Basics of mass spectrometry

1. Mass Spectrometry

2. Definition:
• A technique for measuring and analyzing
molecules, that involves introducing enough
energy into a (neutral) target molecule to
cause its ionization and disintegration. The
resulting primary ions and their fragments are
then analyzed, based on their mass/ charge
ratios, to produce a "molecular fingerprint."

3. Principle:
• 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.

4. Different forms of MS instruments

5. • Atmospheric Pressure Chemical Ionisation
(APCI)
• Chemical Ionisation (CI)
• Electron Impact (EI)
• Electrospray Ionisation (ESI)
• Fast Atom Bombardment (FAB)
• Field Desorption / Field Ionisation (FD/FI)
• Matrix Assisted Laser Desorption Ionisation
(MALDI)
• Thermospray Ionisation (TSP)
Methods of sample ionisation

6. Analysis and Separation of Sample
Ions
• The main function of the mass analyser is
to separate , or resolve , the ions formed in the
ionisation source of the mass spectrometer
according to their mass-to- charge (m/z) ratios.
Eg: quadrupoles , time-of- flight
(TOF) analysers, magnetic sectors , and both Fourier
transform and quadrupole ion traps .
• Tandem (MS-MS) mass spectrometers are
instruments that have more than one analyser and
so can be used for structural and sequencing studies

7. Detection and recording of sample
ions.
• The detector monitors the ion current,
amplifies it and the signal is then transmitted
to the data system where it is recorded in the
form of mass spectra .
• the molecular mass of each component is
plotted against the relative abundance of the
various components in the sample.
eg: photomultiplier , electron multiplier micro-
channel, plate detectors.

8. • Take a substance(analyte) and place it in a
spectrometer.
• This analyte in spectrometry get bambarded
with the beam of high intensity electron. Inturn
it will ionize the analyte and thus converting it
into an ion.
• This will be demonstrated by equation
X + e- → X+ + 2 e-
the result is a ray of electrons this will be run in
magnetic field of different strengths this will
determine the masses of all the ions resulted
from ionizing the analyte.

9. Teminologies
Molecular ion (M): The ion that the spectrometer
analyzes. This is the molecule comprized of lowest
mass atomic isotopes.
M/Z: M stands for mass and Z stands for charge
number of ions. Since Z is almost always 1, the m/z
value is often considered to be the mass.
Molecular mass of C-12, O-16, N-14, F-17.
Eg: carbon has two isotopes 12C and 13C.
M + e- → M+ + 2e- , here M+ is a molecular ion and
is very unstable so it give rise to fragment ions
which are mostly stable.

10. Calculate the m/z
• Eg: Butane C4 H10
• M/Z: (4 x 12) + ( 10 x 1) = 58
• C4 H10 + e- → C4 H10+ + 2e-
• Sometimes molecule disintegrates during the
ionization process forms a fragment ion.
• Since we defined (M) as the molecule with
lowest mass atomic isotopes, we can identify
fragments easily.

11. • Anything with a mass less than the m/z of (M)
we know that M as a lowest possible mass, so
anything to the left of M/Z ratio of M we know it
must be a fragment ion.
• Fragment ion is represented by M- H, M-2H
etc.
• Conversely , as we deal with isotope, there are
heavier variants of the analyte eg: M+1, M+2,
etc.

12. Mass spec plot
The m/z ratio of M is
452
The base peak is 257
It is highest
abundance
(100%)
The relative abundance is the main part of mass
spec analysis. It tells us how different parts of
molecule relate to each other. From these
information we can draw conclusion that what
this molecule is…

13. Relative abundance
• Relative abundances tells us about the analyte.
• M+1: molecular ion with mass 1 higher than M
due to heavier isotope atom.
• Formula : # of carbon atoms= relative intensity of
M+1 peak / 0.011 x (relative intensity of M peak)
• Relative abundance of M+1 = no of carbon X
(natural abundance of 13C/ natural abundance of
12C)

14. Fragmentation reactions
• Each of the fragments are separated according
to mass to charge ratio (m/z)
• Only ions appear in the mass spectrum;
neutral molecules and radicals do not appear.
• Individual ions are detected by instrument
and counted
• When ions are produced by electron impact,
the spectrum is called EI mass spectrum.

15. Isotopic peaks
• There is 1.1% probability that each carbon in
the molecule will be present as 13C
• The abundance of the M+1 peak is then the
number of carbons x 1.1%
• This value is relative to the molecular ion peak
• For eg., cyclopentane’s M+1 peak would be
5.5% the intensity of M peak

17. Let us solve one spectral problem….
43 72
Relativeabundance
m/z
12CH3
12CH2
12CH2
12CH2
12CH3
Pentane
MW= 72 g/mole (M)
12CH3
12CH2
12CH2
12CH2
13CH3
Pentane
MW= 73 g/mole (M+1)
Carbon atoms= Relative intensity of M+1 peak/
.011 X ( relative intensity of M peak)
C=5.5%/ .011 X ( 100%)
C= .055/ .011 X ( 1), so C= 5.

18. How to analyze relative atomic massRelativeabundance
m/z
28 29 30
28P14 : 29P14 : 30P14
92 : 5 : 3 Relative abundances
Relative
Atomic mass= (28 X 92) + (29 X 5) + (30 X3) /
(92 + 5 + 3)
= 28.11

19. THANK YOU