Transcript Mass Spectroscopy

Mass Spectroscopy
Dr. Nikhat Siddiqi
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• Mass spectrometry is a powerful analytical
technique that is used to identify unknown
compounds, to quantify known compounds, and
to elucidate the structure and chemical
properties of molecules.
• Detection of compounds can be accomplished
with very minute quantities (as little as 10-12g, 1015 moles for a compound of mass 1000 Daltons).
This means that compounds can be identified at
very low concentrations (one part in 1012) in
chemically complex mixtures.
Dr. Nikhat Siddiqi
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Uses of Mass Spectroscopy
• Detect and identify the use of steroids in athletes.
• Monitor the breath of patients by
anesthesiologists during surgery .
• Determine whether honey is adulterated with
corn syrup.
• Monitor fermentation processes for the
biotechnology industry.
• Detect dioxins in contaminated fish.
• Determine gene damage from environmental
causes.
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• Identify structures of biomolecules, such as
carbohydrates, nucleic acids and steriods .
• Sequence biopolymers such as proteins and
oligosaccharides .
• Determine how drugs are used by the body.
• Analyze for environmental pollutants.
• Identify and quantitate compounds of
complex organic mixtures.
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Mass Spectrometer
• A mass spectrometer is an instrument that measures the masses of
individual molecules that have been converted into ions, i.e.,
molecules that have been electrically charged.
• Since molecules are so small, it is not convenient to measure their
masses is kilograms, or grams, or pounds.
• In fact, the mass of a single hydrogen atom is approximately 1.66 X
10-24 grams. We therefore need a more convenient unit for the
mass of individual molecules. This unit of mass is often referred to
by chemists and biochemists as the dalton (Da for short), and is
defined as follows: 1 Da=(1/12) of the mass of a single atom of the
isotope of carbon-12(12C).
• This follows the accepted convention of defining the 12C isotope as
having exactly 12 mass units.
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• A mass spectrometer does not actually measure the
molecular mass directly, but rather the mass-to-charge
ratio of the ions formed from the molecules.
• It follows that the charge on an ion is denoted by the
integer number z of the fundamental unit of charge,
and the mass-to-charge ratio m/z therefore represents
daltons per fundamental unit of charge.
• In many cases, the ions encountered in mass
spectrometry have just one charge (z=1) so the m/z
value is numerically equal to the molecular (ionic) mass
in Da.
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PRINCIPLES OF MASS
SPECTROMETRY
• Any moving charged species of mass,m, and
velocity, v, will be deflected by an applied
magnetic field.
• The magnitude of this deflection will depend
on the momentum, μ, of the species which is
given by Equation
µ = m. v
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• Species with large momentum are deflected less than those with
small momentum.
• Thus, if a stream of atoms and small molecules of identical velocity
and charge but different mass in the gas phase is passed through a
magnetic field, the deflection experienced by each atom or
molecule depends on its mass.
• This deflection can therefore provide an accurate measure of mass.
Larger molecules are uncharged in the gas phase so, in order to
deflect these, it is necessary to confer a charge upon them.
• This may be achieved by irradiating the molecules with a beam of
electrons and is called electron impact (EI) ionization.
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• Since the beam of electrons is of quite high energy, this
ionization method can cause extensive breakdown of
molecular structure, splitting a single molecule into a
number of fragment ions of different mass (Figure 4.3)
each of which may be deflected differently in the
magnetic field.
• This generates a mass spectrum (MS) which is
characteristic of the chemical structure of the
molecule. In fact, the complete structure of small
biomolecules may be determined by electron impact
MS (EI/MS) analysis alone.
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Overview of MS Experiment
• A mass spectrometer consists of three distinct
components viz., a source, analyzer and detector all
maintained under a powerful vacuum (∼1 mPa).
• The source is an ionization chamber where the stream
of ions is generated.
• The analyzer maintains either a magnetic or electric
field which accelerates the stream of ions to a single
velocity and then differentially deflects them so that
they can be detected.
• This separates ions based on differences in their m/z
ratio.
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• Provided all ions carry the same charge, they
will deflect in the analyzer according to their
m/z ratio that is according to their different
mass.
• A spectrum of differing mass can therefore be
generated from a single chemical species. It is
possible to generate ions with z > 1, thus
allowing analysis of large mass species.
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• The ion beam may be detected by a
photomultiplier detector and converted into
an electric signal.
• This signal is amplified by a factor of up to 106
resulting in extremely sensitive detection.
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• Formation of gas phase samples
ions is an essential prerequisite to
the mass sorting and detection
processes that occur in a mass
spectrometer.
• The sample, which may be a
solid, liquid, or vapor, enters the
vacuum chamber through an
inlet.
• Depending on the type of inlet
and ionization techniques used,
the sample may already exist as
ions in solution, or it may be
ionized in conjunction with its
volatilization or by other methods
in the ion source.
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• The gas phase ions are sorted
in the mass analyzer according
to their mass-to-charge (m/z)
ratios and then collected by a
detector.
• In the detector the ion flux is
converted to a proportional
electrical current.
• The data system records the
magnitude of these electrical
signals as a function of m/z
and converts this information
into a mass spectrum.
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