentation Handbook, 3rd Ed., Marcel Dekker, 2005,
Chapter 7.
Optional:
http://www.spectroscopynow.com/raman/details/education/sepspec13199education/Introdu
ction-to-Raman-Spectroscopy-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