Mass Spectrometry and Its Applications

Goals:
1) Understanding basic principle of mass
spectrometry.
2) Knowing its important applications
3) As a training tool for future research
4) As an analytical skill for chemical analysis.
Grading: One exam for understanding basics of
MS & its applications (50) & Research Reports
(80). But the final score can not be more
than100.

A Few Reference Books

Mass Spectrometry Basics; Chris. G. Herbert and Robert A. W.

Johnstone
Mass Spectrometry: Principles and Applications; Edmond de Hoffmann
& Vincent Stroobant
The Expanding Role of Mass Spectrometry in Biotechnology; Gary
Siuzdak
Biomedical Applications of Mass Spectrometry; Clarence H. Suelter & J.
Throck Watson
Time-of-Flight Mass Spectrometry; Bob J Cotter
Electrospray Ionization Mass Spectrometry; Richard B. Cole
Quadrupole Ion Trap Mass Spectrometry; Raymond E. March & John F.
J. Todd
Liquid Chromatography-Mass Spectrometry:An Introduction; Bob
Ardrey
Protein Sequencing and Identification Using Tandem Mass
Spectrometry; Michael Kinter & Nicholas Sherman
Mass Spectrometry in the Biological Sciences; edited by A. L.
Burlingame & Steven A. Carr
It is not essential to have any of the above books. Hoffmanns book is
available in local bookstores

Brief Bio-sketch of Tutor

Name:
School:
1969,BS; Chemistry Dept. NTU
1974, Ph.D University of Chicago
Working Experience: 1974 ~ 2005 Oak Ridge National
Laboratory; 2005 ~ Now; GRC, Sinica. Adjunct Professor:
Vanderbilt University; University of Tennessee; Knoxville;
Honorary Professor: Beijing Tsing Hwa University
Possible Advantages: Experience in many different
research fields. Willing to learn from students. Eager to
make friends with young people.
Disadvantages: No much teaching experience; Not at
NTU all the time; No Chinese word processing skill;
Possible Generation Gap (?) and etc.

(?)

Outline on Mass Spectrometry Course

Chung-Hsuan Chen
I.

Introduction:
I.a Basic of Mass Spectrometry
I.b Major Applications of Mass Spectrometry
I.c Vacuum Science and Technology
II. Ionization:
II.a Electron Ionization
II.b Chemical Ionization
II.c Photoionization
II.d Matrix-assisted Laser Desorption/Ionization
II.e Electrospray Ionization
II.f Other Ionization methods: FAB, Field Ionization, SIMS etc.
II.g Major challenges for Future Development
III. Mass Analysis:
III.a Quadrupole
III.b Ion Trap
III.c Time-of-flight
III.d Magnetic Sector
III.e FTICR
III.f Major Challenges for Future Development

Outline of MS Course (continued)

IV

V.

VI

Detection:
IV.a
Charge Collector
IV.b
Charge Amplifier Detector
IV.c
Scientillator Detector
IV.d
Other Detectors and Major Barriers to
overome.
Tandem Mass Spectrometry
V.a
Basic of MS/MS
V.b
Collision induced Dissociation
V.c
Ion-Molecule Interaction
Chromatography/Mass Spectrometer Coupling
VI.a
GC/MS
VI.b
LC/MS
VI.c
CE/MS

Outline of MS Course (continued)

VII

Biomolecule Analysis:
VII.a
VII.b
VII.c
VII.d

Oligonucleotide, Protein, Polysaccharide Analysis

DNA Analysis and Sequencing
Proteomic & Metabolomic Applications
Disease Diagnosis

VIII Other Applications:

VIII.a
VIII.b
VIII.c
VIII.d
IX

Trace Element Analysis

Organic Polymer Analysis
Environmental Application
Material Analysis

Future Challenges:
IX.a
IX.b
IX.c
IX.d
IX.e
IX.f

X. Conclusion:

Ionization Mechanism
Mass to Charge Ratio Extension
Quantitative Measurements
Dynamic Range Extension
Cost Reduction
Size Reduction

What can a Mass Spectrometer do?

1) A mass spectrometer (MS) is a device which
aims to weigh atoms, molecules, cluster, nanoparticle, virus, cell and etc. In general, it can
only determine mass.
2) Indeed, present mass spectrometers can not
even measure mass directly, it can only
measure mass-to-charge ratio (M/Z) for a
particle in gas phase. For most mass
spectrometers, Z is equal to 1 so that mass
can be determined.

Essential Parts of a MS
Ionizer: A MS can only measure M/Z of an
ion so that ionization process is necessary.
Electron ionization, chemical ionization and
photoionization are among examples.
M/Z Separator: Magnetic Sector, Time-ofFlight, quadrupole, ion trap
Detector: charged particle detectors such
as channeltron, electromultiplier and
microchannel plates.

Schematic of Mass Spectrometry

Ionizer

Mass-to-charge ratio Analyzer

Detector

Mass Spectrometry Categorization

Based on ionization:
Matrix-assisted laser desorption/ionization (MALDI) mass
spectrometry
Electrospray ionization (ESI) mass spectrometry
Induction-coupled plasma mass spectrometry (ICP-MS)
Atmospheric Ionization Mass Spectrometry
Secondary Ionization Mass Spectrometry (SIMS)

Based on M/Z Separation:

Quadrupole Mass spectrometry
Ion trap mass spectrometry
magnetic sector mass spectrometry
time-of-flight mass spectrometry
Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Ion Mobility Mass Spectrometry

Mass Spectrometry Categorization

(continued)

Based on Applications:
Environmental Mass Spectrometry
Biological Mass Spectrometry
Cell Mass Spectrometry
Portable Mass Spectrometry
Based on Configuration:
Tandem Mass Spectrometry
Based on Sample Introduction:
GCMS; LCMS, Electrophoresis Mass
Spectrometry

MS Operation
Nearly all mass spectrometers need to operate
under high vacuum condition with the pressure
less than 10 -5 Torr with the only exceptions of an
ion trap mass spectrometer (milli-Torr) and an
ion mobility mass spectrometer (Torr).
Never turn on a mass spectrometer without
knowing the chamber pressure.
A tour to major mass spectrometry facilities in
Genomic Research Center, Sinica will be
arranged.

Mass Ranges for Different Species

One atomic mass unit (A.M.U or u) = 1 Dalton (Da) = 1.66 x
10-24 g
Atom: < 300 Da
Small Molecule: 2 ~ 2,000 Da.
Protein: 500 ~ 10,000,000 Da
Human Chromosome: 109 ~ 1011 Da
Human Genome: 2 x 1012 Da
Microbial Genome: 109 ~ 1010 Da
Nanoparticle: 104 ~ 1012 Da
Microparticle: 1011 ~ 1022 Da
Virus: ~ 109 Da
Cell: 1013 ~ 1015 Da

Isotope Concern
Average Mass: a value based on the stoichimetric
calculation of various isotope. Cl is considered as 35.45
Daltons
Monoisotopic mass: Molecular weight based on each
individual isotope.
A mass spectrometer with high enough mass resolution
should measure monoisotopic masses not average
mass. For example mass spectrum for Cl2 should be
obtained with three peaks as 35Cl2, 37Cl2 and 35Cl37Cl.
Since the natural abundances of each isotope is known,
multiple peaks from the same compound can provide
additional information for molecule determination.

Key Parameters in MS
Mass Accuracy: Internal calibration is often
needed for high mass accuracy
Mass Resolution: M/M
Detection Sensitivity: Ieff x Teff x Deff
Dynamic Range: wish to have 8 orders of
magnitude
Reproducibility: poor reproducibility mostly
comes from the ionization process
Quantitative Measurement: Up to now, MS is not
very good for quantitative measurement.

MS Applications (non-biomedical)

Pollutant Analysis
Trace Metal Analysis
Explosive Analysis
Illegal Drug Detection
Alcohol Analysis
Organic Chemical Analysis
Inorganic Chemical Analysis

MS for Biomedical Applications

Proteomic Analysis
DNA sequencing
DNA fingerprinting for Forensic Applications
Biomolecule structure analysis
Polysaccharide Analysis
Metabolomic Analysis & Pharmacological
Applications
Disease Diagnosis

Basic Vacuum Technology for MS

Gas Phase Collision Kinetics:
Gas Phase collision can destroy existing ions
and /or produce new ions to make MS analysis
difficult. Electronics can be unstable when gas
pressure is too high.
Vacuum Technology:
Good vacuum condition is generally required
for the operation of a mass spectrometer. Basic
knowledge on vacuum technology becomes
essential.

Vacuum Category

Gas Phase Collision Kinetics

Collision frequency () to a surface is
proportional to gas number density and mean
velocity of gas molecules.
= nvave = n (kT/2m)1/2
At room temperature (300 K) and 1 atmosphere
of air, the collision frequency is ~ 2.4 x 1023 /cm2
sec which is equivalent to the gas pumping
speed of 12 liter per second per square
centimeter. The design of outlet pumping flange
should allow the maximum use of the pumping
speed of the pump.

Leak & Outgas Concern

IF there is a leak of 0.01 mm at 1 atmosphere,
the leak rate is about
1.2 x10-3 liter-Atm/sec. If the vacuum is at 10-5
Torr, it needs the pumping speed of ~100,000
liter/sec which is impossible. Thus, no leaks are
allowed for any high vacuum and ultra-high
vacuum chamber. Although outgas rate is
usually much smaller, any material with high
outgas rate can not be allowed in the ultra-high
vacuum chamber either.

Differential Pumping
For certain mass spectrometers, carrier
gas needs to be leaked into the chamber
to carry the sample for analysis,
differential pumping becomes essential.
Addition of differential pumping often adds
the size, cost and alignment difficulty.

Mean Free Path

Mean Free Path (L) can be estimated as
L = vaver/ fcollision = kT/(20.5 2 P) ~ 0.005/P
P: Torr, T: 300K, Ion interaction cross section:
0.7 nm. For large biomolecular ion,
can be much larger. In general, L is
preferred to be longer than 1 m, pressure
needs to be at least 5 x 10 -5 Torr.

Vacuum Pump
Rough Pump (From atmosphere to milli-Torr):
a) Mechanical Rotatory Pump: 20 to 250
liter/sec; Advantage: rugged, convenient and
relatively inexpensive; Disadvantage: Oil
contamination
b) Cryosorption Pump (Liquid nitrogen &
activated charcoal) Advantage:oil free.
Disadvantage: Liquid nitrogen is needed and
activated charcoal needs to be replaced.
c) All high vacuum pumps need to be backed by
a rough pump to pump the chamber down to
milli-Torr region.

Vacuum Pump (continued 1)

Diffusion Pump:
Range: 10-3 ~ 10-9 Torr
Oil jet to carry residual gas down for pumping
Speed: 50 ~ 600 liters/ sec
Advantages: Fast Pumping speed
Disadvantages: Heating, cold trap and cooling
are often required. If electricity is discontinued, it
can become a disaster for the entire experiment.
* Diffusion pump is gradually replaced by ion
pump and turbo pump.

Vacuum Pump ( continued)

Ionization Pump:
(a) An electronic discharge is set up between anode and
cathode plates of which the cathode is fabricated of titanium or
tantalum material.
(b) The discharge is confined by a magnetic field, which
causes the emitted electrons to travel in a long spiral path,
under acceleration of 2000 ~ 5000 volts.

Electron collision with residual gas molecules to produce

ionization.
(d) Ions colliding with cathode to produce clean surface for
further reactions to produce oxide or nitride to remove the
residual gas.
(e) Ion pump only works efficiently under ultra-high vacuum.
(f) Ion pump is not efficient in pumping rare gas atoms such
as He and Ar.

Vacuum Pump (continued)

Sublimation Pump:
(a) Titanium is vaporized to deposit on the wall.
Such deposit of fresh titanium have a great
chemical affinity for all of active gases, including
hydrogen,nitrogen and oxygen.
(b) Sublimation pump has very high pumping
speed under ultra-high vacuum.
Sublimation pump does not work well for
rare gas

Vacuum Pump (continued)

Turbomolecular Pump:
(a) This pump makes use of a multiple-stage high-speed
rotator consisting of a series of flat disks rotating between
fixed disks which accelerate gas molecules by a collision
method and utilize the centrifugal effect to compress
these gas molecules. Such pumps operate at a very high
rotating speed (>= 24000 rpm).
(b) Pumping speed is a couple of hundred liters per
second.
Advantages: no oil and no heating. Suitable for ultrahigh vacuum
(d) Disadvantages: Expensive but privices have come
down. Most modern and expensive mass spectrometers
are using turbo pump.

Turbomolecular Pump (continued)

Most turbomolecular pumps employ
multiple stages consisting of rotor,stator
pairs mounted in series. Gas captured
by the upper stages is pushed into the
lower stages and successively
compressed to the level of the forevacuum pressure. As the gas molecules
enter through the inlet, the rotor,which
has a number of angled blades, impacts
on the molecules. Thus the mechanical
energy of the blades is transferred to the
gas molecules. With this newly acquired
momentum, the gas molecules enter into
the gas transfer holes in the stator. This
leads them to the next stage where they
again collide with the rotor surface, and
this process is continued, finally leading
them outwards through the exhaust.
Turbomolecular pump is less efficient for
very light molecules such as hydrogen
and helium

Vacuum Pump (continued)

Getter Pump:
Similar to Sublimation pump except
only getter process. This pump can be
used for pumping without any external
electricity. The pumping speed is very
low. Nevertheless, it can be used for
environmental mass spectrometers in
field use.

Vacuum Gauge
Thermal Couple Gauge:
Pressure Range: 1 Torr ~ 0.001 Torr
Ionization Gauge:
Pressure Range: 10-3 ~ 10-12 Torr
Mechanical Pressure Gauge: >1 Torr
MKS Baratron: Precision Pressure
Gauge

Mechanical Pressure Gauge

Most
pressure
gauges are
diaphragm
seal
nowadays

Thermal Couple Gauge

The thermal
conductivity for air is
nearly constant down
to 1 Torr. Then it
begins to change
linearly with pressure
down to 1 mTorr. T/C
gauge is based on
thermal conductivity.
It is a gas species
dependent gauge

Ionization Gauge
"

Ionization
Gauge is
based on the
ionization
measurement
. Thus it is
gas species
dependent.

Leak Detector
Helium Leak Detector:
It is a simple mass spectrometer for
measure helium. External helium spray is
needed.
Portable or Hand-held leak detectors:
Based on conductivity or discharge such
as SF6 as a carrier gas instead of He.

Sealing Materials
Rubber Gasket (Viton O-Ring):
High Vacuum
* Copper Gasket: Ultra-high
vacuum

Flow of Gas
Viscous Flow (short mean free path):
C (conductance) = 5.68 (a4/L)(P2 + P1)
a: radius of a tube; L: Tube length
P1: Initial Pressure; P2: Final Pressure
Molecular Flow (long mean free path)
C = 2.64 KA (T/M)1/2; A: area of tube; K is
a function of tube length and geometry
* Transition Flow: Difficult to estimate

Conductance Estimate
Assuming a pipe connecting a pump to a
chamber to be evacuated:
1/S = 1/Cpump + 1/Cpipe
If Cpump = Cpipe, Conductance is reduced by
half. When a small pipe is connected
between a chamber, a large pump will not
help much. Try your best not to use long
and thin pipe for vacuum system.

Pumping Down Time

Viscous Flow:
t (sec) = V/S Ln (P1 / P2);
V: Volume of the system, S: Net pumping
speed,P1: initial pressure,P2 : Final
Pressure.
* Molecular Flow: Controlled by outgas &
leak. t (hour) = Q/S; Q is overall ourgas &
leak rate; S is pumping speed at final
pressure.

Summary
Basic Concept of Mass Spectrometry:
Ionization Mass Analyzer Detection
Potential Applications:
Chemical Analysis; Disease Diagnosis;
Genomic, Proteomic and Metabolomic
Analysis
Vacuum Technology:
Gas Kinetics & Vacuum Instrumentation