Mass spectroscopy -> by Mohit kumar

PRESENTED BY: MOHIT KUMAR
M. PHARMACY
(PHARMACEUTICS)
3rd SEMESTER
SETH G. L. BIHANI S. D. COLLEGE OF TECHNICAl EDUCATION
(INSTITUTE OF PHARMACEUTICAL SCIENCES & DRUG RESEARCH)
GAGAN PATH, SRI GANGANAGAR (RAJ.) 335001, INDIA.

 INTRODUCTION
 HISTORY
 BASIC PRINCIPLE
 EQUATION
 LABELLED DIAGRAMS
 INSTRUMENTATION

 Mass spectrometry (MS) is an analytical
technique that measures the mass-to-charge
ratio of ions.
 The results are typically presented as a mass
spectrum, a plot of intensity as a function of the
mass-to-charge ratio.
 Mass spectrometry is used in many different
fields and is applied to pure samples as well as
complex mixtures.
 A mass spectrum is a plot of the ion signal as a
function of the mass-to-charge ratio.

 In a typical MS procedure, a sample, which may
be solid, liquid, or gaseous, is ionized, for
example by bombarding it with electrons. This
may cause some of the sample's molecules to
break into charged fragments or simply become
charged without fragmenting.
 These ions are then separated according to their
mass-to-charge ratio, for example by accelerating
them and subjecting them to an electric or
magnetic field.

 Ions of the same mass-to-charge ratio will undergo the
same amount of deflection. The ions are detected by a
mechanism capable of detecting charged particles, such
as an electron multiplier.
 Results are displayed as spectra of the signal intensity
of detected ions as a function of the mass-to-charge
ratio. The atoms or molecules in the sample can be
identified by correlating known masses (e.g. an entire
molecule) to the identified masses or through a
characteristic fragmentation pattern.

 In 1886, Eugen Goldstein observed rays
in gas discharges under low pressure that
travelled away from the anode and through
channels in a perforated cathode, opposite to
the direction of negatively charged cathode
rays (which travel from cathode to anode).
 Goldstein called these positively
charged anode rays "Kanalstrahlen"; the
standard translation of this term into English is
"canal rays".

 Modern techniques of mass spectrometry were
devised by Arthur Jeffrey Dempster and F.W.
Aston in 1918 and 1919 respectively.
 In 2002, the Nobel Prize in Chemistry was
awarded to John Bennett Fenn for the
development of electrospray ionization (ESI)
and Koichi Tanaka for the development of soft
laser desorption (SLD) and their application to
the ionization of biological macromolecules,
especially proteins.

 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.
 The first step in the mass spectrometric
analysis of compounds is the production of
gas phase ions of the compound, basically by
electron ionization.

 This molecular ion undergoes fragmentation.
 Each primary product ion derived from the
molecular ion, in turn, undergoes fragmentation,
and so on.
 The ions are separated in the mass spectrometer
according to their mass-to-charge ratio, and are
detected in proportion to their abundance.
 A mass spectrum of the molecule is thus
produced. It displays the result in the form of a
plot of ion abundance versus mass-to-charge
ratio.

 Ions provide information concerning the
nature and the structure of their precursor
molecule.
 In the spectrum of a pure compound, the
molecular ion, if present, appears at the
highest value of m/e (followed by ions
containing heavier isotopes) and gives the
molecular mass of the compound.

 When the ions are accelerated by electric field ,
they acquires the energy E, given by the
equation-
E = e v (eq. 1)
 Where ,
e = charge on the ion
v = applied potential acceleration
 When the ions are emerging out of ion source, at
that time, the potential energy is converted into
the kinetic energy, represented by the equation-
K.E. = ½ m v’2 (eq. 2)

 Where,
m = mass of the ion
v’ = velocity of the ion after acceleration
 We know that ,
Potential energy = Kinetic energy
 So,
e v = ½ m v’2 (eq. 3)
 When an magnetic field or electric field is applied , the
positive charged fragments , which were travelling in a
straight path, now travels in curved path.
 When ions enter the magnetic field , they assume circular
path and are subjected to centripetal (magnetic) force,
given by the equation-
Fm = H e v’ (eq. 4)

 Where,
H = magnetic field strength
e = charge on the ion
 At equilibrium (in order for an ion to move in
a circular path), centripetal force will be
equalled by the centrifugal force, given by the
equation-
Fc = mv’2/r (eq. 5)
 Where,
r = radius of the circular path

 At equilibrium,
Fm = Fc
H e v’ = mv’2/r
v’ = H e r / m (eq. 6)
 Substituting the value of v’ in eq. 3 ,
e v = ½ m ( H e r / m)2
m/e = H2 r2 / 2v - This the fundamental
eq. of mass spectroscopy.

 Molecular chamber
 Ionization chamber
 Acceleration zone
 Magnetic field / Electric field
 Ionization techniques
 Mass analyzers (Ion separators)
 Detectors (Transducers)

High temperature and negative pressure (10-2
torr) is maintained so as to convert the analyte
into vapour form.

 Contains thin sheet of gold with a pinhole /
orifice called Molecular Peak.
 From molecular peak , one molecule can pass.
 Have lower negative pressure (10-6torr).
 Positive charged ions are produced.

 Divided into different acceleration plates.
 High potential is applied between these
accelerated plates (103 to 104 volts), which
cause acceleration of ions in a straight path.

 Magnetic field / electric field is applied on
the positive charged ions and fragments.
 The ions or fragments which were travelling
in straight path , now travels in curved path.

 The positive charged molecule ions /
fragments are collected using a collecting slit
/ collector and allowed to fall on a detector
and mass spectra is recorded.
 Therefore, these positive charged ions are
separated according to their m/e ratio (m/e α
r2 α H α 1/v).

 In ion source, a sample is ionized, usually to cations by loss of an electron.
1) Gas phase sources
 Electron impact (EI)
 Chemical ionization (CI)
 Field ionization (FI)
2) Desorption sources
 Field desorption (FD)
 Atmospheric Pressure Ionization (API)
 Electro-spray Ionization (EI)
 Atmospheric Pressure Chemical Ionization (APCI)

3) Fast Atom Bombardment (FAB)
4) Matrix Assisted Laser Desorption Ionization (MALDI)
5) Laser Ionization Mass Spectroscopy (LIMS)
6) Secondary Ion Mass Spectroscopy (SIMS)
7) Thermal Ionization Mass Spectroscopy (TIMS)
8) Plasma Desorption (PD)
9) Atmospheric Pressure Photoionization Ionization (APPI)

• Electron impact ionisation occurs in a stainless steel chamber at a pressure
of less than 6 x10-7 mmHg (i.e. vacuum conditions) achieved by means of a
diffusion oil pump or a turbo-molecular pump.
• At 2000 degree celcius by thermoelectronic effect, electrons emitted by a
rhenium filament are accelerated to the anode by a 5–100 V potential
difference.

 0.01% yield.
 The electron kinetic energy generates the
pulling out of an electron, resulting in a
positive molecular ion, also provided with a
single electron:
M + e-  M+ + 2 e-
 The ease of electron removal from the
molecule depends on its nature, n > π > σ.

 Chemical Ionisation is the second of the
techniques to be considered a 'classical'
ionisation method.
 It is still widely use today for the analysis of low-
mass, volatile, thermally stable organic
compounds especially when coupled with gas
chromatography (GC-MS).
 Chemical ionisation is a lower energy
alternative to EI for volatile analytes.
 In chemical ionisation, there is a reagent gas
(usually ammonia or methane) in the ion
chamber.

 Fast atom bombardment (FAB) is an ionization technique used in mass
spectrometry in which a beam of high energy atoms strikes a surface to create ions.
 It was developed by Michael Barber at the University of Manchester in 1980.

 The atoms are typically from an inert gas such
as argon or xenon.
 Common matrices
include glycerol, thioglycerol, 3-nitrobenzyl
alcohol (3-NBA), 18-crown-6 ether, 2-
nitrophenyloctyl
ether, sulfolane, diethanolamine,
and triethanolamine. This technique is similar
to secondary ion mass
spectrometry and plasma desorption mass
spectrometry.

 Equationally,
 Xe + e-  Xe+ + 2e-
 Xe+
acceleration Xe+ (cations of higher
translational energy obtained by providing
acceleration of 6 – 10 kev)
 Xe+ + Xe  Xe+ + Xe (resonant electron
exchange reaction)
 Xe + M  Xe + M+ + e- (reaction with
compound)

• In mass spectrometry, matrix-assisted laser
desorption/ionization (MALDI) is an ionization technique that uses a laser
energy absorbing matrix to create ions from large molecules with minimal
fragmentation.
• It has been applied to the analysis of biomolecules (biopolymers such
as DNA, proteins, peptides and sugars) and large organic molecules (such
as polymers, dendrimers and other macromolecules), which tend to be fragile
and fragment when ionized by more conventional ionization methods.

• MALDI methodology is a three-step process-
1. First, the sample is mixed with a suitable matrix
material and applied to a metal plate
2. Second, a pulsed laser irradiates the sample,
triggering ablation and desorption of the sample
and matrix material.
3. Finally, the analyte molecules are ionized by
being protonated or deprotonated in the hot
plume of ablated gases, and then they can be
accelerated into whichever mass spectrometer is
used to analyse them.

 The matrix consists of crystallized molecules, of which the three most
commonly used are 3,5-dimethoxy-4 hydroxycinnamic acid (sinapinic
acid), α-cyano-4-hydroxycinnamic acid (α-CHCA, alpha-cyano or
alpha-matrix) and 2,5-dihydroxybenzoic acid (DHB).
 A solution of one of these molecules is made, often in a mixture of highly
purified water and an organic solvent such as acetonitrile (ACN)
or ethanol.

 A counter ion source such as Trifluoroacetic
acid (TFA) is usually added to generate the
[M+H] ions. A good example of a matrix-
solution would be 20 mg/mL sinapinic acid in
ACN:water:TFA (50:50:0.1).
 The matrix solution is mixed with the analyte
(e.g. protein-sample).

 A mixture of water and organic solvent allows
both hydrophobic and water-soluble (hydrophilic)
molecules to dissolve into the solution.
 This solution is spotted onto a MALDI plate (usually
platinum plate designed for this purpose). The
solvents vaporize, leaving only the recrystallized
matrix, but now with analyte molecules embedded into
MALDI crystals. The matrix and the analyte are said to
be co-crystallized.
 Co-crystallization is a key issue in selecting a proper
matrix to obtain a good quality mass spectrum of the
analyte of interest.

• Atmospheric pressure chemical ionization (APCI) is an ionization
method used in mass spectrometry which utilizes gas-phase ion-molecule
reactions at atmospheric pressure (105 Pa), commonly coupled with high-
performance liquid chromatography (HPLC).
• APCI is a soft ionization method similar to chemical ionization where
primary ions are produced on a solvent spray.

 The main usage of APCI is for polar and
relatively less polar thermally stable
compounds with molecular weight less than
1500 Da.
 The application of APCI with HPLC has
gained a large popularity in trace analysis
detection such as steroids, pesticides and also
in pharmacology for drug metabolites.

 A typical APCI usually consists of three main
parts:
1. A nebulizer probe which can be heated to
350-500oC,
2. An ionization region with a corona
discharge needle.
3. An ion-transfer region under intermediate
pressure

 Electrospray ionization (ESI) is a technique
used in mass spectrometry to produce ions
using an electrospray in which a high voltage
is applied to a liquid to create an aerosol.
 It is especially useful in producing ions
from macromolecules because it overcomes
the propensity of these molecules to fragment
when ionized.

 It may produce multiple-charged ions.
 ESI is a so-called 'soft ionization' technique,
since there is very little fragmentation.
 Advantage of ESI is that solution-phase
information can be retained into the gas-phase

IONIZATION MECHANISM
 The liquid containing the analyte(s) of interest
is dispersed by electrospray, into a fine aerosol.
 Because the ion formation involves extensive
solvent evaporation (also termed desolvation).
 The droplet undergoes Coulomb fission,
whereby the original droplet 'explodes' creating
many smaller, more stable droplets.
 The new droplets undergo desolvation and
subsequently further Coulomb fissions.

 Atmospheric pressure photoionization
ionization (APPI) is a soft ionization
technique for liquid chromatography-mass
spectrometry (LC-MS) that uses
photochemical action to ionize samples in the
gas phase.
 The APPI facilitates the analytical detection
of weakly polar and non-polar compounds
by mass spectrometry

In APPI, the solvent and sample from the liquid chromatography first form
a gaseous analyte (M), which is ionized to interact with photons emitted
by the light source, and the ions are introduced into the mass spectrometer
for analysis.

 During this process, both the analyte and the
solvent may be excited, and the analyte may
also acquire protons from the protic solvent:
M + hv  M+ + e- (eq.1)
M + S  [ M + H]+ + [S-H]. ( eq.2)
 Wherein M represents an analyte and S
represents a solvent.

 The APPI light source can be derived from an
argon lamp, a xenon lamp, a xenon lamp, or
the like.
 Among them, xenon lamps are used most
frequently, and the red light energy emitted
by xenon lamps is between common solvents
and most compounds.
 The method in which the analyte in the
formula (1) is directly ionized is called direct
APPI.

 The medium that can help the analyte to ionize is
called a dopant.
 The typical dopant involved in the APPI process
is as follows (D represents the dopant):
D + hv  D+ + e- (eq. 3)
D+ + M  M+ + D (eq. 4)
D+ + S  [D-H]. + [ S + H ]+ (eq. 5)
[S + H]+ + M  [ M + H ]+ + S (eq. 6)
 The APPI process in which the dopant is involved
is referred to as dopant-assisted APPI (DA-
APPI).

 Magnetic sector analysers
 Time of flight
 Quadrupole analyzers

• In Magnetic Sector Mass Analyzer, ions are accelerated so that
they have the same kinetic energy.
• All the ions are accelerated into a focused beam. And then the
ions are deflected by the magnetic field according to masses of
ions.
• The lighter ions have more deflection than the heavier ones.

 A time of flight analyzer consists of a pulsed ion
source, an accelerating grid, a field-free flight
tube, and a detector.
 The flight time needed by the ions with a
particular mass to charge, accelerated by a
potential voltage, to reach the detector placed at a
distance, can be calculated from a formula.
 Pulsing of the ion source is required to avoid the
simultaneous arrival of ions of different m/e at
the detector.

 At high masses, not all the ions of the same
m/e values reach their ideal velocities.
 To fix this problem, often a reflectron which
consists of a series of ring electrodes with high
voltage is added to the end of the flight tube.

• The quadrupole mass analyzer (QMS) is one type of mass analyzer
used in mass spectrometry.
• It is also known as a transmission quadrupole mass
spectrometer, quadrupole mass filter, or quadrupole mass
spectrometer.

 As the name implies, it consists of four
cylindrical rods, set parallel to each other.
 In a quadrupole mass
spectrometer the quadrupole is the mass
analyzer - the component of the instrument
responsible for selecting sample ions based on
their mass-to-charge ratio (m/e).
 Ions are separated in a quadrupole based on the
stability of their trajectories in the
oscillating electric fields that are applied to the
rods.

 Electron multiplier detectors (EMD)
 Faraday cups
 Scintillation counter
 Photographic plate
 Cryogenic detectors


 Boris L. Milman, (2015) General principles of identification by mass spectrometry. Trends in
Analytical Chemistry 69 , 24-33 [Online]. Available at
<https://www.researchgate.net/publication/274461248>
 B.L. Milman, I.K. Zhurkovich (2015) Mass spectrometry analysis of medical samples
for clinical diagnostics, J. Anal. Chem. 70
 Galen P.M., et al. (2016) Mass Spectrometry. Instrumental Analysis in (Bio)Molecular
Chemistry
 Nicolescu T.O. Interpretation of Mass Spectra. Intech [Online] Available at
< http://www.intechopen.com/books/mass-spectrometry >
 Griffiths I.W. (1997) The centenary of his discovery of the electron and of his invention of
mass spectrometry. Rapid Communications in Mass Spectrometry. 5,
1-16
 Hoffmann E.D. and Stroobant V. (eds.)(2007) Mass Spectrometry: Principles and
Applications. England, Antony Rowe Ltd [Online]. Available at
<www.wileyeurope.com or www.wiley.com >

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:614302612475955@1523472564519/A-schematic-of-matrix-assisted-laser-desorption-
ionisation-source-adapted-from.png >
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%2C%20ion%20trap>
 < https://science.widener.edu/svb/massspec/intro_to_ms/magnet.htg/.gif>
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Mass spectroscopy -> by Mohit kumar

Mass spectroscopy -> by Mohit kumar

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