Transcript Mass Spectrometry - Villanova University

Mass Spectrometry and Related
Techniques 1
Lecture Date: February 25th, 2008
Ion and Particle Spectrometry 1 - Outline
 Atomic and Molecular Mass Spectrometry
– Skoog et al. Chapter 11 (atomic) and Chapter 20 (Molecular).
– Cazes Chapter 14
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
m/z
Up to m/z = 100000!
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
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
EAccel!
 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
 See also Fig. 20-3, pg.
502 in Skoog, et al.
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
ions! (makes structural identification difficult!)
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:
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
 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.
 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
 APCI (diagram from Agilent)
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!
Diagram from John B. Fenn (Nobel Lecture), 2002
Picture from http://www.newobjective.com/electrospray/electrospray.html
Electrospray Ionization (ESI)
 An ESI source:
Diagram from Agilent Technologies
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 – complementary techniques:
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 can ionize things that ESI and APCI can’t:
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 mostly 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 – see Table 20-4 (pg. 509) in Skoog et al.
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 and DART
 Desorptionelectrospray
ionization (DESI)
 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)
 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
Mass Analyzers - 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
Properties of Mass Analyzers
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 Analyzers
 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)
Magnetic Sector Mass Analyzers
Ion kinetic energy:
T  zeV  12 mv
Forces:
Fm  BzeV
Fc 
mv
2
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
m is the mass
2
r
Only ions with equal
forces will pass:
Fc  Fm
Therefore:
m
z

2
2
B r e
2V
Diagram from Strobel and Heineman, Chemical Instrumentation, A
Systematic Approach, Wiley, 1989.
Electrostatic Sector Mass Analyzers
Ion kinetic energy:
T  zeV  12 mv
2
Forces:
Fm  eV
Fc 
mv
2
r
Only ions with equal
forces will pass:
Fc  FM
Therefore:
m
z

V can be varied to bring ions of
different KE (and different m/z
ratio to the exit)
reV
v
2
Diagram from Strobel and Heineman, Chemical Instrumentation, A
Systematic Approach, Wiley, 1989.
Double-Focusing Sector Mass Analyzers
 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 doublefocusing MS: the NierJohnson geometry
Diagram from Strobel and Heineman, Chemical Instrumentation, A Systematic Approach, Wiley, 1989.
Double-Focusing Sector Mass Analyzers
 Another design, the Mattauch-Herzog geometry
 This geometry is analogous to CCD-based optical
electronic spectroscopy systems, while Nier-Johnson
instruments are similar in nature to traditional scanning
monochromator spectrometers.
Diagram from Strobel and Heineman, Chemical Instrumentation, A Systematic Approach, Wiley, 1989.
Time-Of-Flight (TOF) Mass Analyzers
 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:
T  12 mv
2
– Typical flight times are 1-30 us
– Lighter ions arrive at the detector first
M. Guilhaus; Journal of Mass Spectrometry, 30; 1995, p1519.
Time-Of-Flight (TOF) Mass Analyzers
 Delayed extraction – anything you can do to
tighten the KE spread will help a TOF
instrument
T  12 mv  zeEs
2
m/z is mass-to-charge ratio of the ion
m
z

2eEs
v
2
t 
 2eEs 
z
d
m
E is the extraction pulse potential
(V)
2
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
M. Guilhaus; Journal of Mass Spectrometry, 30; 1995, p1519.
Time-Of-Flight (TOF) Mass Analyzers
 The reflectron – a method of compensating for different ion KE’s
Figure from http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html
Time-Of-Flight (TOF) Mass Analyzers
 The reflectron – a method of compensating for different ion KE’s
Figure from http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html
Quadrupole Mass Analyzers
 The quadrupole (named for its “electrical structure”) is one
of the simplest and most effective mass spectrometers.
Diagrams from Skoog et al.
Quadrupole Mass Analyzers
 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 from Skoog et al.
Quadrupole Mass Analyzers
 Another view – and the concept
of the mass scan…
Light ion:
(ex. m/z = 100)
Dragged by AC
Heavy ion:
(ex. m/z = 500)
Dragged by DC
Just right:
Dragged by both,
But equally balanced
Images from http://www.jic.bbsrc.ac.uk/SERVICES/metabolomics/lcms/single1.htm
Ion Trap Mass Analyzers



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 Mattieu’s
work on 2nd order linear
differential equations (in the
1860’s), and on Wolfgang
Paul’s Nobel Prize winning
implementations
R. E. March and R. J. Hughes, Quadrupole Storage Mass Spectrometers, Wiley, 1989.
See also Chem. Eng. News 1991; 69(12):26-30, 33-41
Figure from W. Paul Nobel Lecture, December 8, 1989.
Ion Trap Mass Analyzers
 The stability region of an
ion trap – based on
differential equations
0  U  V cos(t )
az 
qz 

 8eU
mr0 
2
2
4eV
mr0 
2
2
Most ITMS systems don’t
use DC (U), i.e. only qz is
controlled
R. E. March and R. J. Hughes, Quadrupole Storage Mass Spectrometers, Wiley, 1989.
Ion Trap Mass Analyzers
 Layout of an ion trap mass analyzer:
M a in R F
R in g
L o w A m p litu d e D ip o le F ie ld
(1 /3 fre q u e n cy o f m a in R F )
E n d ca p
L e n se s
C o n ve rsio n D in o d e
+
+
+
+
+
+
+
E le ctro n M u ltip lie r
S h u tte r
O cto p o le
F o cu s
End Cap
O p tim ize d A sym p to te A n g le
Diagram courtesy of M. Olsen, GlaxoSmithKline
Ion Trap Mass Analyzers
 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 resonance” occurs.
When an ion travels through a strong magnetic field, it
starts circulating in a plane perpendicular to the field
with an angular frequency c:
c 
v
r

zeB
m
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 packet’s 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

=  B
qB
=
m
Picture courtesy Prof. Alan Marshall, FSU/NHMFL
The OrbitrapTM: 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
Finnigan LTQ™ Linear Ion Trap
API Ion source
Linear Ion Trap
Differential pumping
C-Trap
Orbitrap
Differential pumping
Image/animation from Thermo Electron Inc. See A. Makarov et al., Anal. Chem. 2006, 78, 2113-2120.
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/animation from Thermo Electron Inc. See A. Makarov et al., Anal. Chem. 2006, 78, 2113-2120.
Frequencies and Masses
The axial oscillation frequency follows the formula
Where
 = oscillation frequency
k
= instrumental constant
m/z = mass-to-charge ratio

Many ions in the Orbitrap generate a complex
signal whose frequencies are determined using a
Fourier Transformation
Image/animation from Thermo Electron Inc. See A. Makarov et al., Anal. Chem. 2006, 78, 2113-2120.
k
m/ z
Multiple-Stage MS: MS-MS, and MSn
 Also known as Tandem MS or MSn
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 MSn
experiments in an ion trap.
Comparison of Mass Analyzers
 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
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
Figure from D. W. Koppenaal, et al.; Anal. Chem., 77; 2005, 418A-427A.
Detectors: Electron Multipliers
 Electron multiplier (EM): most common design in current
use
High gain (107), low noise, good dynamic range (104-106)

 Several designs:
Figure from D. W. Koppenaal, et al.; Anal. Chem., 77; 2005, 418A-427A.
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
Figure from D. 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
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 Matrixassisted 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: 1091–1112
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”, 6th 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.