Mass spectrometry(MS)

Mass Spectrometry
PREPARED BY:-AJAY KUMAR

History of Mass Spectrometry
2
Year Scientist Discovery/Award
1886 E. Goldstein Discovers anode rays (positive gas ions) in
gas discharge
1897 J.J.Thomson Discovers the electron and determines its
m/z ratio. Nobel Prize in 1906
1898 W.Wien Analyzes the anode rays by magnetic
deflection, and establishes that they carry a
positive charge.
Nobel Prize in 1911
1909 R.A. Millikan &
H. Fletcher
Determine the elementary unit of charge

Mass Spectrometry
3
1912 J.J.Thomson First Mass Spectrometer. In 1913 J. J.Thomson
separated the isotopes 20Ne and 22Ne
1919 A.J. Dempster Electron ionization and magnetic sector MS
1942 Atlantic
Refining
Company
First commercial use
This technique resolves ionic species by their m/e
ratio
1953 W. Paul and
H.S.
Steinwedel
Quadrupole and the ion trap.
Nobel Prize to Paul in 1989.

Mass Spectrometry
4
1956 First GC-MS
1968 First commercial quadrupole
1975 First commercial GC-MS
1990s Explosive growth in biological MS, due to ESI
& MALDI
2002 Fenn &
Tanaka
Nobel Prize to Fenn &Tanaka for ESI & MALDI
2005 Commercialization of Orbitrap MS

Mass Spec - Introduction
 Very different from IR and NMR
 Absorption of electromagnetic energy
 Sample can be recovered and reused
 Mass spectrometry
 Records what happens when an organic molecule is hit
by a beam of high-energy electrons
 Sample is completely destroyed

Mass Spec - Introduction
What does a mass spectrum tell us?
1. Molecular weight
2. Molecular formula
 Either directly or in conjunction with other kinds
of spectra such as IR or NMR
3. Fragmentation pattern
 Key pieces of what the molecule looks like (such
as methyl, ethyl, phenyl, or benzyl groups

 Ms spectrometry gives composition of sample.
 Structure of inorganic, organic & biological sample
 Qualitative & quantitative composition of solid surfaces
 Isotopic ratios of atoms in samples
 Atomic or Molecular weight expressed in terms of
atomic mass unit (amu) or daltons (Da).
Introduction to Mass Spectrometry

 The amu is based upon the relative scale in which the
reference is carbon isotope C-12.
 Thus amu is defined as 1/12 the mass of the one neutral
C-12
 Molecular weight can be obtained from a very small sample.
 It does not involve the absorption or emission of light.
 A beam of high-energy electrons breaks the molecule apart.
The masses of the fragments and their relative abundance
reveal information about the structure of the molecule.
8

Separation of Ions
 Only the cations are deflected by the magnetic field.
 Amount of deflection depends on m/z.
 The detector signal is proportional to the number of ions
hitting it.
 By varying the magnetic field, ions of all masses are
collected and counted.
9

Atomic MS Acronym Atomic ion
source
Typical Ms
analyzer
Inductive coupled
plasma
ICPMS High temp. argon
plasma
Quadruple
Direct current
plasma
DCPMS High temp. argon
plasma
Quadruple
Microwave
induced plasma
MIPMS High temp. argon
plasma
Quadruple
Spark source SSMS Radio frequency
electric spark
Double focusing
Thermal
ionization
TIMS Electrically
heated plasma
Double focusing
Glow discharge GDMS Glow discharge
plasma
Double focusing
Laser microprobe LMMS Focused laser
beam
Time of flight
Secondary ion SIMS Accelerated ion
bombardment
Double focusing

- Used quantitatively and qualitatively
(identification)
 Useful for both organic and inorganic compounds
 Can measure ~ 75 elements
 Rapidly evolving technology
 Expensive and complex
11
General Characteristics and Features

12
A B + e
molecular ion =
cation-radical
(high energy)
electron beam;
~5000 kJ/mol
fragmentation
cations + neutral species (radicals)
: A B+
+ 2 e
Mass Spectrometry…
Sample is ionized (an electron is removed)  M
.+
Ionization frequently fragments molecules 
bonds most likely to break are the weakest -> form cations & radicals
Modern techniques can be used to study non-volatile
molecules such as proteins and nucleotides

MS perform three functions:
 Creation of ions – the sample molecules are subjected
to a high energy beam of electrons (70 eV), converting
some of them to ions
 Separation of ions – as they are accelerated in an
electric field, the ions are separated according to mass-
to-charge ratio (m/z)
 Detection of ions – as each separated population of
ions is generated, the spectrometer needs to qualify
and quantify them
 All type of MS need very high vacuum (~ 10-6 torr),

source analyzer
ion
detection
data
system
Vacuum pumps
Sample
Introduction
Ion Formation Ion Sorting Ion Detection
Data Handling
Data Output
Mass spectrum
Basic Components of a MS

Components of MS
1. Sample Introduction System
 Volatilizes the sample and introduces it to the
ionization chamber under high vacuum
2. Ion Source
 Ionizes the sample (fragmentation may occur) and
accelerates the particles into the mass analyzer
3. Mass Analyzer (or Mass Separator)
 Separates ionized particles based on their mass-to-
charge ratio (m/e-)
16

Components of MS
4. Detector - Ion Collector
 Monitors the number of ions reaching detector per
unit time as a current flow
5. Signal Processor
 Amplifies the current signal and converts it to a DC
Voltage
6. Vacuum Pump System
 A very high vacuum (10-4 to 10-7 torr) is required so
that the generated ions are not deflected by
collisions with internal gases
17

Mass Spectrometry
II. The Mass Spectrometer
B. Single Focusing Mass Spectrometer
 A small quantity of sample is injected and vaporized under
high vacuum
 The sample is then bombarded with electrons having 70-80
eV of energy
 A valence electron is “punched” off of the molecule, and an
ion is formed

Mass Spectrometry
B. The Single Focusing Mass Spectrometer
4. Ions (+) are accelerated using a (-) anode towards the focusing magnet
5. At a given potential (1 – 10 kV) each ion will have a kinetic energy:
½ mv2 = eV
As the ions enter a magnetic field, their path is curved; the radius of the
curvature is given by:
r = mv
eH
If the two equations are combined to factor out velocity:
m/e = H2r2
2V
m = mass of ion
v = velocity
V = potential difference
e = charge on ion
H = strength of magnetic field
r = radius of ion path

Mass Spectrometry
B. Single Focusing Mass Spectrometer
6. At a given potential, only one mass would have the correct
radius path to pass through the magnet towards the detector
7. “Incorrect” mass particles would strike the magnet

Ion Sources
Purpose: create gaseous ions out of the sample
components
Two types:
1. Molecular sources
 gas phase
 desorption sources
2. Elemental sources
21

Ion Sources MS (cont.)
Type S.No Name and Acronym Ionizing Process
Gas Phase 1 Electron Impact (EI) Exposure to electron
stream
2 Chemical Ionization (CI) Reagent gaseous
ions
3 Field Ionization (FI) High potential
electrode
Desorption 1 Field Desorption (FD) High potential
electrode
2 Electrospray Ionization (ESI) High electric field
3 Matrix-assisted desorption
ionization (MALDI)
Laser beam
4 Plasma Desorption (PD) Fission fragments
from 252Cf
5 Fast Atom Bombardment (FAB) Energetic atomic
beam
6 Secondary Ion Mass
Spectrometry (SIMS)
Energetic beam of
ions
7 Thermospray Ionization (TS) High temperature22

Electron Impact Ionization
 Ionization methods required for gaseous sample.This method is
not useful for non volatile or thermally unstable molecule.
 In desorption technique sample directly converted in to gaseous
ions.
 We hit an organic molecule with a beam of electrons (usually 70-75
eV)
M + e–  M+ + e– + e– ionization
M+  A+ + B fragmentation
 That removes an electron from the molecule resulting in the
molecular ion (a radical cation)
 The molecular ion then fragments in smaller radicals and cations
 The cations are detected by the MS instrumentation

Chemical Ionization
25
 Gaseous sample
atoms are ionized by
collision with
positively charged
“reagent” gases (e.g.
CH4
+).
 The reagent ions are
produced by electron
bombardment
A0 (g) + CH4
+ (g) -------> A+ (g) + CH4
0 (g)

Chemical Ionization (CI)
A modified form of EI
Higher gas pressure in ioniation cavity (1 torr)
Reagent gas (1000 to 10000-fold excess) added; usual
choice is methane, CH4
•
•A “soft ionization” technique
• Reagent gases are ionized
o methanol, methane, ammonia, others
• Sample molecules collide with the ionized reagent gas
o usually results in a proton transfer from the reagent gas to
the sample compound
o so M+1 ions are common

Chemical Ionization
Reactions
 Reagent gas ionization:
CH4  CH4
+ +e– (also CH3
+, CH2
+)
 Secondary reactions:
CH4
+ + CH4  CH5
+ + CH3
CH3
+ + CH4  H2 + C2H5
+ (M+29)
 Tertiary reactions
CH5
+ + MH  CH4 + MH2
+ (M+1) proton exchange
CH3
+ + MH  CH4 + M+ (M–1) hydride exchange
CH4
+ + MH  CH4 + MH+ (M) charge exchange
27

Field Ionization and
Desorption
 Intense electric field
(107-108 V/cm)
 Electrons “tunnel”
into pointed electrode,
yielding positive ions
with little excess energy
 Very gentle; little fragmentation
 In Field Desorption, anode coated with analyte
 Not as efficient as EI sources by an order of
magnitude
 Waller 1972, Mc Fadden 1973, Beckey 1969
29

Electrospray Ionization
Source
30
How it Works
 Small, electrically charged
droplets are formed from a
solution flowing out of a
hollow needle into a chamber
under low vacuum
 The charged droplets are
attracted to an electrode
across an open volume by
the application of an
electrical field in the open
chamber

Electrospray Ionization Source
 Some of the solvent is evaporated (and concentration occurs)
during transit across the chamber
 As the droplets shrink, ions are forced closer together. At
some point the repulsive forces between the ions is greater
than surface tension and small droplets break off the larger
drops.
 This process continues several times as the droplets transit
across the chamber
 Eventually the solvent disappears and ions are generated, a
process called ion evaporation& analysed by quadrupole Mass
analyser
31

Matrix-Assisted Laser
Desorption/Ionization (MALDI)
 Analyte mixed with radiation-absorbing material such as
Nicotinic acid, Benzoic acid deriv., Pyrazine –carboxylic acid,
cinnamic acid deriv., Nitrobenzyl alcohol
 The resulting solution was evaporated on the metallic probe
surface and dried
 Sample mixture was exposed to pulsed laser beam, which
result in the sublimation of analyte ion and were drawn into
time-of-flight (TOF) analyser for analysis
 Excellent for larger molecules, e.g. peptides, polymers
32

Inductively Coupled Plasma (ion
source)
 Plasma
 An electrically conducting gaseous
mixture containing cations and anions
 ∑ cation(s) charge = ∑ electron charge
 Argon Plasma
 Ar is the principal conducting species
 Temperatures of 10,000 K possible
 Powered by radiofrequency energy (2
kW @ 27 Mhz)
33

source)
An ICP “torch” consists of:
 Three concentric quartz tubes
through which a stream of argon
flows at a rate of 5-20 L/min
 The three concentric rings are
constructed to eliminate
atmospheric gases from contacting
the sample stream in the inner-
most ring
34

source)
 An argon plasma is
generated by a water-
cooled induction coil
through which a radio-
frequency energy (0.5 to 2
kW of power at 27-41 MHz)
is transmitted
 Ionization of the flowing
argon must be “initiated”
by aTesla coil
35
Radiofrequency
Induction Coil
Argon Plasma

source)
 Sample is introduced through
the inner-most ring in the torch
as a “mist” carried by the argon
stream
 The “mist” is generated by a
nebulizer
36
Sample Inlet Tube
Cetac Ultrasonic Nebulizer

source)
 Analytes are ionized in the argon
plasma and the ionized gas (i.e.
plasma) is positioned on the
entrance to the mass spectrometer.
 The interface consists of a series of
metal (Pt, or Ni) cones with a small
hole permitting the ions to be
drawn in by the large vacuum on
the inside.
 Can measure 90% of the elements
in the periodic table can be
simultaneously measured
37
MS Interface

Fast Atom Bombardment
 Ion source for
biological molecules
 Ar ions passed
through low pressure
 Ar or Xe gas to produce
beam of neutral ions
 Atom-sample collisions
produce ions as large as 25,000 Daltons
38

Fast Atom Bombardment Ionization
Source
39
Ar+* + Ar0 ----------------> Ar+ + Ar0*
Production of “fast atoms”
Charge transfer
Accelerated
argon ion
from “ion
gun”
Ground
state
argon
atom
“slow ion” “fast atom”
(a) The atom gun
(b) The atom beam
(c) Metal sample holder
(d) The end of the probe arm used to insert the
sample into the chamber
(e) The sample in the low volatility solvent
(f) The sample ion driven from the surface
(g) Ion extraction plate-select positive ions for
mass analysis
(h) Ion lens system leading to mass analyser

FAB Characteristics
 Used with high molecular weight organic molecules
 The fast atom interacts with analyte on a “target” to produce
ions by “sputtering” (i.e. transfer of energy from argon to
analyte)
 Analyte ions are accelerated into the MS by application of an
electric field (ion extraction plate and lenses)
40

Thermal Ionization (Ion) Source
41
How it Works
 It employ two wire filaments (usually
tungsten or rhenium) closely spaced
(~ 1 mm) situated in a chamber under
high vacuum
 The sample is coated (usually in a
silica gel matrix with phosphoric acid
coated on top) on one wire filament
that is heated gently
 The second filament, the ionizing
filament is heated to incandescence
 The analyte desorbs from the
filament and become ionized by the
second filament.
 Sample ions are accelerated into the
MS by application of an electric field
Characteristics
Old ionization method (70+
years)
Used primarily for very high
precision isotopic ratio studies
of the elements

Example Resolution Calculation
1. What is the resolution required to separate
particles having masses of 999 and 1001?
500
2
1001)/2
(999
R 


42
(1 part in 500)
2. For Masses of 28.0061 (N2
+) and 27.9949
(CO+)
2500
0.00112
28.0005
27.9949)
-
(28.0061
27.9949)/2
(28.0061
R 



(1 part in 2500)

LC-MS Inlets
 Direct inlet
 Moving-belt inlet
 Thermo spray inlet
43

Sample Introduction -Direct MS
Inlet
Four BasicTypes:
1. Batch Inlet
 Sample is volatilized externally
and allowed to “leak” into the
ion source
 Good for gas and liquid
samples with boiling points <
500 °C
 Major interface problem –
carrier gas dilution
44
Purpose: Introduce the sample (as a gas) into the
ion source under high vacuum- GC MS

Direct Probe
 Good for non-volatile liquids, thermally unstable
compounds and solids
 Sample is held on a glass capillary tube, fine wire or
small cup
45
A mixture of compounds is separated by gas chromatography,
then identified by mass spectrometry (GC-MS Inlets)

Thermospray- Inductively Coupled
Plasma (ICP)
 Operates somewhat like a
nebulizer in an AAS
 Also ionizes the sample in
argon stream (at very high
temperatures, >6000 °C)
 Only a small amount of
analyte is utilized (< 1%)
48

Mass Analyzer
The function of the MS analyzer like monochromator in
an optical spectrometer.
Transducer converts the beam of ions to an electrical
signal that can be then Processed, stored in memory.
MS require an elaborate vacuum system to maintain a
low pressure in all of the components except signal
processor

Mass Analyzers
Type Mass Range Resolution Sensitivity Advantage Disadvantage
Magnetic
Sector
1-15,000
m/z
0.0001 Low High
resolution
Expensive
Quadrupole 1-5000 m/z Unit High Easy to use;
inexpensive
Low res; low
mass
Ion trap 1-5000 m/z Unit High Easy to use;
inexpensive
Low res; low
mass
Time of
Flight
Unlimited 0.0001 High High mass;
simple design
Fourier
Transform
Up to 70
kDa
0.0001 High Very high res
and mass
Very expensive
Silverstein, et. al., Spectrometric Identification of Organic Compounds, 7th Ed, p 13.
Single Focus
Double Focus

Mass Analyzers
 There are several methods for separating different
masses
 Elemental analysis -Want to distinguish between
individual mass units

particles)
two
(of
mass
in
difference
particles)
two
(of
mass
average
Resolution 
51

Single Focus
Determine m/e by
varying H, r, or V
R = 500-5000
52
V
r
H
e
m
2
2
2


Magnetic Sector Mass Spectrometer
Carey, Chapter 13

Single Focusing Magnetic Sector
Mass Analyzer
54
 Masses are
separated in a
(single) magnetic
field
 Ions are deflected
60-180°
 Varying the
magnetic field
separates the
masses

Double Focus
 Ion source produces ions
with a certain spread of
Kinetic Energy (K.E.).
 Electrostatic field and exit
slit select only ions with
same K.E.
 Net effect is to increase R to
2500-150000
 Can distinguish very similar
ions, e.g., C2H4
+ (28.0313)
and CO+ (27.9949)
55

Double Focusing Magnetic Sector
Mass Analyzer
56
 A “double focusing” analyzer
has two regions of mass
separation
 Magnetic Field preceded
by an electrostatic field
 The electrostatic field
helps to isolate particles of
a specific kinetic energy
 Ion sources which produce
particles of variable kinetic
energy have low resolution

Quadrupole Mass Analyzers
57
 Mass separation is achieved using 4
electrically connected rods (two “+”
and two “-”)
 Both DC and AC signals are passed
through the rods to achieve
separation
 Scans are achieved by varying the
frequency of the (AC) radio-
frequency or by varying potentials of
the two sources while keeping their
ratio and frequency constant

resonant ion
nonresonant ion
detector
source
focusing lens
quadrupole rods
Quadrupole Mass Analyzers
Mass filters

Quadrupole Analyzer
 Ions forced to wiggle
between four rods whose
polarity is rapidly
switched
 Small masses pass
through at high
frequency or low voltage;
large masses at low
frequency or high voltage
 Very compact, rapid (10
ms)
 R = 700-800
59

Merit and Demerit
Classical mass spectra
Good reproducibility
Relatively small/ compact,
Relatively low-cost systems
Limited resolution
Peak heights variable as a function of mass (mass
discrimination). Peak height vs. mass response must be
'tuned'.
Not well suited for pulsed ionization methods
60

Quadrupole Ion Trap
 Ions follow complex
trajectories between two
pairs of electrodes that
switch polarity rapidly
 Ions can be ejected from
trap by m/z value by
varying the frequency of
end cap electrodes
61

Time of Flight Mass Analyzer
62
Operation Characteristics
 Separates ions based on flight time in drift tube
 Positive ions are produced in pulses and accelerated in an
electric field (at the same frequency)
 All particles have the same kinetic energy but the velacities
vary with mass of the ions
 Lighter ions reach the detector first
 Typical flight times are 1-30 µsec

Time-of-Flight Analyzer
 Pulsed ion source
 Arrival of ions at detector is timed,typically 1- 30 ms
63
m
t
1


Time of Flight Mass Analyzer
Separation Principle
 All particles have the same kinetic energy
 In terms of mass separation principles:
 Vparticle = Her/m
 Hold H,e, and r constant
 Vparticle = 1/m (constant)
 Vparticle is inversely proportional to mass
64

Inductively Coupled Plasma Mass
Spectrometer
65

Detectors for MS
 Two BasicTypes
1. Electron Multipliers
2. Faraday Cup
 Time of Flight (TOF) and FourierTransform Ion-
Cyclotron Resonance (FTICR) instruments can
separate more than one m/e- ratio
simultaneously
 Multiple detectors are required in this case
66

Discrete Dynode Electron
Multiplier
 Operates somewhat like a
PMT tube
 Each dynode is at successively
higher potential
 Produces a cascade of electrons
67

Channel (or Continuous) Dynode
Electron Multiplier
 A glass tube that is coated
with lead or a conductive
material
 A potential of ~ 2000V is
applied between the
opening and the end of the
tube
 The curved shape helps to
reduce electrical noise by
preventing positive ions
returning upstream.
68

Dynode-Based Detectors
 A disadvantage of dynode-based detectors is
that the number of secondary electrons released
in a detector depends on the type of primary
particle, its energy and its incident angle,
 Mass discrimination occurs when ions enter the
detector with different velocities.
69

Electron Multiplier Detector
Tilted so that ions do not pass through undetected
70

Faraday Cup
71
How it Works
 Ions exiting the analyzer strike the
collector electrode
 The faraday cage prevents escape
of reflected ions and ejected
secondary electrons
 The inclined collector reflects ions
away from the entrance
 The collector is connected to
ground via a large resistor
 Positive ions are neutralized on the
surface of the collector by a flow of
electrons (from ground) through
the resistor
 The potential energy difference
across the resistor is amplified

Faraday Cup
Characteristics
 Inexpensive
 Low sensitivity
 Slow response
 A metal or carbon cup
 Produces a few micro amps of current (that is
then amplified)
 Current is directly proportional to number of
ions entering
 Detector response is independent of ion
 Kinetic energy
 Mass
 Chemical nature
 Does not exhibit mass discrimination
 Used in isotope ratio MS
72

Application of MS
1. Drug discovery, combinatorial chemistry, Drug
testing/Pharmacokinetics
2. Antiterror/Security (e.g. bomb molecule ‘sniffers’)
3. Environmental Analysis (e.g. water quality testing)
4. Quality Control (food, pharmaceuticals)
5. Medical Testing (various blood illnesses and… cancer?)
6. Validation of art/History/Anthropology etc.
7. Validation during chemical synthesis
8. Biochemical research (proteomics, interact…omics)
9. Tissue imaging (with MALDI)
10. Analysis of Proteins, peptides, olegonucleotides
11. Clinical testing etc 73
-

Mass spectrometry(MS)

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