SUMMER 2020
MASS SPECTROMETRY
Professor Dr. Hasina Yasmin
Department of Pharmacy
Four spectroscopic methods are important for the structure
elucidation of organic compounds.
These are-
i.
UV-VIS spectroscopy
ii.
IR spectroscopy
iii.
NMR Spectroscopy and
iv.
Mass Spectrometry
Spectroscopic
methods
Structural information
UV-VIS
spectroscopy
Indicates the presence of
chromophore only.
IR spectroscopy Indicates the presence of
functional groups.
Mass
spectrometry
Gives✓Exact Mol. weight,
✓Mol. formula and
✓presence of structural unit
NMR
spectroscopy
It indicates the no. of protons
and carbons & their chemical
environment and others.
Application in Pharmaceutical Industry
Quantitative
Identification of compounds
determination
Many compounds having the Widely used in
same chromophore usually Pharmaceutical
have the same UV spectrum. Industry.
Play a negligible role.
Different compounds have
Not widely used.
diff. IR spectra. Therefore,
widely used for identification
purpose.
Mass spectrometry can be
Not used.
used for identification
purpose.
NMR spectrum can be used
for identification purpose.
Not used.
MASS SPECTROMETRY
Mass spectrometry: An analytical technique by which chemical
substances are identified by sorting of gaseous ions of the
substance in electric and magnetic fields according to their mass
to charge (m/z) ratio.
It is 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.
BASIC PRINCIPLE OF MASS SPECTROMETRY
• A mass spectrometer generates multiple ions from the sample
under investigation. Then it separates them according to their
specific mass-to-charge ratio (m/z) and then records the
relative abundance of each ion type. Mass spectrometer
measures the masses of individual molecules that have been
converted into ions. Therefore the molecule must be in the
vapor state.
5 Stages of mass spectrometry
3
2
1
VAPORIZATION
4
5
https://www.chemguide.co.uk/analysis/masspec/howitworks.html
STAGE 1: VAPORIZATION
• Gases and sample with high vapor pressure are directly
introduced into the mass spectrometer
• Liquid and solids are heated to increase the vapor pressure
• If sample is thermolabile or does not have sufficient vapor
pressure, directly ionized from condensed phase. For example,
direct ionization of proteins and polymers (MW>100000 Da)
3
2
1
VAPORIZATION
4
5
Stage 2: Ionization
The vaporized sample is introduced into the ionization chamber
of Mass Spectrometer for the production of gas phase ions of
the compound. The molecule is ionized by knocking one or more
electrons off to give a positive ion. This molecular ion undergoes
fragmentation. Each primary product ion derived from the
molecular ion, in turn undergoes fragmentation and so on.
Ionization
A
Fragmentation
A+ ▪
-e-
A+▪
B+
+
▪
C
neutral
molecule
C+
+
▪
Bneutral
molecule
D+
+
neutral molecule
If fragmented ion B or
C has sufficient energy,
they may fragment
further
Stage 3: Acceleration
The simplest way to accelerate ions is to place them between a set of
charged parallel plates. The ions are repelled by one plate, attracted to
the other, and if we cut a hole in the second plate the ions emerge
with a kinetic energy determined by the potential difference between
the plates. The ions are accelerated so that they all have the same
kinetic energy.
Stage 4: Deflection
The ions are then deflected by a magnetic
field according to their masses. The lighter
they are, the more they are deflected.
1
Stage 5: Detection
The beam of ions passing through the
machine is detected electrically.
3
2
VAPORIZATION
4
5
Mass Spectrometry Basics
+▪
̶
+▪ ▪
+
+
̶
̶
+▪ + +
+
+ +
▪
̶
Ionization of molecules by
high energy electron beams
̶
̶
̶
Acceleration by
applying negative
potential
̶
+
Deflection by
magnetic field
+
+
Detection
Mass spectrum: A mass spectrum is a graphical plot of measured ion
abundance vs. m/z.
A+▪
B+
+
▪
C
neutral
molecule
C+
+
▪
Bneutral
molecule
D+
+
neutral molecule
▪
100
100
M+
O
RA
M+
OMe
0
136
m/z
C8H8O2
Methyl benzoate
RA
0
31
105
77
m/z
136
Relative abundance (RA)
Base peak
Molecular ion peak
m/z
• Molecular ion peak: The molecular ion peak is usually the heaviest peak
in the mass spectrum.
• Base peak: The most abundant ions give rise to the tallest peak in the
mass spectrum. This peak is known as the base peak and is arbitrarily
assigned a value of 100 percent.
Mass Spectrometer: Block diagram
Ion
http://www.premierbiosoft.com/tech_notes/mass-spectrometry.html
Mass Spectrometer
Direct
vapor inlet
SAMPLE INLET
• Gases and sample with high vapor pressure are directly introduced into the
mass spectrometer
• Liquid and solids are heated to increase the vapor pressure
• If sample is thermolabile or does not have sufficient vapor pressure, directly
ionized from condensed phase.
• Various sample inlet systems include:
• Direct vapor inlet
• GC
• HPLC
• Direct insertion probe
IONIZATION TECHNIQUES
M
M + H+
-e-
M+ ▪
MH+
• M+ ▪ is the radical cation produced by removing a single electron from the molecule.
It is called the molecular ion. M is the molecule, + is the charge of the cation,
and ▪
is the remaining unpaired electron of the radical.
• Adduct ions are produced by a chemical reaction between an ion and a neutral
molecule. Many of these reactions cause the addition of a proton (H+) to the
molecule (M) and produce an adduct ion (MH+).
• Factors
-Physical state of the analyte
-Ionization energy
IONIZATION TECHNIQUES
A) Volatile materials
a) Electron Ionization (EI)
b) Chemical Ionization (CI)
B) Non-volatile materials
a) Field desorption
b) Desorption ionization by particles or radiation
i) Matrix Assisted Laser Desorption Ionization (MALDI)
ii) Fast Atom Bombardment (FAB)
c) Electrospray Ionization (ESI)
ELECTRON IONIZATION (EI)
(HARD IONIZATION)
• Electron ionization plays an important role in the routine
analysis of small molecules. Samples are introduced into the ion
source of the mass spectrometer with the help of a probe.
• Readily volatile samples like liquids and low molecular weight
solids are volatilized by heating under low pressure outside the
ion source and then allowed to diffuse into the source.
Electrons are accelerated from a hot filament to an anode.
70eV
• Due to bombardment with high energy electrons (70eV) molecules are
ionized by removal of an electron and the radical cation is formed M·+.
M + e- → M·+ + 2e• An radical anion does not produce to a significant amount since the
bombarding electrons have such high energies that they cannot be
captured to form the anion.
M + e- → M-·
ELECTRON IONIZATION (EI)
M + e-
M+ ▪ + 2e-
The molecular ions produced by EI usually have sufficient energy and undergo
extensive fragmentation which has both an advantage and a disadvantage.
• The advantage is that it gives rise to a pattern of fragment ions which can
help to characterize the compound.
• The disadvantage is the frequent absence of a molecular ion peak.
• Other advantages and disadvantages are given below:
Advantages
i)
Sensitivity
ii)
Availability of vast computer data base
Disadvantages
i)
Analysis of small and volatile materials only.
ii)
Thermal decomposition