A detailed presentation on Mass spectrometry.

2. Dr. P.Samuel
Assistant Professor of Biotechnology
Ayya Nadar Janaki Ammal College (Autonomous)
Sivakasi

3. Definition:
 Mass spectrometry (MS) is an analytical
technique that ionizes complex chemical species
(Proteins) and sorts the ions based on
their mass-to-charge ratio.
 In simpler terms, a mass spectrum measures
the masses within a sample. Mass spectrometry
is used in many different fields and is applied to
pure samples as well as complex mixtures.

4.  Spectrophotometry is a method to measure
how much a chemical substance absorbs light
by measuring the intensity of light as a beam
of light passes through sample solution. The
basic principle is that each compound
absorbs or transmits light over a certain
range of wavelength.

5.  The term spectroscopy is normally reserved
for measurements of the electromagnetic
spectrum. Words ending in -scopy mean
"looking at" whereas words in -metry mean
"measurement of". In practice, the two are
used interchangeably, with the exception of
mass spectrometry where the term
"mass spectroscopy" is discouraged.

6.  The mass spectrometer was invented by F. W.
Aston shortly after World War I. By 1927 Aston
had built an instrument that was accurate to
more than 1 part in 10,000, and mass
spectrometry became the method of choice for
measuring the relative mass of an atom or
molecule.

7.  A mass spectrometer produces charged
particles (ions) from the chemical substances
that are to be analyzed. The mass
spectrometer then uses electric and
magnetic fields to measure
the mass ("weight") of the charged particles.

8.  Basic Principle.
 A mass spectrometer generates multiple ions
from the sample under investigation, it then
separates them according to their specific mass-
to-charge ratio (m/z), and then records the
relative abundance of each ion type. ... This
molecular ion undergoes fragmentation.

9. Year Contribution
1886 Eugen Goldstein observes canal rays.
1898 Wilhelm Wien demonstrates that canal rays can be deflected using
strong electric and magnetic fields. He shows that the mass-to-
charge ratio of the particles have opposite polarity and is much
larger compared to the electron. He also realizes that the particle
mass is similar to the one of hydrogen particle.
1898 J. J. Thomson measures the mass-to-charge ratio of electrons.
1901 Walter Kaufmann uses a mass spectrometer to measure the
relativistic mass increase of electrons.
1905 J. J. Thomson begins his study of positive rays.
1906 Thomson is awarded the Nobel Prize in Physics "in recognition of
the great merits of his theoretical and experimental investigations on
the conduction of electricity by gases"

10. Year Contribution
1913 Thomson is able to separate particles of different mass-to-charge
ratios. He separates the 20Ne and the 22Ne isotopes, and he correctly
identifies the m/z = 11 signal as a doubly charged 22Ne particle.
1919 Francis Aston constructs the first velocity focusing mass spectrograph
with mass resolving power of 130.
1922 Aston is awarded the Nobel Prize in chemistry "for his discovery, by means
of his mass spectrograph, of isotopes, in a large number of non-radioactive
elements, and for his enunciation of the whole-number rule."
1931 Ernest O. Lawrence invents the cyclotron.
1934 Josef Mattauch and Richard Herzog develop the double-focusing
mass spectrograph.
1936 Arthur J. Dempster develops the spark ionization source.
1937 Aston constructs a mass spectrograph with resolving power of 2000.
1939 Lawrence receives the Nobel Prize in Physics for the cyclotron.
1942 Lawrence develops the Calutron for uranium isotope separation.
1943 Westinghouse markets its mass spectrometer and proclaims it to be
"A New Electronic Method for fast, accurate gas analysis".
1946 William Stephens presents the concept of a time-of-flight mass
spectrometer.

11. Year Contribution
1954 A. J. C. Nicholson (Australia) proposes a hydrogen transfer reaction
that will come to be known as the McLafferty rearrangement.
1959 Researchers at Dow Chemical interface a gas chromatograph to a
mass spectrometer.
1964 British Mass Spectrometry Society established as first dedicated mass
spectrometry society. It holds its first meeting in 1965 in London.
1966 F. H. Field and M. S. B. Munson develop chemical ionization.
1968 Malcolm Dole develops electrospray ionization.
1969 H. D. Beckey develops field desorption.
1974 Comisarow and Marshall develop Fourier Transform Ion Cyclotron
Resonance mass spectrometry.
1976 Ronald MacFarlane and co-workers develop plasma desorption mass
spectrometry.
1984 John Bennett Fenn and co-workers use electrospray to ionize
biomolecules.
1985 Franz Hillenkamp, Michael Karas and co-workers describe and coin
the term matrix-assisted laser desorption ionization (MALDI).

12. Year Contribution
1987 Koichi Tanaka uses the “ultra fine metal plus liquid matrix method” to
ionize intact proteins.
1989 Wolfgang Paul receives the Nobel Prize in Physics "for the
development of the ion trap technique".
1999 Alexander Makarov presents the Orbitrap mass spectrometer.
2002 John Bennett Fenn and Koichi Tanaka are awarded one-quarter of the
Nobel Prize in chemistry each "for the development of soft desorption
ionisation methods ... for mass spectrometric analyses of biological
macromolecules."

14. Mass spectrometry uses an instrument called a
mass spectrometer. The main components of
a mass spectrometer are:
Inlet system (LC, GC, Direct probe etc...)
Ion source (EI, CI, ESI, APCI, MALDI, etc...)
Mass analyzer (Quadrupole, TOF, Ion Trap,
Magnetic Sector)
Detector (Electron Multiplier, Micro Channel Plates
MCPs)

15. 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...).
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.
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, Magnetic, Quadrupole, Ion trap,
Fourier Transform, Time of Flight, etc...The ion beam exiting the analyzer
assembly is then detected and the signal is registered.
Common ionization method acronyms include:
EI=Electron Impact;
CI=Chemical Ionization;
SIMS=Secondary Ions Mass Spec;
FAB=Fast Atom Bombardment;

16. Ionization Methods
Selection of the proper ionization method for the analysis of your sample
is extremely important. Although we can offer suggestions, it is your
responsibility to understand and select the method(s) appropriate for
your research compounds.
Electron Impact EI Ionization
Chemical Ionization CI
Negative Ion Chemical Ionization
Electrospray IonizationTechniques
Matrix Assisted Lazer Desorption
Atmospheric Pressure Chemical Ionization APCI

17. Analysis of Ions
It is possible to use several different physical parameters to achieve mass
separation. Common types of mass analyzers are discussed below. Each
has advantages and disadvantages.
Magnetic Sector Mass Spectrometer

18. Ion source: There are several types of ionization methods in mass
spectrometry. The physical basis of ionization methods are very complex
and outside the scope of the course. Most common methods are:
(a) Matrix-assisted laser desorption/ionization (MALDI)
This method of ionization is a soft ionization method and results in minimum
fragmentation of sample.
This method is used for non-volatile, and thermally labile compounds such as
proteins, oligonucleotides, synthetic polymers.
Sample is mixed with 1000 times molar excess of sample and spotted onto a
metal plate and dried.
 Matrix plays a key role in this technique by absorbing the laser light energy
and causing a small part of the target substrate to vaporize.
Although, the process of forming analyte ions is unclear, it is believed that
matrix which has labile protons, such as carboxylic acids, protonates neutral
analyte molecules after absorbing laser light energy.

20. (b) Electrospray Ionization (ESI)
Electrospray Ionization (ESI) is a preferred method of ionization when the sample is
in liquid form.
This is also a soft method of ionization and results in less fragmentation. ESI is a
very valuable method for analysis of biological samples.
The method was developed by John Fenn and he shared 2002 Nobel prize in
chemistry for this work.
The analyte is introduced either from a syringe pump or as the eluent flow from
liquid chromatography with a flow rate 1µl min-1 .
The analyte solution passes through the Electrospray needle (Stainless steel capillary
with 75-150 µm internal diameters) that has a high potential difference (with respect to
the counter electrode) applied to it (typically in the range from 2.5 to 4 kV).
This forces the spraying of charged droplets from the needle with a surface charge of
the same polarity to the charge on the needle.
As droplet moves towards counter electrode cone (which passes it to analyzer),
solvent evaporation occurs and droplet shrinks until it reaches the point that the surface
tension can no longer sustain the charge (the Rayleigh limit) and at that point droplets
break.
This produces smaller droplets and the process is repeated. Finally after all solvent
evaporated, charge is passed on to analyte.