Introduction, Basic Principles, Terminology, Instrumentation, Ionization techniques (EI, CI, FAB, MALDI, and ESI), Mass Analyzer (Magnetic sector instruments, Quadrupole, TOF, and ICR ), and Applications of Mass Spectrometry.

1. MASS SPECTROMETRY-I
Dr. BASAVARAJAIAH S. M.
Assistant Professor and Coordinator
P.G. Department of Chemistry
Vijaya College
Bangalore-560 004

2. Contents:
1.Introduction
2.Basic Principle
3.Instrumentation
4.Applications

3. INTRODUCTION
Mass spectrometry is a powerful analytical technique used to;
Quantify known materials
To identify unknown compounds
To elucidate the structure
Chemical properties of different molecules
The complete process involves the conversion of the sample
into gaseous ions, with or without fragmentation, which are then
characterized by their mass to charge ratios (m/z) and relative
abundances.
This technique basically studies the effect of ionizing energy on
molecules.

4. In this technique, molecules are bombarded with a beam of
energetic electrons.
The molecules are ionized and broken up into many fragments,
some of which are positive ions. Each kind of ion has a particular
ratio of mass to charge, i.e. m/e ratio (value).
For most ions, the charge is one and thus, m/e ratio is simply
the molecular mass of the ion.
The ions pass through magnetic and electric fields to reach
detector where they are detected and signals are recorded to
give a mass spectra.
BASIC PRINCIPLE

5. Ex: Neopentane

6. Terminology:
•Molecular ion or Parent ion: The ion obtained by the loss of an
electron from the molecule.
•Base Peak: The most intense peak in the MS, assigned 100%
intensity.
•Fragment ions: Lighter cations formed by the decomposition of
the molecular ion.
•M+: Symbol often given to the molecular ion.
•Radical cation: +ve charged species with an odd number of
electrons.
•Metastable ion* peak: Metastable ion peaks are usually broad
peaks, and they frequently appear at nonintegral values of m/e.

7. Nitrogen rule: This rule states that if a compound has an even
number of nitrogen atoms (or no nitrogen atoms), its molecular ion
will appear at an even mass value. On the other hand, a molecule
with an odd number of nitrogen atoms will form a molecular ion
with an odd mass.
Mass:100
Heptane
Mass:78
Benzene
NH2
Mass:115
Heptane-1-amine
NO2
Mass:123
Nitrobenzene
NH2
NH2
Benzene-1,2-diamine
Mass:108
Cl
Chlorobenzene
Mass:112
Cl
Cl
1,2-Dichlorobenzene
Mass:146
Ex:

8. Natural Abundance of Common Elements and their Isotopes
Element Relative Abundance
Hydrogen 1H 100 2H 0.016
Carbon 12C 100 13C 1.08
Nitrogen 14N 100 15N 0.38
Oxygen 16O 100 17O 0.04 18O 0.20
Fluorine 19F 100
Silicon 28Si 100 29Si 5.10 30Si 3.35
Phosphorus 31P 100
Sulfur 32S 100 33S 0.78 34S 4.40
Chlorine 35Cl 100 37Cl 32.5
Bromine 79Br 100 81Br 98
Iodine 127I 100

9. Halogen
Relative Intensities
M M+2 M+4 M+6
Br 100 97.7
Br2 100 195.0 95.4
Br3 100 293.0 286 93.4
Cl 100 32.6
Cl2 100 65.3 10.6
Cl3 100 97.8 31.9 3.47
BrCl 100 130.0 31.9
Br2Cl 100 228.0 159.0 31.2
Cl2Br 100 163.0 74.4 10.4
Relative Intensities of Isotope Peaks for Various
Combinations of Bromine and Chlorine

10. INSTRUMENTATION
In order to measure the characteristics of individual molecules, a
mass spectrometer converts them to ions so that they can be
moved about and manipulated by external electric and magnetic
fields.
The three essential functions of a mass spectrometer, and the
associated components, are:
 The Ion Source: A small sample is ionized, usually to cations by loss
of an electron.
 The Mass (Ion) Analyzer: The ions are sorted and separated
according to their mass and charge.
 The Detector :The separated ions are then measured, and the results
displayed on a chart.

11. Figure: Components of a Mass Spectrometer

12. Inlet: Samples can be introduced to the mass spectrometer
directly via solids probe, or in the case of mixtures, by the
intermediary of chromatography device (e.g. Gas
chromatography, Liquid chromatography, Capillary
electrophoresis, etc...).
Ion Source: Once in the source, sample molecules are subjected
to ionization. Ions formed in the source (molecular and fragment
ions) acquire some kinetic energy and leave the source. Common
ionization methods include:
EI=Electron Impact; FAB=Fast Atom Bombardment; MALDI=Matrix Assisted
Laser Desorption Ionization; CI=Chemical Ionization; SIMS=Secondary Ions
Mass Spec; TS=Thermospray; PDMS=Plasma Desorption Mass Spec;
AS=Aerospray; ESMS=Electrospray Mass Spec.

13. Ion analyzer: A calibrated analyzer then analyzes the passing
ions as a function of their mass to charge ratios. Different kind of
analyzer(s) can be used;
Quadrapole Mass Spectrometers
Time of Flight Mass Spectrometer
Ion Cyclotron Resonance
Double Focussing Analysers
Magnetic Sector-MS
Ion Trap
Detector: The ion beam exiting the analyzer assembly is then
detected and the signal is registered.

14. IONIZATION METHODS
The large number of ionization methods, some of which are
highly specialized, precludes complete coverage.
The most common ones in the three general areas of;
Gas-Phase: EI, CI
Desorption: FAB, MALDI, FD, PDI
Evaporate Ionization: Thermospray, Electrospray

15. Electron Impact Ionization:
Electron impact (EI) is the most widely used method for
generating ions for mass spectrometry.
Firstly the analyte must be vaporized.
This is usually achieved by heating the probe tip containing a
droplet of the analyte in solution. If the sample is thermally
unstable, this will often be the first cause of sample
fragmentation.
Once in the gas-phase, the analyte passes into an EI chamber
where it interacts with a homogeneous beam of electrons
typically at 70 electron volts energy.

16. The electron beam is produced by a filament (rhenium or
tungsten wire) and steered across the source chamber to the
electron trap.
A fixed magnet is placed, with opposite poles slightly off-axis,
across the chamber to create a spiral in the electron beam.
This is to increase the chance of interactions between the beam
and the analyte gas.
There are no actual collisions between analyte molecules and
electrons, ionization is caused by electron ejection from the
analyte or by analyte decomposition.

17. Figure: EI Ionization chamber

18. Electron Ionization Mass Spectra of 3,4-Dimethoxy acetophenone

19. CHEMICAL IONIZATION
Chemical Ionization (CI) is especially useful technique when no molecular
ion is observed in EI mass spectrum, and also in the case of confirming the
mass to charge ratio of the molecular ion.
Chemical ionization technique uses virtually the same ion source device as
in electron impact, except, CI uses tight ion source, and reagent gas.
Reagent gas (e.g. ammonia or methane) is first subjected to electron impact.
Sample ions are formed by the interaction of reagent gas ions and sample
molecules. This phenomenon is called ion-molecule reactions.
Reagent gas molecules are present in the ratio of about 100:1 with respect
to sample molecules. Positive ions and negative ions are formed in the CI
process. Depending on the setup of the instrument (source voltages,
detector, etc...) only positive ions or only negative ions are recorded.

20. In CI, ion molecule reactions occur between ionized reagent gas
molecules (G) and volatile analyte neutral molecules (M) to
produce analyte ions. Pseudo-molecular ion MH+ (positive ion
mode) or [M-H]- (negative ion mode) are often observed.
Unlike molecular ions obtained in EI method, MH+ and [M-H]-
detection occurs in high yield and less fragment ions are
observed.
Positive ion mode:
GH+ + M ------> MH+ + G
Negative ion mode:
[G-H]- + M ------> [M-H]- + G

21. Figure: CI Ionization chamber

22. Chemical Ionization Mass Spectra of 3,4-Dimethoxy acetophenone

23. FAST ATOM BOMBARDMENT IONIZATION
Fast atom bombardment (FAB) uses high-energy Xenon and Argon
atoms (6-10 keV) to bombard samples dissolved in a liquid of low vapor
pressure (Matrix) (e.g., glycerol, m-nitro benzyl alcohol, diethanolamine
etc.).
The matrix protects the sample from excessive radiation damage. A
related method, liquid secondary ionization mass spectrometry. LSIMS,
is similar except that it uses somewhat more energetic cesium ions (10-
30 keV).
In both methods, positive ions (by cation attachment) ([M+1]+ or
[M+Na]+) and negative ions (by deprotonation [M-1]+) are formed; both
types of ions are usually singly charged and, depending on the FAB is
used primarily with large nonvolatile molecules, particularly to determine
molecular weight.

24. Figure: FAB Ionization chamber

25. MATRIX-ASSISTED LASER DESORPTION/IONIZATION
(MALDI)
MALDI is a soft ionization that involves a laser striking a matrix
of small molecules to make the analyte molecules into the gas
phase without fragmenting or decomposing them.
Some biomolecules are too large and can decompose when
heated, and traditional techniques will fragment or destroy
macromolecules.
MALDI is appropriate to analyze biomolecules like peptides,
lipids, saccharides, or other organic macromolecules.

26. Figure: MALDI Ionization chamber
The analyte is embedded in a very large excess of a matrix
compound deposited on a solid surface called a target, usually
made of a conducting metal and having spots for several
different samples to be applied. After a very brief laser pulse, the
irradiated spot is rapidly heated and becomes vibrationally
excited.

27. The matrix molecules energetically ablated from the surface of the
sample, absorb the laser energy and carry the analyte molecules into
the gas phase as well.
During the ablation process, the analyte molecules are usually
ionized by being protonated or deprotonated with the nearby matrix
molecules.
The most common MALDI ionization format is for analyte
molecules to carry a single positive charge.
Lasers of both ultraviolet (UV) and infrared (IR) wavelengths are in
use, but UV lasers are by far the most important light sources in
analytical MALDI. Among these, nitrogen lasers and frequency-tripled
or quadrupled Nd: Yag lasers often serve for the majority of
applications.

28. Commonly used MALDI matrix substance;
In general, highly polar analytes work better with highly polar
matrices, and nonpolar analytes are preferably combined with
nonpolar matrices. Different matrixes have been sought and
widely used. Currently, the most commonly used matrixes are
α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid,
3,5-dimethoxy-4-hydroxycinnamic acid, and 2,6-dihydroxy
acetophenone.

29. ELECTROSPRAY IONIZATION
Among the most used spray ionization techniques is Electrospray Ionization
(ESI). This technique continues to be the method of choice for analyzing thermo
labile chemicals.
It uses an electrical stress between the ESI probe exit (e.g. capillary) and the
counter electrode, which is located few millimeters from the probe.
The process results in the generation of highly charged droplets directly from
the infused solution. Multiply and/or singly charged analyte molecules desorbed
from the sprayed droplets and sampled through the rest of the mass
spectrometer.
ESI has been distinguished for its ability to produce multiply charged
molecular ions from a large variety of polymers such as protein and DNA
fragments; it allows also sensitive detection of singly charged low molecular
weight polar species such as drugs and drug metabolites.

30. The formation of positive or negative ions (depending on the sign of the applied
electrical field) occurs in high yield. In the positive ion mode protonated and/or alkali
adduct analyte molecules generally observed in the mass spectra.
In the negative ion mode operation peaks corresponding to deprotonated analyte
molecules are observed. ESI is described as a very "soft" ionization technique where the
surrounding bath gas has a moderating effect on the internal and translational energies
of desorbed ions.
Figure: ESI Ionization chamber

31. Advantages of ESI:
Soft ionization process so intact molecular ions are observed
ESI allows production of multiply charged ions. This results in the
ability of analyzing very high molecular weight species using the
most available mass analyzers (e.g. quadrupoles).
ESI is an atmospheric pressure process. This makes it easy to
use and easy to interface with HPLC and CE separation
techniques.

32. MASS (ION) ANALYZER
The mass analyzer, which separates the mixture of ions that are generated
during the ionization step by m/e in order to obtain a spectrum, is the heart of
each mass spectrometer, and there are several different types with different
characteristics. Each of the major types of mass analyzers is described below.
Magnetic Sector-MS
Quadrapole Mass Spectrometers
Time of Flight Mass Spectrometer
Ion Cyclotron Resonance
Double Focussing Analysers
Ion Trap

33. MAGNETIC SECTOR-MS
Ions enter the instrument from the source where they are initially
focussed.
They enter the magnetic sector through the source slit where they
are deflected according to the left-hand rule.
Higher-mass ions are deflected less than lower-mass ions.
Scanning the magnet enables ions of different masses to be
focussed on the monitor slit.
At this stage, the ions have been separated only by their masses.
To obtain a spectrum of good resolution where all ions with the
same m/z appear coincident as one peak in the spectrum, ions have
to be filtered by their kinetic energies.

34. After another stage of focussing the ions enter the electrostatic
sector where ions of the same m/z have their energy distributions
corrected for and are focussed at the double focussing point on
the detector slit.

35. QUADRUPOLE MASS SPECTROMETERS
A quadrupole mass filter consists of four parallel metal rods
arranged as in the figure below.
Two opposite rods have an applied potential of (U+Vcos(wt))
and the other two rods have a potential of -(U+Vcos(wt)), where U
is a dc voltage and Vcos (wt) is an ac voltage.
The applied voltages affect the trajectory of ions traveling down
the flight path centered between the four rods.
For given dc and ac voltages, only ions of a certain mass-to-
charge ratio pass through the quadrupole filter and all other ions
are thrown out of their original path.

36. A mass spectrum is obtained by monitoring the ions passing
through the quadrupole filter as the voltages on the rods are
varied. There are two methods: varying w and holding U and V
constant, or varying U and V (U/V) fixed for a constant w.

37. Time-of-flight mass spectrometry (TOFMS) is a method
of mass spectrometry in which an ion's mass-to-charge ratio is
determined via a time of flight measurement.
Ions are accelerated by an electric field of known strength. This
acceleration results in an ion having the same kinetic energy as
any other ion that has the same charge.
The velocity of the ion depends on the mass-to-charge
ratio (heavier ions of the same charge reach lower speeds,
although ions with higher charge will also increase in velocity).
TIME-OF-FLIGHT MASS SPECTROMETRY

38. The time that it subsequently takes for the ion to reach a
detector at a known distance is measured.
This time will depend on the velocity of the ion, and therefore is
a measure of its mass-to-charge ratio. From this ratio and known
experimental parameters, one can identify the ion.

39. ION CYCLOTRON RESONANCE
Ion cyclotron resonance mass spectrometry is a type of
mass analyzer (or mass spectrometer) for determining the mass-
to-charge ratio (m/z) of ions based on the cyclotron frequency of
the ions in a fixed magnetic field.
The ions are trapped in a Penning trap (a magnetic field with
electric trapping plates), where they are excited (at their resonant
cyclotron frequencies) to a larger cyclotron radius by an
oscillating electric field orthogonal to the magnetic field.
After the excitation field is removed, the ions are rotating at
their cyclotron frequency in phase (as a "packet" of ions).

40. These ions induce a charge (detected as an image current) on
a pair of electrodes as the packets of ions pass close to them.
The resulting signal is called a free induction decay (FID),
transient or interferogram that consists of a superposition of sine
waves.
The useful signal is extracted from this data by performing
a Fourier transform to give a mass spectrum.

41. High Resolution Mass Spectrometry
High resolution mass spectrometry (HR-MS) allows detection of
analytes to the nearest 0.001 atomic mass units.
Examples of HR-MS instruments are time-of-flight (TOF)
and Fourier transform ion cyclotron resonance (FTICR), which
forms the basis of the orbit rap technology.
Generally, these instruments measure the exact mass of
analytes without fragmentation, however, they can be combined
with a quadrupole in which case fragmentation is also possible
and can add more selectivity to the method.

42. The major advantage of this technique is that it is very selective
since it measures the exact mass of a compound allowing even
minor changes in structure to be distinguished.
For example, we can distinguish at a unit mass of CO, N2,
C2H4, and CH2N. The exact masses are as below:
CO= 28.0062, N2=28.0062, C2H4=28.0312, and CH2N=28.0187

43. Exact Masses of Isotopes

44. Applications of Mass Spectrometry (MS)
Environmental monitoring and analysis (soil, water and air
pollutants, water quality, etc.).
Geochemistry – age determination, soil and rock composition,
oil and gas surveying.
Chemical and Petrochemical industry – Quality control
Identify structures of biomolecules, such as carbohydrates,
nucleic acids.
Sequence biopolymers such as proteins and oligosaccharides.
Determination of molecular mass of peptides, proteins, and
oligonucleotides.
Monitoring gases in patients breath during surgery.

45. Identification of drugs abuse and metabolites of drugs of abuse
in blood, urine, and saliva.
Analyses of aerosol particles.
Determination of pesticides residues in food.

46. SUGGESTED BOOKS:
Spectrometric identification of organic compounds, R. M.
Silverstien, F X Webster, D J Kiemle and D L Bryce, 8th Wiley
student edition, New Delhi,2015.
Introduction to spectroscopy, 4th edition, D. L. Pavia, G. M.
Laupman and G. S. Kriz, Harcourt College Publishers, 2009.
Mass spectrometry a foundation course, K Downard, RSC,
Cambridge,2004.
Organic spectroscopy, W. Kemp, Macmillan, London, 2011.
Spectroscopy of Organic Compounds By P S Kalsi · 2007.