PRESENTATION OF

BIOPHYSICAL CHEMISTRY

MASS SPECTROSCOPY
 Mass Spectroscopy
Mass spectrometry is an analytical tool
used for measuring the molecular mass of
a sample. For large samples such as
biomolecules, molecular masses can be
measured to within an accuracy of 0.01%
of the total molecular mass of the sample
i.e. within a 4 Daltons (Da) or atomic
mass units (amu) error for a sample of
40,000 Da. This is sufficient to allow
minor mass changes to be detected, e.g.
the substitution of one amino acid for
another or a post-translational
An outline of what happens
in a mass spectrometer
Atoms can be deflected by magnetic fields -
provided the atom is first turned into an ion.
Electrically charged particles are affected by a
magnetic field although electrically neutral ones
aren't.
 Stage 1: Ionization
The atom is ionized by knocking one or more
electrons off to give a positive ion. This is true
even for things which you would normally
expect to form negative ions (chlorine, for
example) or never form ions at all (argon, for
example). Mass spectrometers always work
with positive ions.
Stage 2: Acceleration

The ions are accelerated so that they all have

the same kinetic energy.
 Stage 3: Deflection

The ions are then deflected by a magnet field

according to their masses. The lighter they are,
the more they are deflected.
The amount of deflection also depends on the
number of positive charges on the ion - in other
words, on how many electrons were knocked off
in the first stage. The more the ion is charged,
the more it gets deflected
 Stage 4: Detection

The beam of ions passing through the

machine is detected electrically. The
chart recorder further displays further
all the details.
The need for a vacuum

It's important that the ions produced in the

ionization chamber have a free run through the
machine without hitting air molecules.
Ionization
Diagrammatic view
 Where are mass
spectrometers
Mass spectrometers are usedused?
in industry
and academia for both routine and research
purposes. The following list is just a brief
summary of the major mass spectrometric
applications:
Biotechnology: the analysis of proteins,
peptides, oligonucleotides
Pharmaceutical: drug discovery,
combinatorial chemistry,
pharmacokinetics, drug metabolism
Clinical: neonatal screening, hemoglobin
analysis, drug testing
Environmental: water quality, food
contamination
Geological: oil composition
How can mass spectrometry
help biochemists?
Accurate molecular weight measurements:
sample confirmation, to determine the purity of
a sample, to verify amino acid substitutions, to
detect post-translational modifications, to
calculate the number of disulphide bridges
Reaction monitoring:
to monitor enzyme reactions, chemical
modification, protein digestion
Raman spectroscopy
 PRINCIPLE OF RAMAN
SPECTROSCOPY
νi ≠ νs
Raman: photon energy change
on interaction (‘collision’) with a molecule

E=h ν
Ei >Es
Ei ≠ Es
 Basic theory
The Raman effect occurs when light impinges
upon a molecule and interacts with the electron
cloud of the bonds of that molecule. The incident
photon excites one of the photons into a virtual
state. For the spontaneous Raman effect, the
molecule will be excited from the ground state
to a virtual energy state, and relax into a
vibrational excited state, which generates
Stokes Raman scattering. If the molecule was
already in an elevated vibrational energy state,
the Raman scattering is then called anti-Stokes
Raman scattering.
 Basic theory
A change in the molecular polarization
potential — or amount of deformation of
the electron cloud —is required for the
molecule to exhibit the Raman effect. The
amount of the polarizability change will
determine the Raman scattering intensity,
whereas the Raman shift is equal to the
vibrational level that is involved.
we are interested in the energy (wave
number) difference between the excitation
and the Stokes lines, the excitation source
should be monochromatic. This forms the
characteristic of a substance.
The difference can be represented as
E’-E=h(f-f’)
This difference in frequency is called
RAMAN SHIFT. It falls in the range of 100-
4000 per cm.
Basic experimental set up
Intense monochromatic radiation from a source
consisting of a large spiral discharge tube with
mercury electrode is allowed to fall on the cell
containing the sample
When the electric discharge passes through the tube
mercury emits lines in its spectrum, the most
intense of which at 43.58 nm serves as the exciting
line.
The scattered light is observed at right angles to the
direction of incident radiation
The detector is either a photographic plate or a
photo multiplier.
 Applications
Raman spectroscopy is commonly used in
chemistry, since vibrational information is specific
for the chemical bonds in molecules. It therefore
provides a fingerprint by which the molecule can be
identified. For instance, the vibrational frequencies
of SiO, Si2O2, and Si3O3 were identified and
assigned on the basis of normal coordinate analyses
using infrared and Raman spectra
find the crystallographic orientation of a sample.
As with single molecules, a given solid material has
characteristic photon modes that can help an
experimenter identify it.
 Applications
can be used to discover counterfeit drugs
without opening their internal packaging, and
for non-invasive monitoring of biological tissue
Raman spectroscopy can be used to investigate
the chemical composition of historical
documents and contribute to knowledge of the
social and economic conditions at the time the
documents were produced
Raman spectroscopy is being investigated as a
means to detect explosives for airport security
 U !
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W i s
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Be M e !!