1

Lecture Date: February 25

th
, 2008
Mass Spectrometry and Related
Techniques 1
Ion and Particle Spectrometry 1 - Outline
- Atomic and Molecular Mass Spectrometry
Skoog et al. Chapter 11 (atomic) and Chapter 20 (Molecular).
Cazes Chapter 14
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
Up to m/z =100000! m/z
Ion
Ion
Ion
General Notes on Atomic and Molecular Mass
- Helpful units and conversions:
1 amu =1 Da =1/12 the mass of a neutral
12
C 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
3
Mass Spectrometers
- A block diagram of a generic mass spectrometer:
Ionization
Source
Mass
Analyzer
Detector
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)
Seealso Table20-1in Skoog, et al.
Desorption
Gas Phase
Ionization
Source
Mass
Analyzer
Detector
4
EI: Electron Ionization/El ectron 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/RheniumFilament
Accel!
E-
Vaporized
Molecules
70 eV
Ions
To
Mass
Analyzer
EI: Electron Ionization/El ectron 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
- See also Fig. 20-3, pg.
502 in Skoog, et al.
DiagramfromF. W. McLafferty, Interpretation of Mass Spectra, 3rd Ed., University ScienceBooks, Mill Valley, CA (1980).
5
EI: Electron Ionization/El ectron 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 millionmolecules undergo the reaction:
M+e
-
M
+
+2e
-
EI: Electron Ionization/El ectron Impact
- Advantages:
Results in complex mass spectra with fragment ions, useful for
structural identification
- Disadvantages:
Can produce too much fragmentation, leading to no molecular
ions! (makes structural identification difficult!)
6
CI: Chemical Ionization
- Chemical ionization (CI) is a form of gas-phase
chemistry that is softer (less energetic) than EI
Ionization via proton transfer reactions
- A gas (ex. methane, isobutane, ammonia) is introduced
into the source at ~1 torr.
- Example: CH
4
reagent gas
CH
4
EI
CH
4
+
CH
4
+
+CH
4 CH
5
+
+CH
3
AH +CH
5
+
AH
2
+
+CH
4
Strong acid
SeeB. 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
7
APCI: Atmospheric-Pressure Chemical
Ionization
- A form of API (atmospheric pressure ionization) a
range of ionization techniques that operate at higher
pressures, outside the vaccuum MS regions.
- APCI a form of chemical ionization using the liquid
effluent in a spray chamber as the reagent
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.
-
63
Ni 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)
SeeA. P. Bruins, Mass Spec. Rev., 10, 53-77 (1991).
8
APCI: Chemical Ionization
- APCI (diagram from Agilent)
DiagramfromAgilent Technologies
760 torr
10
-6
torr
APCI: Chemical Ionization
- An APCI mass spectrum:
DiagramfromAgilent Technologies
9
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
- orthe analyte ion is evaporated from a small enough droplet
Electrospray Ionization (ESI)
- A picture of two ideas for the electrospray process:
Diagramfrom J ohn B. Fenn (Nobel Lecture), 2002
Picturefromhttp://www.newobjective.com/electrospray/electrospray.html
Note ions which are
surface-active will be
preferentially ionized
this can lead to ion
suppression!
10
Electrospray Ionization (ESI)
- An ESI source:
DiagramfromAgilent Technologies
Typical ESI Spectra
- An ESI mass spectrum:
DiagramfromAgilent Technologies
11
Typical ESI Spectra
- An ESI Mass Spectrum of a 14.4 kDa enzyme:
Diagramfromhttp://www.nd.edu/~masspec/ions.html
ESI and APCI
- ESI and APCI complementary techniques:
FigurefromAgilent Instruments
12
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 [M+H]
+
Singly-charged ions,
[M+H]
+
and [M-H]
-
Atmospheric Phase Photo-ionizati on
- APPI can ionize things that ESI and APCI cant:
13
Atmospheric Phase Photo-ionizati on
- APPI can ionize things that ESI and APCI cant:
Comparison of Ionization Methods
- How to choose an ionization technique:
FigurefromAgilent Instruments
14
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 mostly used for
large biomolecules and
polymers.
DiagramfromKoichi 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 see Table 20-4 (pg. 509) in Skoog et al.
15
MALDI at Atmospheric Pressure
- Advantages: fast, easy and sensitive
- Disadvantages: no LC, matrix still needed
S. Moyer and R. Cotter, Atmospheric PressureMALDI, 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.
K. L. Rinehart, J r., Science, 218, 254(1982)
K. Biemann, Anal. Chem., 58, 1288A, (1986).
Xe
e
-
Xe
+
+2e
-
- Advantages:
Rapid sample heating reduced fragmentation
A glycerol solution matrix is often used to make it easier to
vaporize ions
Xe
+
accel
Xe
+
(high KE)
Xe
+
(high KE) +Xe Xe (high KE) +Xe
+
16
SIMS: Secondary Ion MS
- Focused Ion Beam
3
He
+
,
16
O
+
,
40
Ar
+
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 atomicand molecular analysis
Sensitive to low levels, picogram, femtogram and lower
- Will discuss more in surface analysis/microscopy talk
Desorption Electrospray: DESI and DART
- Desorption-
electrospray
ionization (DESI)
- A new technique
for desorbing
ions using
supersonic jets
of solvents
(charged like in
electrospray)
FromZ. Takats et al., Science, 2004, vol 306, p471.
17
Inductivel y Coupled Plasma (ICP)
- The inductively-coupled
plasma serves as an
atomization and
ionization source (two-in-
one!) for elemental
studies.
Photo bySteve Kvech, http://www.cee.vt.edu/program_areas/environmental/teach/smprimer/icpms/icpms.htm#Argon%20Plasma/Sample%20Ionization
- See optical electronic lecture
for more details
- Solution flow rates up to: 50-
100 mL/min
Mass Anal yzers - Outline
- Sector Mass Analyzers (Magnetic and Electrostatic)
- Quadrupole Analyzers
- Ion Traps
- Ion Cyclotron Resonance
- Time-of-Flight
- and many more.
Ionization
Source
Mass
Analyzer
Detector
18
Properties of Mass Anal yzers
Resolution(R):
R = m/m
m =mass difference of two adjacent resolved peaks
(typically
m=mass of first peak or average
Example: R =500 (low resolution)
resolves m/z=50 and 50.1, and m/z=500 and 501
Example: R =150000 (high resolution)
resolves m/z=50 and 50.0003, and m/z=500 and
500.0033
Sector Mass Anal yzers
- Basic Features
A sector: a geometrical construction that has
two arcs inside of one another.
(Technically, a pie slice!)
- Types:
Magnetic
Electrostatic
Combination (e.g. double-focusing)
19
Magnetic Sector Mass Anal yzers
Ion kinetic energy:
V
e r B
z
m
2
2 2
=
2
2
1
mv zeV T = =
BzeV F
m
=
r
mv
F
c
2
=
m c
F F =
Forces:
Only ions with equal
forces will pass:
Therefore:
Where:
T is kinetic energy
z is charge on ion
e is electron charge (1.60 x 10
-19
C)
B is magnetic field (T)
v is velocity (m/s)
V is the accelerating voltage
mis the mass
DiagramfromStrobel and Heineman, Chemical Instrumentation, A
Systematic Approach, Wiley, 1989.
Electrostatic Sector Mass Anal yzers
2
v
reV
z
m
=
Therefore:
Ion kinetic energy:
2
2
1
mv zeV T = =
eV F
m
=
r
mv
F
c
2
=
M c
F F =
Forces:
Only ions with equal
forces will pass:
V can be varied to bring ions of
different KE (and different m/z
ratio to the exit)
DiagramfromStrobel and Heineman, Chemical Instrumentation, A
Systematic Approach, Wiley, 1989.
20
Double-Focusing Sector Mass Anal yzers
- If a batch of ions of equal
m/z but with different
kinetic energies enters a
magnetic sector
instrument, this will result
in a spread-out beam
- Soution: minimize
directional and energy
differences between ions
of the same m/z.
- Example of a double-
focusing MS: the Nier-
J ohnson geometry
DiagramfromStrobel and Heineman, Chemical Instrumentation, A Systematic Approach, Wiley, 1989.
Double-Focusing Sector Mass Anal yzers
- Another design, the Mattauch-Herzog geometry
- This geometry is analogous to CCD-based optical
electronic spectroscopy systems, while Nier-J ohnson
instruments are similar in nature to traditional scanning
monochromator spectrometers.
DiagramfromStrobel and Heineman, Chemical Instrumentation, A Systematic Approach, Wiley, 1989.
21
Time-Of-Flight (TOF) Mass Anal yzers
- The principle of Time-of-flight mass analysis:
A batch of ions is introduced into a chamber by an
pulse of accelerating current.
This chamber has no fields, and is a drift tube
Since the ions have the same kinetic energy, their
velocities vary inversely with their mass during their
drift.
- Notes:
Typical flight times are 1-30 us
Lighter ions arrive at the detector first
2
2
1
mv T =
M. Guilhaus; Journal of Mass Spectrometry, 30; 1995, p1519.
Time-Of-Flight (TOF) Mass Anal yzers
- Delayed extraction anything you can do to
tighten the KE spread will help a TOF
instrument
M. Guilhaus; Journal of Mass Spectrometry, 30; 1995, p1519.
m/z is mass-to-charge ratio of the ion
E is the extraction pulse potential
(V)
s is the length of flight tube over which E is applied
d is the length of field free drift zone
t is the measured time-of-flight of the ion
zeEs mv T = =
2
2
1
2
2
v
eEs
z
m
=
2
2 |
.
|

\
|
=
d
t
eEs
z
m
22
Time-Of-Flight (TOF) Mass Anal yzers
- The reflectron a method of compensating for different ion KEs
Figurefromhttp://www.abrf.org/ABRFNews/1997/J une1997/jun97lennon.html
Time-Of-Flight (TOF) Mass Anal yzers
- The reflectron a method of compensating for different ion KEs
Figurefromhttp://www.abrf.org/ABRFNews/1997/J une1997/jun97lennon.html
23
Quadrupole Mass Anal yzers
- The quadrupole (named for its electrical structure) is one
of the simplest and most effective mass spectrometers.
Diagrams fromSkooget al.
Quadrupole Mass Anal yzers
- How a quadrupole works:
Most important points:
- It is easier for an applied AC field to deflect a
light ion than a heavier ion
- Conversely, it is easier for an AC field to
stabilize a light ion
Using this knowledge a combined AC/DC
potential is applied to the rods. Via the DC,
the ion is attracted to one set of rods and
repelled by the other
The DC serves to stabilize heavy ions in one
direction (high pass filter). The AC serves to
stabilize light ions in the other direction (low
pass filter).
The ion must pass through the quadrupole to
make it to the detector
Diagrams fromSkooget al.
24
Quadrupole Mass Anal yzers
- Another view and the concept
of the mass scan
Images fromhttp://www.jic.bbsrc.ac.uk/SERVICES/metabolomics/lcms/single1.htm
Light ion:
(ex. m/z =100)
Dragged by AC
Heavy ion:
(ex. m/z =500)
Dragged by DC
J ust right:
Dragged by both,
But equally balanced
Ion Trap Mass Anal yzers
- Ion trap: a device for trapping
ions and confining them for
extended periods using EM
fields
- Used as mass analyzers
because they can trap ions and
eject them to a detector based
on their mass.
- Theory is based on Mattieus
work on 2
nd
order linear
differential equations (in the
1860s), and on Wolfgang
Pauls Nobel Prize winning
implementations
R. E. MarchandR. J. Hughes, QuadrupoleStorageMassSpectrometers, Wiley, 1989.
Seealso Chem. Eng. News 1991; 69(12):26-30, 33-41
FigurefromW. Paul Nobel Lecture, December 8, 1989.
25
Ion Trap Mass Anal yzers
- The stability region of an
ion trap based on
differential equations
2 2
0
8
O

=
mr
eU
a
z
2 2
0
4
O
=
mr
eV
q
z
) cos(
0
t V U O + =
- Most ITMS systems dont
use DC (U), i.e. only q
z
is
controlled
R. E. MarchandR. J. Hughes, QuadrupoleStorageMassSpectrometers, Wiley, 1989.
Ion Trap Mass Anal yzers
- Layout of an ion trap mass analyzer:
Diagramcourtesyof M. Olsen, GlaxoSmithKline
+
Main RF
Ring
Endcap
Lenses
Octopole
Optimized Asymptote Angle
End Cap
Shutter
Focus
Electron Multiplier
Conversion Dinode
Low Amplitude Dipole Field
(1/3 frequency of main RF)
+
+
+
+
+
+
26
Ion Trap Mass Anal yzers
- The Bruker
Esquire ESI
ITMS - a typical
ion-trap LC-MS
system:
Photo courtesy of M. Olsen, GlaxoSmithKline
Ion Cyclotron Resonance
- FT-ICR: a FT-based mass spectral method that offers
higher S/N, better sensitivity and high resolution
- Also contains a form of ion trap, but one in which ion
cyclotron resonanceoccurs.
- When an ion travels through a strong magnetic field, it
starts circulating in a plane perpendicular to the field
with an angular frequency e
c
:
m
zeB
r
v
c
= =
27
Ion Cyclotron Resonance
- How ICR works:
The ions are circulated in a field
An RF field is applied to match the cyclotron frequency of the ions
this field brings them into phase coherence (forming ion packets)!
The image current is produced as these little packets of ions get
near the plates. The frequency of the image current is characteristic
of the ion packets m/z ratio.
http://www-methods.ch.cam.ac.uk/meth/ms/theory/fticr.html
Ion Cyclotron Resonance and Magnetic Field
- Parallels between NMR/EPR and ICR:
B

B
=

q B
m
=

B
PicturecourtesyProf. AlanMarshall, FSU/NHMFL
28
The Orbitrap
TM:
A Hybrid Trap Between IT and
ICR
- The Orbitrap is a recently developed
electrostatic ion trap with FT/MS read-out of
image current, coupled with MS/MS
- Advantages
Ease of use
Resolving power (superior to TOF)
Precision and accuracy
Versatility, dynamic range
- A lower-resolution, more economical ICR
LTQ Orbitrap schematic
API Ion source Linear Ion Trap C-Trap
Orbitrap
Finnigan LTQ Linear Ion Trap
Differential pumping
Differential pumping
Image/animationfromThermo ElectronInc. SeeA. Makarov et al., Anal. Chem. 2006, 78, 2113-2120.
29
LTQ Orbitrap Operation Principle
1. Ions are stored in the Linear Trap
2. . are axially ejected
3. . and trapped in the C-trap
4. . they are squeezed into a small cloud and injected into the Orbitrap
5. . where they are electrostatically trapped, while rotating around the central electrode
and performing axial oscillation
The oscillating ions induce an image current into the two
outer halves of the orbitrap, which can be detected using
a differential amplifier
Ions of only one mass generate a sine
wave signal
Image/animationfromThermo ElectronInc. SeeA. Makarov et al., Anal. Chem. 2006, 78, 2113-2120.
The axial oscillation frequency follows the formula
Where =oscillation frequency
k =instrumental constant
m/z =mass-to-charge ratio
z m
k
/
=
Frequencies and Masses
Many ions in the Orbitrap generate a complex
signal whose frequencies are determined using a
Fourier Transformation
Image/animationfromThermo ElectronInc. SeeA. Makarov et al., Anal. Chem. 2006, 78, 2113-2120.
30
Multiple-Stage MS: MS-MS, and MS
n
- Also known as Tandem MS or MS
n
Mass
Analyzer
Mass
Analyzer
- Multiple quadrupoles are very common (e.g. triple-quad or
QQQ systems, EB for double-focusing, Q-TOF for quad
time-of-flight)
- Why tandem MS? Because of the possibility of doing CID
collisionally induced dissociation. Ions are allowed to
collide with a background gas (He) for several
millliseconds, prior to analysis. Allows for MS
n
experiments in an ion trap.

Comparison of Mass Anal yzers

- A brief overview of the properties of common mass
analyzers
Analyzer Cost Scan speed Resolution
Double-focusing High Slow High
Quadrupole Low Medium Low-medium
Trap Low Medium Medium
TOF Medium Medium Medium-high
ICR High Fast High
31
Detectors for Mass Spectrometry
- Electron multipliers: like a photomultiplier
tube. Ions strike a surface, cause
electron emission. Each successive
impact releases more electrons
- Faraday Cups: Ions striking a cup cause
charge to flow across a load. The
potential across the load is monitored.
- See pg 257 of Skoog et al. for more
details.
Ionization
Source
Mass
Analyzer
Detector
FigurefromD. W. Koppenaal, et al.; Anal. Chem., 77; 2005, 418A-427A.
Detectors: Electron Multipliers
- Electron multiplier (EM): most common design in current
use
- High gain (10
7
), low noise, good dynamic range (10
4
-10
6
)
- Several designs:
FigurefromD. W. Koppenaal, et al.; Anal. Chem., 77; 2005, 418A-427A.
32
Detectors: Others
- Super-conducting tunner junction high mass range,
used with MALDI
Can detect fmol of 150 kDa proteins
Can measure both energy and arrival time (2D MS plots of m/z
vs. kinetic energy)
- Focal-plane array detectors/CCD
Like in electronic spectroscopy, much more challenging to design
for ion detection
Would combine well with mini-traps or other small MS systems
FigurefromD. W. Koppenaal, et al.; Anal. Chem., 77; 2005, 418A-427A.
MS-Chromatography Interfaces
- GC-MS: gas eluent from a column is piped directly to the
MS source
- LC-MS: the ionization methods themselves serve as
interfaces techniques like ESI, APCI and APPI work on
liquid phase samples. The methods are generally
tolerant to RP LC solvents and some NP solvents. Some
buffers can quench ionization of analytes though:
Bad: Phosphate leaves a solid upon evaporation. Also ionizes
preferentially
Bad: any other non-volatile additives are also bad
Good: TFA, ammonium acetate, formic acid
Good: lower concentrations, <50 mM
33
Homework
- Choose one of these references to read:
R. E. March, "An Introduction to Quadrupole Ion Trap Mass
Spectrometry", J . Mass. Spec., 1997, 32, 351-369.
D. H. Russell and R. D. Edmondson, "High-resolution Mass
Spectrometry and Accurate Mass Measurements with Emphasis
on the Characterization of Peptides and Proteins by Matrix-
assisted Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry", J . Mass. Spec., 1997, 32, 263-276.
R. Aebersold and D. R. Goodlett, "Mass Spectrometry in
Proteomics", Chem. Rev., 2001, 101, 269-295.
L. Sleno and D. A. Volmer, Ion activation methods for tandem
mass spectrometry, J . Mass Spectrom. 2004; 39: 10911112
References
- Note: see Mass Spectrometry and Related Techniques
Part 2 for applications of MS, and theory/applications of
Ion Mobility Spectrometry
- R. M. Silverstein, et al., Spectrometric Identification of
Organic Compounds, 6
th
Ed., Wiley, 1998.
- R. E. March and R. J . Hughes, Quadrupole Storage
Mass Spectrometers, Wiley, 1989.
- F. W. McLafferty, Interpretation of Mass Spectra, 3rd Ed.,
University Science Books, 1980.