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OUTLINE
Introduction to Mass Spectrometry
Ionization Methods
Mass Analyzer
Fragmentation and MS Interpretation
Chemical Identification
Comparison of
Physical Properties
Elemental Analysis
Burn the compound and
Boiling Point
Melting Point
Density
Optical rotation
Appearance
Odor
measure the amounts of
CO2, H2O and other
components that are
produced to determine the
empirical formula
Spectroscopic Methods for
Structure Determination
Ultraviolet-Visible (UV/Vis) spectroscopy:
determination of solutions of transition metal ions and highly
conjugated organic compounds
Infrared (IR) spectroscopy:
Functional groups
Mass spectrometry (MS):
Molecular mass and formula and structure information
Nuclear magnetic resonance (NMR) spectroscopy:
Map of carbon-hydrogen framework
Definition of Mass Spectrometry
Mass spectrometry (MS) :
An analytical technique by using mass spectrometry
for the determination of the composition of a sample
or molecule and elucidation of the chemical
structures of molecules, such as peptides and other
chemical compounds.
Mass spectrometry has been described as the
smallest scale in the world, not because of the mass
spectrometer’s size but because of the size of what it
weighs -- molecules.
What information can be determined?
• Molecular weight
• Molecular formula (HRMS)
• Structure (from
fragmentation fingerprint)
• Isotopic incorporation /
distribution
• Protein sequence (MS-MS)
MS Principles
• Find a way to “charge” an atom or molecule
(ionization)
• Place charged atom or molecule in a magnetic
field or subject it to an electric field and measure
its speed or radius of curvature relative to its
mass-to-charge ratio (mass analyzer)
• Detect ions using microchannel plate or
photomultiplier tube
General principles on mass spectrometry
Computer
system
Sample
introduction
Resulting chromatogram
Ionization of
sample in the
ion source
Separation of
ions by mass
analyzer
Detector
Analysis by GC-MS
Mixture
GC
MS
Separation
Identification
B
m/z
A
m/z
C
m/z
A+B+C
B
A
C
8
Instrumentation
Components of a mass spectrometry
1) Inlet system : introduce a very small amount of sample ( micromole or less)
into the mass spectrometer, where its components are converted to gaseous
ions.
2) Ion source :
converts the components of a sample into ions by
bombardment with electrons, ions, molecules, or photon.
3) Mass analyzer : separates the analyte ions according to their m/z ratios.
4) Detector : converts the beam of ions into an electrical signal(currents) ; the
detector output can be displayed or stored, to yield the mass spectrum.
5) Electronics of power supply and control of the systems.
6) Vacuum systems : maintain low pressures ( 10–5 to 10–8 torr); rotary vacuum
oil pump, diffusion pump, turbomolecular pump.
Three Components of an MS
• A typical mass spectrometer contains
– Ionizer
– Mass analyzer
– Detector
• Ion source charges the to-be-measured molecules.
– Charge can be negative but often positive.
– Two common types: MALDI and ESI.
– John B. Fenn & Koichi Tanaka 2002 Nobel Prize in Chemistry
for Electrospray and MALDI
• Mass analyzer separates ions according to the mass to
charge ratio (m/z) of the ions.
– Iontrap, TOF, Quadrupole, FTICR.
• Detector detects the ions.
Ionization Methods
• Electron bomb Ionization EI
• Chemical Ionization
• Matrix Assisted Laser Desorption Ionization
• Fast atom bombardment
• Electro Spray Ionization
Sample introduction / ionization method:
Ionization
method
Typical
Analytes
Sample
Introduction
Mass
Range
Electron Impact (EI)
Relatively
small
volatile
GC or
liquid/solid
probe
to
1,000
Daltons
Method
Highlights
Hard method
versatile
provides
structure info
Chemical Ionization (CI)
Relatively
small
volatile
GC or
liquid/solid
probe
to
1,000
Daltons
Soft method
molecular ion
peak [M+H]+
Liquid
Electrospray (ESI)
Peptides
Proteins
nonvolatile
Chromatography
or syringe
to
200,000
Daltons
Soft method
ions often
multiply
charged
Carbohydrates
Organometallics
Peptides
nonvolatile
Sample mixed
in viscous
matrix
to
6,000
Daltons
Soft method
but harder
than ESI or
MALDI
Peptides
Proteins
Nucleotides
Sample mixed
in solid
matrix
to
500,000
Daltons
Soft method
very high
mass
Fast Atom Bombardment
(FAB)
Matrix Assisted Laser
Desorption
(MALDI)
Electron Ionization ( EI )
Sample is heated and energized by a beam of electrons, usually gives a
molecular ion (M+) and a lot of fragments。
H H
H C C H
H H
H H
e- +
H H
H C C H
H C C+
H H
H H
H
(M-R2)+
Mass Spectrum (M-R )+
1
+
M+
(M-R3)
H C+
H
H
C H
H
H
Electron Impact Ionization (EI)
• Most widely used method
• Analytes are bombared with
high-energy electrons (usually
70eV)
• As a result of collision, an
electron is removed from the
analytes (M), generating a
molecular ion M+ (radical
cation)
M + e- M+ + 2e-
Electron Impact Ionization (EI)
• Due to excess internal energy, fragmentation
of the molecular ion will occur.
• The fragmentation is reproducible and
characteristic of the compund.
• It is also possible to predict the fragmentation
on the basis of chemical structures which is
why MS is good tool for structure elucidation
of unknown compounds
Chemical Ionization (CI)
• Softer ionization technique Less fragmentation  Easier to find
molecular ions.
• Two different modes: Negative chemical ionization (NCI) and Positive
Chemical Ionization (PCI).
• NCI is used for analytes that are able to form stable negative ions, for
example samples containing acidic groups or halogens. NCI is often used
to analyze pesticides (contains Cl or Br).
• PCI is used for samples that can form positive ions (most compounds).
Chemical Ionization (CI)
•
The principle for NCI and PCI is similar:
•
Reagent gas (usually methane, isobutane or ammonia) is introduced into the source where it is
ionized:
PCI (simplified):
CH4 + e- CH4+ +2eCH4 + CH4+ CH3 + C H5+
NCI:
•
CH4 + e- CH4-
The ionized gas collide with the sample molecules generating a [M+H]+ or [M+H]- ion that is
detected:
PCI:
CH5+ + M  [M+H]+ + C H4
NCI:
CH4- + M  [M+H]- + C H4
Field ionization (FI)
Field ionization (FI) is a method that uses very strong electric
fields to produce ions from gas-phase molecules.
阳极
+
+
+
+
+ +
d<1mm
+
+
+
+ + +
+
阴极
Field ionization (FI)
+
+
+
- - +
- + - +
+ - + + - ++ +
+ +
- + + - +
-
+
+
+
+ + +
+ + + +
+
+
+
+ +
+
+
+ +
+
+ + + +
+
+
+
+
+
+ +
+ +
+
Matrix Assisted Laser Desorption
Ionization (MALDI)
sample is co-crystallized with a matrix and then irradiated
with laser.
MALDI is achieved in two steps. In the first step, the
compound to be analyzed is dissolved in a solvent
containing in solution small organic molecules, called
the matrix. The second step occurs under vacuum
conditions inside the source of the mass spectrometer.
Properties of MALDI
Good solubility
Vapour pressure must be sufficiently low to maintain vacuum conditions
Viscosity must allow diffusion of the analyte from the bulk to the surface
Polar : to solvate and separate preformed ion
Less Sensitive to Salts
Lower PRACTICAL detection limits
Easier to interpret spectra (less multiple charges)
Quick and easy
Higher mass detection
Higher Throughput (>1000 samples per hour)
Principle of MALDI
MALDI mass spectrometry has become a powerful analytical
tool for both synthetic polymers and biopolymers.
ElectroSpray Ionization (ESI)
Electrospray is abbreviated to ESI ，ample is sprayed out of
a narrow nozzle in a high potential field. Generates positive
(M+nH)n+ and negative (M - nH)n- ions and almost no
fragmentation. Generates multiple charged ions.
Electrospray (Detail)
Electrospray Ionization
• Can be modified to “nanospray” system with flow < 1
mL/min
• Very sensitive technique, requires less than a picomole
of material
• Strongly affected by salts & detergents
• Positive ion mode measures (M + H)+ (add formic acid to
solvent)
• Negative ion mode measures (M - H)- (add ammonia to
solvent)
Fast atom bombardment ( FAB)
Softer than EI and CI. Ions are produced by bombardment with
heavy atoms. Gives (M+H)+ ions and litle fragmentation.
Good for more polar compounds.
Ar + e
Ar+ + Ar
fast
slow
Ar+
acceleration (5-15 KeV)
Ar + Ar+
+ 8 KeV
fast slow
Properties of FAB
Advantages
Parent Ion
High Mass Compounds
(10,000 amu)
Thermally Labile
Compounds (R.T.)
Disadvantages
No Fragment Library
Solubility in Matrix
(MNBA, Glycerol)
Quantitation Difficult
Needs Highly Skilled
Operator
Relatively Low Sensitivity
Mass analyzers
• After analytes have been ionized they are separated according to
their mass-to-charge ratio (m/z) in a mass analyzer (mass filter).
• Quadrupoles and ion traps are common mass filters in GC-MS
systems.
• Time of flight (TOF) mass filter is very much used nowadays in LCMS systems.
Computer
system
Sample
introduction
Ionization of
sample
Separation of ions
by mass analyzer
Detector
Transport of ions to the mass filter
• The ionization takes place in the ion source.
• Ions are then transported to the mass filter by
focusing lenses. These have a voltage running
through them and by either attracting or
repelling the ions they guide them into the
mass filter.
Different Mass Analyzers
• Magnetic Sector Analyzer (MSA)
– High resolution, exact mass, original MA
• Quadrupole Analyzer (Q)
– Low (1 amu) resolution, fast, cheap
• Time-of-Flight Analyzer (TOF)
– No upper m/z limit, high throughput
• Ion Trap Mass Analyzer (QSTAR)
– Good resolution, all-in-one mass analyzer
• Ion Cyclotron Resonance (FT-ICR)
– Highest resolution, exact mass, costly
Magnetic Sector Analyzer
Magnetic sector analyzer – Uses electric and/or
magnetic fields to separate ions
Advantages
Double focusing magnetic sector mass analyzers are the
"classical" model against which other mass analyzers are
compared.
Classical mass spectra
Very high reproducibility
Best quantitative performance of all MS analyzers
High resolution
High sensitivity
10,000 Mass Range
Linked scan MS/MS does not require another analyzer
• Disadvantages
Requires Skilled Operator
Usually larger and higher cost than other mass analyzers
Difficult to interface to ESI
Low resolution MS/MS without multiple analyzers
• Applications
All organic MS analysis methods
Accurate mass measurements
Quantitation
Isotope ratio measurements
Time of Flight Analyzer
TOF analyzer – ions are accelerated through a flight tube
and the time of light to the detector is measured
Ions are accelerated and their time of flight to the
detector is measured.
Principle of TOF Analyzer
• Uses a pulse of ion mixtures, not steady stream
• Ions accelerated into drift tube by a pulsed electric
• field called the ion-extraction field
• Drift Tube is usually 1-2 m long, under vacuum
• Ions traverse the drift tube at different speeds
• ( L / t ) = v = ( 2zV / m )½
Advantages of TOF Analyzer
Good for kinetic studies of fast reactions and for
use with gas chromatography to analyze peaks
from chromatograph
High ion transmission
Can register molecular ions that decompose in the
flight tube
Extremely high mass range (>1MDa)
Fastest scanning
•
Disadvantages
Requires pulsed ionization method or ion beam
switching (duty cycle is a factor)
Low resolution (4000)
Limited precursor-ion selectivity for most MS/MS
experiments
• Applications
Almost all MALDI systems
Very fast GC/MS systems
Quadrupole Analyzers
Quadrupole analyzers – ions
are filtered or trapped in a
device consisting of several
metal rods using specifically
tailored electromagnetic
fields
Quadrupole Analyzers
• Electric/magnetic fields trap, store, eject ions
• Requires an in-line quadrupole to act as
mass pre-filter
• Contains a single ring electrode and a top
and bottom cap electrode
• Varying RF frequency will vary the m/z ratios
that are trapped
• Additional fragmentation can be performed
on ions stored in the ion trap
• Advantages
Easy to use ,simple construction,fast
Good reproducibility
Relatively small and low-cost systems
Quadrupoles are now capable of routinely
analyzing up to a m/q ratio of 3000,which is
useful in electrospary ionization of biomolecules,
which commonly produces a charge distribution
below m/z 3000
MS detectors
• Many different types available
• Electron multipliers (EM) are often used
• Continuous –Dynode Version mainly in GC-MS
Computer
system
Sample
introduction
Ionization of
sample
Separation of ions
by mass analyzer
Detector
Continious dynode EM
•
•
The EM multiplies incident charges,
thereby amplifying the signal. The
current is measured that is
proportional to the amount of
analyte in the sample.
EMs have limited lifetime which is
dependent on the number of ions
that hits the device, i.e., the amount
of samples introduced and number
of samples analyzed.
+Fast response
+ High sensitivity
Watson, Introduction to Mass Spectrometry, 4th ed
Ions are detected with a microchannel plate
primary ion
-1000V
+
ee-
L
e- e
-100V
D
L >> D
What does a mass spectrometer do?
1. It measures mass better than any other technique.
2. It can give information about chemical structures.
What are mass measurements good for?
To identify, verify, and quantitate: metabolites,
recombinant proteins, proteins isolated from natural
sources, oligonucleotides, drug candidates, peptides,
synthetic organic chemicals, polymers
Applications of Mass Spectrometry
Pharmaceutical analysis
Bioavailability studies
Drug metabolism studies, pharmacokinetics
Characterization of potential drugs
Drug degradation product analysis
Screening of drug candidates
Identifying drug targets
Biomolecule characterization
Proteins and peptides
Oligonucleotides
Environmental analysis
Pesticides on foods
Soil and groundwater contamination
Forensic analysis/clinical
The mass spectrum shows the results
MALDI TOF spectrum of IgG
MH+
Relative Abundance
40000
30000
(M+2H)2+
20000
10000
(M+3H)3+
0
50000
100000
Mass (m/z)
150000
200000
ESI Spectrum of Trypsinogen (MW 23983)
M + 15 H+
1599.8
M + 16 H+
M + 14 H+
1499.9
1714.1
M + 13 H+
1845.9
1411.9
1999.6
2181.6
m/z
Mass-to-charge ratio
Protein Identification
1. Peptide Mass Finger Printing (PMF)
from MS data
2. Database search using fragment ion masses
from MS/MS data
3. Sequence Tags
from MS/MS data
PROBLEM
Bank President
Biologist
Who robbed the bank?
What protein was isolated?
GATHER EVIDENCE
Police Officer
1. Interview witnesses
2. Dust for fingerprints
Mass Spectrometrist
1. Interview biologist who
isolated the protein
2. Cleave protein to obtain
peptide mixture
enzyme
3. Analyze peptide mixture by
MS to obtain peptide
molecular masses!
DATABASE SEARCH
Police Officer
Height: 5’7”
Weight: 160 lbs
Gender: male
Age: 35-40
Fingerprints
search
DATABASE OF
KNOWN FELONS
Mass Spectrometrist
Approx. molecular weight: 30,000
Origin: bovine liver
Peptide mass list from MS analysis:
975.4832, 1112.5368, 632.3147,
803.4134, 764.3892
search
PEPTIDE MASS
DATABASE
OF KNOWN
PROTEINS
DATABASE SEARCH RESULTS
Police Officer
Mass Spectrometrist
Identifies the robber
Identifies the protein
Anthony J. Felon
bovine carbonic anhydrase