Molecular Mass Spectrometry

Introduction:
Mass spectrometry is a separate technique in which the analyte in its gaseous form is
fragmented and separated on the basis of mass to charge ratio.
Mass spectrometry is perhaps the most widely applicable of all the analytical tools available
because the technique can provide information about
● The elemental composition of samples of matter
● The structures of inorganic organic and biological molecules
● the qualitative and quantitative composition of complex mixtures
● Isotopic ratios of atoms in samples

Theory:

Principle:
In this technique the sample is first vaporized and then bombarded with a stream of
electrons that led to the loss of an electron by the analyte molecule and formation of the
molecular ion.the collision between energetic electrons in analyte molecule usually imparts
enough energy to the molecules to leave them in the excited state.

The excited molecules relax by fragmentation and produce ions of lower masses. These ions
are then attracted through the slit of a mass spectrometer/analyser where they are sorted
according to their mass to charge ratios and displayed in the form of a mass spectrum.

Mass spectrum:
A mass spectrum is plotted as m/e ratio versus relative abundance of ions. The plot is in the
form of a bar graph. The largest peak in a spectrum is termed as base peak, and is arbitrarily
assigned a value of 100. The heights of the remaining peaks are then computed as a
percentage of the base peak height. Figure shows the mass spectrum of ethylbenzene.
Types of spectra
The appearance of mass spectra for a given molecular species strongly depends on the
method used for ion formation.

Hard source spectra

the hard ionization sources to reduce extensive fragmentation and The spectrum appears as
shown

The many pieces in the spectra correspond to the number of fragments formed and the
height determines their abundance.quite frequently the base peaks in EI spectra correspond
to fragments and not the molecular ion. Hardt source spectrum provides useful information
about the kinds of functional groups and thus structural information about the analyte.
However, with certain types of molecules fragmentation is so complete that the molecular ion
is not detected.without a molecular ion important information for determining the molecular
mass of the analyte is lost.

Soft source spectra

Soft ionization sources cause little fragmentation; the mass spectrum from a soft ionization
source consists of a molecular ion peak and only a few if any other peaks.The base peak in
soft spectra correspond to the molecular ion.
Soft source spectra are useful because they supply accurate information about the
molecular mass of the analyte molecule.

Instrumentation
Several types of instruments are currently used for molecular mass spectrometric
measurements.a distinguishing feature of mass spectrometer is the requirement of an
elaborate vacuum system to create low pressure 10 power minus 2 to 10 power minus 7
Pascal in all the instrument components except the signal processor and re dost the need for
a high vacuum arises to prevent the in frequent collisions with atmospheric components.

Components of mass spectrometer

Figure shows the block diagram of major components of the mass spectrometer.

Sample inlet systems

The purpose of inlet system is to introduce a very small amount of sample into the iron
source with minimal loss of vacuum

Batch inlet systems

These are the conventional and simplest inlet system in which the sample is wallet analysed
externally and then allowed to leak into the evacuated ionization region
The first step is to wipe off any residue from a previous sample and then the current sample
is introduced. For gaseous samples a measured amount is introduced into the reservoir. For
liquid samples with boiling point greater than 150 degree c the reservoir and tubing are
maintained at an elevated temperature by means of a heating tape.the sample which is now
in gas phase is leaked into the ionization area by means of a metal or glass die from
containing one or more pinholes.
Iron sources
Mass spectrometric analysis relies on the formation of gaseous analyte ions.the iron sources
can be classified as
● Hard sources
● Softp sources
● Desorption sources
● Atmospheric pressure ionization

Hard ionization sources

Electron ionization
Historically ions for mass analysis are produced by electron ionization. Figure shows a
schematic diagram of an EI source.

Electrons are emitted from a heated tungsten filament and accelerated by applying a 70V
between the filament and anode. The path of the electrons in molecules are at right angles.
The primary product is a singly charged positive ion formed when electrons collide with the
molecule. The electron approaches molecules closely enough to cause them to lose
electrons by electrostatic repulsion. The primary reaction can be written is

Here m represents the analyte molecule and M is its molecular ion.The positive ions
produced by electron ionization are attracted through the slits of the accelerating plate by
applying a potential difference between the plates and the repellers. The applied potential
difference gives the ions their final velocity before they enter the mass analyser.

Isotope peaks
It is interesting to note that the spectra of a hard source contains some peaks that occur at a
mass to charge ratio greater than that of the molecular ion.these peaks are attributable to
ions having the same chemical formula but different isotopic composition. The size of the
various peaks depends on the relative natural abundance of the isotopes.
Merits and demerits
IE sources are convenient to use; they produce high iron currents which lead to good
sensitivities. the extensive fragmentation and resulting large number of peak is also an
advantage because it often makes an unambiguous identification of analytes possible
extensive fragmentation can also be a disadvantage there it results in the disappearance of
the molecular ion peak so that the molecular mass of analytes cannot b be
measured.another limitation of resource is the need to volatilize the simple which may result
in thermal degradation of some analytes before ionization can occur.

Soft ionization sources

Chemical ionization
In chemical ionization the electron beam does not strike the sample directly, instead the
gaseous atoms of the sample are ionized by colliding with the ions of the agent gas
produced by the bombardment of the electron beam with the reagent molecule. The gas
used reagent is introduced into the ionization region in an amount much greater than that of
the sample. because of the large disintegration difference the electron beam reacts nearly
exclusively with the reagent molecule
one of the most common reagents for chemical ionization is methane which gives the
following ions.these ions react rapidly with additional methane molecules

The collisions between the analyte molecules m and reagent ions result in one of the
following reactions.

The proton transfer reaction gives M + h + ion where the hydride transfer produces an iron
with a mass one less than the analyte MH - h plus.
The chemical ionization spectra generally contains well-defined m plus h plus speak for MH
minus x + peak resulting from the addition or abstraction of a proton in the presence of a
reagent ion.
Desorption sources
Desorption ionization methods are useful for the non volatile or thermally unstable sample
that cannot be ionized with EI or CI methods.these methods have enabled mass spectra to
be obtained for thermally delicate biomolecules and species with higher molecular weight
about 100,000 Da.

Matrix assisted laser desorption ionization MALDI

In this technique a low concentration of the analyte is uniformly dispersed in a solid or liquid
matrix placed on a metal plate.The plate is then placed in a vacuum chamber in which the
laser beam is focused onto the sample. The MALDI matrix must strongly absorb the laser
radiation.The matrix and analyte are then dissolved in ionization creating an ion plume.
The mechanism of formation of MALDI ion plume is thought to involve absorption of the laser
beam by the matrix followed by transfer of the energy from the matrix to the analyte.
Desorption of the analyte and the matrix then occurs. The dissolved analyte is then passed
through the mass analyser.the most common type of mass analyser used with MALDI is the
time of flight (TOF) analyser.
Figure shows a mass spectrum from a mult-TOFi instrument.

Note that the spectrum is characterized by very low background noise in a complete
absence of fragmentation of the large analyte ion. multiple charged ions are present as well
as speech for dimer and trimer species.

Fast atom bombardment sources

With this type of source sample in a condensed state usually in a viscous solution methods
are ionized by bombardment with energetic xenon or argon atoms. Both positive and
negative analyte ion are desorbed from the surface of the sample.
abhi mean of first atoms is obtained by passing accelerated argon or xenon ions from an ion
source through hm per containing argon or xenon atoms at a pressure of about 10 power
minus 3 p a.the high velocity ions undergo a resonant electron exchange reaction with the
atoms. Thus a beam of energetic atoms is formed. The lower energy ions from the exchange
are readily removed by electrostatic deflector.
However the drawbacks of FAB include
● LIMITED MOLECULAR MASS
● Need for larger sample quantities
● Necessity for finding an appropriate matrix in which the analyte is soluble.
Atmospheric pressure ionization methods

Electrospray ionization
electrospray ionization takes place under atmospheric pressure and temperatures in the
apparatus shown in figure

a solution of the sample is pumped through a stainless steel capillary needle at a rate of few
microliters per minute.the needle is enclosed in a cylindrical electrode maintained at several
kilo volts.the resulting charged spray of fine droplets then passes through a desolvating
capillary where evaporation of the solvent in attachment of charge to the analyte molecules
takes place.is the droplet becomes smaller after the evaporation of the solvent their charge
density becomes greater until the Coulombic explosion occurs at Rayleigh limit(where
surface tension can no longer support the charge). The droplet is torn apart into smaller
droplets.The small droplets can repeat the process until all the solvent is removed from the
analyte leaving a multiply charge analyte molecule.
The mass spectra obtained from this ionization source is shown

In these spectra adjacent peaks are for analyte ions that differ by one or more charges. A
striking feature of the spectra is that the average charge state increases in approximately
linear fashion with the molecular mass. The charge state corresponding to each peak can be
determined from pig distribution making it possible to determine the molecular mass of a
protein from the spectrum.
An advantage of of electricity process is that little fragmentation of large and thermally fragile
biomolecules occur because little energy is retained by the analyte upon ionization.another
advantage is that the ions formed are multiplied charged so that their master charge values
are small enough to make them detectable with the quadrupole instrument with a range of
1500 or less.
Mass analyser
Several devices are available for separating ions with different mass to charge ratios. the
analyser should be capable of distinguishing minute mass differences.

Resolution of mass spectrometer

the capability of a mass spectrometer to differentiate between masses is termed as
resolution R. It is defined as

Where Dell M is the mass difference between two adjacent peaks of equal intensity that I
just resolved and M is the nominal mass of the first peak. has a spectrometer with a
resolution of 4000 would resolve peaks at mass to charge values of 400.0 and 400.1.

Types of mass analyser

A mass spectrometer might be equipped with one of the several types of mass analyser.

1. Magnetic analyser
Figure illustrates when form of magnetic analyser

magnetic mass analysis rely upon the effect of a magnetic field on a moving ions to separate
the ions.the accelerated ions that leave the ionic source possess nearly identical kinetic
energies regardless of their masses.the ionic beam is directed between the poles of an
electromagnet where are the field causes the ionic beam to assume a curved path. The
mass to charge ratio is related to other parameters of instrument by the equation

it is clear from the equation that for a fixed set of instrumental parameters the radius of
curvature of an iron in the magnetic field increases with mass to charge ratio.heavier ions of
a fixed charge are deflected less by the magnetic field then lighter elements.consequently
the ions emerging from the magnetic field are separated in space according to their mass to
charge ratio.
 2. Double focusing analysis
A double focusing analyser uses a cylindrical electric sector between the ionic source and
the magnetic mass analyser to restrict the energies of ions that enter the magnetic analyser
to a relatively low band.
in the ionic source not all of the ions are formed at the same location.consequently the ions
do not experience identical accelerating potential from the accelerating electrodes of the
ionic source. Also the ions do not possess identical kinetic energy at the time of their
formation. The combination of these two factors causes the ions to enter the mass analyzer
with slightly different kinetic energies that could lead to peak broadening because the value
of e slightly where is between the ions.
Figure shows a diagram of a double focusing analyser

the ions from the source pass through a slit that ko limits the beam prior to entering the
electric sector.an electric potential is applied between two plates of the sector such that the
entering ions are repelled by the outer electron and attracted towards the inner electrode.the
ions having nearly identical kinetic energies are focused on slit to between the electric and
magnetic sectors after passing through slit to the ions enter the magnetic sector and is
separated based on their mass to charge ratio.

3. Quadrupole mass analyser

quadrupole mass analyser consists of four parallel metallic rods arranged as shown

diagonally opposite rods are electrically connected, that is rod 1 is connected to 2 rod 3 and
rod to is connected to rod 4. ADC potential difference is applied between the two groups of
rods that compels the electron to move forward.simultaneously a radio frequency AC
potential is also applied that continuously changes the relative charge on the two sets of the
rods.
the accelerated ionic beam from the source passes through a collimating whole that is
aligned within the space between the four rods.positive ions that enter the space between
the electrodes are repelled by the rods that are momentarily positively charged and attracted
to the rods that are negatively charged.because the relative charge on the sets of rods is
continuously changing the ions follow and irregular oscillating path between the rods. only
those ions that can pass through the space between the rods strike the exit hole and are
measured by the detector

Time of flight analyser TOF

A diagram of a time of flight mass spectrometer is shown

The operation of a time of flight analyser is relatively easy to understand. Ions that exit from
the ionic source have essentially identical kinetic energies.because the masses of the ions
differ the velocities must also differ.if a group of ions that have different masses
simultaneously and toured the mass analyser the heavier I don't have a velocity that is less
than that of lighter ions.consequently the time that is required for an iron to travel a fix
distance in the analyser where is with the mass of the iron.lighter I on strike the detected
before the heavier ions. The separation in time of different ions which write the detector is
generally less than a microsecond.

MS Detectors and Readouts:

A mass spectrometer contains a transducer (for ions) that converts the beam of ions into an
electrical signal that can then be processed, stored in the memory of a computer, and
displayed or recorded in a variety of ways.

Applications of MS

1. Determination of molecular mass and molecular formula

The molecular ion peak gives mass to charge of the molecular ion which is the molecular
mass of the compound.determination of molecular formula is possible using high resolution
mass spectrometer.
Elucidation of structure
Study of fragmentation processes show their presence of certain structural units in points of
their attachment in the molecule.it gives an idea about the structure of the molecule.
Mass spectrometry in drug Discovery
Drug Discovery involves a number of areas including targeted identification, lead
identification, small molecule optimisation and preclinical and clinical development. MS is a
useful tool in drug Discovery it helps in
● Detection of impurities in active pharmaceutical ingredients
● ADME studies of a drug
● Preclinical development

2. Metabolites analysis
Determination of metabolic pathway and different metabolites of a drug aur xenobiotic is very
important to assess its different parameters of pharmacokinetics drug metabolism
transformations involve changes in the molecular weight. These changes can be accurately
measured by MS.

3. Mass spectrometry in quality control of pharmaceutical

products
NS provides an excellent means for quality control of recombinant proteins some of which
are now used as drugs for example human insulin interference tissue plasminogen activating
factor.the variations in protein structure such as degree of glycosylation or in the terminal
amino acids of proteins can be seen clearly using MS

4. Phytochemical analysis
mass spectrometry is widely employed in phytochemical analysis due to its capability to
identify and measure metabolites having low molecular weight at very low concentration
ranges below nanogram per ml.a variety of analyte separation techniques like gas
chromatography and high performance liquid chromatography United with mass
spectrometry for simultaneous separation and determination of analytes.

5. Applications in forensic sciences

Inference excellence is the use of MS is becoming significant because of an increase in the
demand to investigate the use of illegal drugs through analysing body fluids and tissues.The
sample of forensic in drug abuse is mainly urine, hair and blood.