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Mass spectrometric methods
Assistant of the
pharmaceutical chemistry department
Burmas Nataliya Ivanivna
e-mail: [email protected]
Plan
1.
2.
3.
4.
The concept of mass-spectrometric
analysis.
The mass spectrometer
Quantitative and qualitative analysis
Applications of mass-spectrometry
1.
The concept of mass-spectrometry analysis
Mass spectrometry (MS) is an
analytical technique for the
determination of the elemental
composition of a sample or molecule. It
is also used for elucidating the chemical
structures of molecules, such as peptides
and other chemical compounds.
a)
b)
c)
d)
e)
In a typical MS procedure:
a sample is loaded onto the MS instrument, and
undergoes vaporization.
the components of the sample are ionized by one of a
variety of methods (e.g., by impacting them with an
electron beam), which results in the formation of
charged particles (ions)
the positive ions are then accelerated by an electric
field
computation of the mass-to-charge ratio (m/z) of the
particles based on the details of motion of the ions as
they transit through electromagnetic fields, and
detection of the ions, which in step 4 were sorted
according to m/z.
Analysers characteristics
The mass resolving power - is the measure of the
ability to distinguish two peaks of slightly different
m/z.
The mass accuracy - is the ratio of the m/z
measurement error to the true m/z. Usually
measured in ppm or milli mass units.
The mass range - is the range of m/z amenable to
analysis by a given analyzer.
The linear dynamic range - is the range over which
ion signal is linear with analyte concentration.
2. The mass spectrometer
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 word spectrograph has been used since 1884 as
an "International Scientific Vocabulary". The
linguistic roots are a combination and removal of
bound morphemes and free morphemes which relate
to the terms spectrum and photographic plate. Early
spectrometry devices that measured the mass-tocharge ratio of ions were called mass spectrographs
which consisted of instruments that recorded a
spectrum of mass values on a photographic plate. A
mass spectroscope is similar to a mass spectrograph
except that the beam of ions is directed onto a
phosphor screen.
1.
2.
3.
The three essential functions of a mass
spectrometer, and the associated components,
are:
A small sample of compound is ionized, usually
to cations by loss of an electron. (the ion
source)
The ions are sorted and separated according to
their mass and charge. (the mass analyzer).
The separated ions are then detected and tallied,
and the results are displayed on a chart. (the
detector)
The analyser and detector of the mass spectrometer, and
often the ionisation source too, are maintained under high
vacuum to give the ions a reasonable chance of travelling
from one end of the instrument to the other without any
hindrance from air molecules. The entire operation of the
mass spectrometer, and often the sample introduction
process also, is under complete data system control on
modern mass spectrometers.
The technique has both qualitative and
quantitative uses. These include identifying
unknown compounds, determining the
isotopic composition of elements in a
molecule, and determining the structure of a
compound by observing its fragmentation.
Other uses include quantifying the amount
of a compound in a sample or studying the
fundamentals of gas phase ion chemistry
(the chemistry of ions and neutrals in a
vacuum).
Replica of an early mass spectrometer
Because ions are very reactive and short-lived,
their formation and manipulation must be
conducted in a vacuum. Atmospheric pressure is
around 760 torr (mm of mercury). The pressure
under which ions may be handled is roughly 10-5 to
10-8 torr (less than a billionth of an atmosphere).
Each of the three tasks listed above may be
accomplished in different ways. In one common
procedure, ionization is effected by a high energy
beam of electrons, and ion separation is achieved
by accelerating and focusing the ions in a beam,
which is then bent by an external magnetic field.
The ions are then detected electronically and the
resulting information is stored and analyzed in a
computer.
When a high energy electron collides with a
molecule it often ionizes it by knocking away one
of the molecular electrons (either bonding or nonbonding). This leaves behind a molecular ion
(colored red in the following diagram). Residual
energy from the collision may cause the molecular
ion to fragment into neutral pieces (colored green)
and smaller fragment ions (colored pink and
orange). The molecular ion is a radical cation, but
the fragment ions may either be radical cations
(pink) or carbocations (orange), depending on the
nature of the neutral fragment.
Many mass spectrometers work in either
negative ion mode or positive ion mode. It is
very important to know whether the
observed ions are negatively or positively
charged. This is often important in
determining the neutral mass but it also
indicates something about the nature of
molecules.
4-methyl-3-pentene-2-one
N, N-diethylmethylamine
bromine
Bromine: 50.50% 79Br and 49.50% 81Br
Isotopes
Since a mass spectrometer separates and detects ions of slightly
different masses, it easily distinguishes different isotopes of a
given element. This is manifested most dramatically for
compounds containing bromine and chlorine, as illustrated by
the following examples. Since molecules of bromine have only
two atoms, the spectrum on the left will come as a surprise if a
single atomic mass of 80 amu is assumed for Br. The five peaks
in this spectrum demonstrate clearly that natural bromine
consists of a nearly 50:50 mixture of isotopes having atomic
masses of 79 and 81 amu respectively. Thus, the bromine
molecule may be composed of two 79Br atoms (mass 158 amu),
two 81Br atoms (mass 162 amu) or the more probable
combination of 79Br-81Br (mass 160 amu). Fragmentation of
Br2 to a bromine cation then gives rise to equal sized ion peaks
at 79 and 81 amu.
vinyl chloride
Chlorine: 75.77% 35Cl and 24.23% 37Cl
methylene chloride
3. Quantitative and qualitative analysis
Mass analyzers separate the ions according to their
mass-to-charge ratio. The following two laws govern
the dynamics of charged particles in electric and
magnetic fields in vacuum:
(Lorentz force law)
where
F is the force (in newtons)
E is the electric field (in volts per meter)
B is the magnetic field (in teslas)
q is the electric charge of the particle (in coulombs)
v is the instantaneous velocity of the particle (in meters per second)
× is the vector cross product
Newton's second law of motion in nonrelativistic case, i.e. valid only at ion
velocity much lower than the speed of
light.
Here
F is the force applied to the ion,
m is the mass of the ion,
a is the acceleration
Many mass spectrometers work in either
negative ion mode or positive ion mode. It is
very important to know whether the observed
ions are negatively or positively charged.
This is often important in determining the
neutral mass but it also indicates something
about the nature of molecules. Different
types of ion source result in different arrays
of fragments produced from the original
molecules.
By understanding the origin of a sample,
certain expectations can be assumed as to
the component molecules of the sample and
their fragmentations. A sample from a
synthesis/manufacturing process will
probably contain impurities chemically
related to the target component. A relatively
crudely prepared biological sample will
probably contain a certain amount of salt,
which may form adducts with the analyte
molecules in certain analyses.
4. Application of mass-spectrometry
Mass spectrometry is also used to determine
the isotopic composition of elements within a
sample. Differences in mass among isotopes of
an element are very small, and the less
abundant isotopes of an element are typically
very rare, so a very sensitive instrument is
required. These instruments, sometimes
referred to as isotope ratio mass spectrometers
(IR-MS), usually use a single magnet to bend a
beam of ionized particles towards a series of
Faraday cups which convert particle impacts
to electric current.
A fast on-line analysis of deuterium content of
water can be done using Flowing afterglow
mass spectrometry, FA-MS. Probably the most
sensitive and accurate mass spectrometer for
this purpose is the accelerator mass
spectrometer (AMS). Isotope ratios are
important markers of a variety of processes.
Some isotope ratios are used to determine the
age of materials for example as in carbon
dating. Labeling with stable isotopes is also
used for protein quantification.
Thank you for attention!