Mass Spectroscopy and its applications

This slide discusses the principle, instrumentation, process, detectors, sample ,solvents used in mass spectroscopy and also its applications and limitations.

1. Mass Spectrometry

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

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

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

5. 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

6. 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

7. 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

8. 8
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.

9. 9
Separation of Ions
Only the cationsare 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.

10. 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 dischargeGDMS Glow discharge
plasma
Double focusing
Laser microprobeLMMS Focused laser
beam
Time of flight
Secondary ion SIMS Accelerated ion
bombardment
Double focusing

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

12. 12A 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

13. 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),

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

15. 15
Mass Spectrometer

16. 16
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
-
)

17. 17
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

18. 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 eVof energy
A valence electron is “punched”off of the molecule, and
an ion is formed

19. Mass Spectrometry
II.The Mass Spectrometer
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 mv
2
= 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 = H
2
r
2
2V
m = mass of ion
v = velocity
V = potential difference
e = charge on ion
H= strength of magnetic field
r = radius of ion path

20. Mass Spectrometry
II.The Mass Spectrometer
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

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

22. 22
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 ElectrosprayIonization (ESI) High electric field
3 Matrix-assisted desorption
ionization (MALDI)
Laser beam
4 Plasma Desorption (PD) Fission fragments
from
252
Cf
5 Fast Atom Bombardment (FAB) Energetic atomic
beam
6 Secondary Ion Mass
Spectrometry (SIMS)
Energetic beam of
ions
7 ThermosprayIonization (TS) High temperature

23. 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
+
+ Bfragmentation
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 cationsare detected by the MS instrumentation

24. 24
Electron Impact Ion Source

25. 25
Chemical Ionization
Gaseous sample
atoms are ionized
by collision with
positively charged
“reagent” gases
(e.g. CH
4
+
).
The reagent ions
are produced by
electron
bombardment
A
0
(g) + CH
4
+
(g) -------> A
+
(g) + CH
4
0
(g)

26. Chemical Ionization (CI)
A modified form of EI
Higher gas pressure in ioniationcavity (1 torr)
Reagent gas (1000 to 10000-fold excess) added; usual
choice is methane, CH
4
•
•A “soft ionization” technique
•Reagent gases are ionized
omethanol, methane, ammonia, others
•Sample molecules collide with the ionized reagent gas
ousually results in a proton transfer from the reagent gas
to the sample compound
oso M+1 ions are common

27. 27
Chemical Ionization Reactions
Reagent gas ionization:
CH
4CH
4
+
+e
–
(also CH
3
+
, CH
2
+
)
Secondary reactions:
CH
4
+
+ CH
4 CH
5
+
+ CH
3
CH
3
+
+ CH
4 H
2 + C
2H
5
+
(M+29)
Tertiary reactions
CH
5
+
+ MH CH
4+ MH
2
+
(M+1) proton exchange
CH
3
+
+ MH CH
4+ M
+
(M–1) hydride exchange
CH
4
+
+ MH CH
4+ MH
+
(M) charge exchange

28. 28
Comparison
of EI and CI

29. 29
Field Ionization and
Desorption
Intense electric field
(10
7
-10
8
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, Beckey1969

30. 30
Electrospray Ionization Source
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

31. 31
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& analysedby quadrupole
Mass analyser
Electrospray Ionization Source

32. 32
Matrix-Assisted Laser
Desorption/Ionization (MALDI)
Analytemixed with radiation-absorbing material such as
Nicotinic acid, Benzoic acid deriv., Pyrazine–carboxylic
acid, cinnamicacid deriv., Nitrobenzylalcohol
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 analyteion and were
drawn into time-of-flight (TOF) analyserfor analysis
Excellent for larger molecules, e.g.peptides, polymers

33. 33
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)

34. 34
Inductively Coupled Plasma (ion 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

35. 35
Inductively Coupled Plasma (ion 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 a Tesla coil
Radiofrequency
Induction Coil
Argon Plasma

36. 36
Inductively Coupled Plasma (ion 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
Sample Inlet Tube
Cetac Ultrasonic Nebulizer

37. 37
Inductively Coupled Plasma (ion source)
Analytesare 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
MS Interface

38. 38
Fast Atom Bombardment
Ion source for
biological molecules
Arions passed
through low pressure
Aror Xegas to produce
beam of neutral ions
Atom-sample collisions
produce ions as large as 25,000 Daltons

39. 39
Fast Atom Bombardment Ionization Source
Ar
+
* + Ar
0
----------------> Ar
+
+ Ar
0
*
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

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

41. 41
Thermal Ionization (Ion) Source
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 analytedesorbs 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

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

27.9949) - (28.0061
27.9949)/2 (28.0061
R 


(1 part in 2500)

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

44. 44
Sample Introduction -Direct MS Inlet
Four Basic Types:
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
Purpose:Introduce the sample (as a gas) into the
ion source under high vacuum-GC MS

45. 45
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
A mixture of compounds is separated by gas chromatography,
then identified by mass spectrometry (GC-MS Inlets)

46. 46
Moving-Belt LC-MS Inlet

47. 47
ThermosprayLC-MS Inlet

48. 48
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
analyteis utilized (< 1%)

49. Mass Analyzer
ThefunctionoftheMSanalyzerlikemonochromatorin
anopticalspectrometer.
Transducerconvertsthebeamofionstoanelectrical
signalthatcanbethenProcessed,storedinmemory.
MSrequireanelaboratevacuumsystemtomaintaina
lowpressureinallofthecomponentsexceptsignal
processor

50. Mass Analyzers
Type Mass
Range
ResolutionSensitivityAdvantage Disadvantage
Magnetic
Sector
1-15,000
m/z
0.0001 Low High
resolution
Expensive
Quadrupole1-5000 m/zUnit High Easy to use;
inexpensive
Low res; low
mass
Ion trap 1-5000 m/zUnit High Easy to use;
inexpensive
Low res; low
mass
Time of
Flight
Unlimited0.0001 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, 7
th
Ed, p 13.
Single Focus
Double Focus

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

52. 52
Single Focus
Determine m/e
by varying H,
r, or V
R = 500-5000V
rH
e
m
2
22


53. Magnetic Sector Mass Spectrometer
Carey, Chapter 13

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

55. 55
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.,C
2H
4
+
(28.0313) and CO
+
(27.9949)

56. 56
Double Focusing Magnetic Sector
Mass Analyzer
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

57. 57
QuadrupoleMass Analyzers
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

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

59. 59
QuadrupoleAnalyzer
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

60. 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

61. 61
QuadrupoleIon Trap
Ions follow complex
trajectories between two
pairs of electrodes that
switch polarity rapidly
Ions can be ejected from
trap by m/zvalue by
varying the frequency of
end cap electrodes

62. 62
Time of Flight Mass Analyzer
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
velacitiesvary with mass of the ions
Lighter ions reach the detector first
Typical flight times are 1-30 µsec

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

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

65. 65
Inductively Coupled Plasma Mass
Spectrometer

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

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

68. 68
Channel (or Continuous) Dynode
Electron Multiplier
A glass tube that is coated
with lead or a conductive
material
A potential of ~ 2000 V 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.

69. 69
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.

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

71. 71
Faraday Cup
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

72. 72
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

73. 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
-

74. 74
Thank You