Mass Spectrometry

Mass Spectrometry (MS)

MS is the generation, separation and
characterization of gas phase ions .
Molecules in a test sample are converted to
gaseous ions that are subsequently separated in a
mass spectrometer according to their mass-tocharge (m/z) ratio & detected.
The mass spectrum is a plot of the (relative)
abundance of the ions at each m/z ratio.
Chemical substances are identified by the sorting
of gaseous ions in electric and magnetic
fields according to their mass-to-charge

Introduction to Mass Spectrometry

MS was first used in the biological sciences to
trace heavy isotopes through biological systems
and later to sequence oligonucleotides and
peptides and analyze nucleotide structure
The development of methods of
macromolecule ionization, including
electrospray ionization (ESI) and matrixassisted laser desorption/ionization
(MALDI), enabled the study of protein structure
by MS

Mass spectrometry:
Mass spectrometer measures molecular
masses.
Molecular mass and formula and structure
information
Ionization: - generate similar ions from all
molecules in the mixture.
Different molecule same charge but
different mass.
Basis of separation: mass / charge ratio
(m/z) =mass of ion.

 The essential features of mass spectrometers are :

o Production of ions in the gas phase;
o Acceleration of the ions to a specific velocity in
an electric field;
o Separation of the ions in a mass analyser; and
o Detection of each species of a particular m/z
ratio.

Mass spectrometry:

Mass Spectrometer
A mass spectrometer needs to perform three
functions:
1. Creation of ions sample molecules are
subjected to a high energy beam of electrons,
converting them in to ions
2. Separation of ions as they are accelerated in
an electric field, the ions are separated according
to mass-to-charge ratio (m/z)
3. Detection of ions as each separated
population of ions is generated, the spectrometer
needs to qualify and quantify them

Principle:
Instrument is mass spectrometer
Separates gas phase ionized atoms, molecules,
and fragments of molecules
Separation is based on the difference in mass-tocharge ratio (m/z)
m = unified atomic mass units (u)
1 dalton (Da) = 1 u = 1.665402 x 10-27 kg
z = charge on the ion (may be positive or
negative)

Process:
A small quantity of sample is injected and vaporized
under high vacuum
The sample is then bombarded with electrons having
25-80 eV of energy
A valence electron is punched off of the molecule,
and an ion is formed

Process:
ions are accelerated to a constant velocity in
vacuum by a series of electrostatic plates.
Then subjected to strong magnetic field
Deflection of ions from its path-least mass more
reflection.
This will lead to separates of ions based on mass
Detector measures the ion flow.
If there is a mixture of different molecules in a
sample, all the masses are measured
simultaneously. So you get a mass spectrum.

The Mass Spectrum:

Each peak corresponds to a different type of molecule in
your sample

INSTRUMENTATION
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Components of a mass spectrometer

A high vacuum
system
A sample inlet
An ion source
A mass
filter/analyser:
A detector:

1. Vacuum system
Vacuum minimises the collisions between ions
and air molecules. Without a high vacuum, the
ions produced in the source will not reach the
detector
Two vacuum pump types are used:
Rotary vane pump
Turbo molecular pump or Diffusion pump.
Rotary vane pump can be an oil pump to provide
initial vacuum (approximately1torr)
Turbo molecular pump provides working high
vacuum(1mtorr to1 ntorr).

Sample introduction:
Two things must be
achieved here
1) Sample must be
introduced into
vacuum
2) Vaporization of
sample prior to
ionization

SAMPLE INPUT METHODS

1. Gas Expansion:
Useful for gas samples and liquids with
sufficiently high vapor pressures
The gas or vapor expands into an
evacuated and heated vessel

Sample leaks through holes in a gold foil

seal into the ionization source

SAMPLE INPUT METHODS

2. Direct Insertion Probe
For liquids with high boiling points and
solids with sufficiently high vapor pressure
The probe (with the sample in a glass
capillary at the tip) is inserted into the
ionization source
The probe is electrically heated to vaporize
the sample

SAMPLE INPUT METHODS

Direct Exposure Probe
Sample is first dissolved in a solvent
A drop of solution is placed at the rounded
glass tip of the probe
Solvent evaporates leaving a thin film of
sample
The tip is inserted into the ionization
source and heated to vaporize sample
Less likely to be contaminated

SAMPLE INPUT METHODS

Chromatography and Electrophoresis Systems
Chromatographic instruments are used to
separate mixtures of gases and liquids
Separated components are introduced into a
mass spectrometer for detection
The GC-MS system
LC-MS system is used for nonvolatile organic
compounds
Capillary electrophoresis (CE) can also be
coupled to MS

IONIZATION SOURCE
Electron Ionization (EI) :

Electro Spray Ionization (ESI)

Matrix Assisted Laser Desorption Ionization
(MALDI)

1. Electron Ionization (EI) :

Most common type of
ionisation.
Sample in the gas phase
delivered in to EI region
Electrons are emitted from a
heated tungsten filament
cathode
Electrons are accelerated
towards the anode with a
potential of about 50 100 V
Electrons meet at right
angles with the sample
molecules

1. Electron Ionization (EI) :

When the sample passes through the electron
stream, the high energy electrons in the stream
knock electrons out of the sample to form ions.

2. Electro Spray Ionization (ESI)

Produce ions directly from aqueous/ aqueous organic
solutions
Analyte solution is passed, at atmospheric pressure,
through a narrow needle kept at high potential (3.5 kV).
The voltage on the needle cerate a spray of highly
charged droplets .
The ions produced are singly or multiply charged.
Used for mixtures of nonvolatile high molecular weight
compounds
It is suited for LC- MS interface
Fine spray of positively or negatively charged droplets

3. Matrix Assisted Laser Desorption

Ionization (MALDI)
sample is co-crystallized with a matrix and then
irradiated with laser
Matrix:-weak organic acids- absorb UV range and is vaporized, together with the sample
Analyte Ionisation:
A pulse of UV laser beam- matrix absorbs UV
& is vaporized, the analyte also vaporized.
MALDI works well with proteins, nucleic acids ,
carbohydrates & lipids.

Matrix-Assisted Laser Desorption Ionization (MALDI)

3. Matrix-Assisted Laser Desorption

Ionization (MALDI)
Matrix Examples
2,5- dihydroxybenzoic acid- used for
proteins, oligonucleotides, peptides
Cyano-4- hydroxycinnamic acid used for
peptides & glycopeptides.
Sinapinic acid for proteins & peptides

Mass analyzer

MAGNETIC SECTOR MASS ANALYZER

Uses electric and/or magnetic fields to separate
ions
Gas phase molecules are ionized by a beam of high
energy electrons
Electrons may be ejected from molecules
(ionization) or bonds in molecules may rapture
(fragmentation)
Ions are then accelerated from source region in to
magnetic sector at 1-12 kV
Ion beam is bend in an arc by the magnetic field
Radius of arc depends on m/z
Ions with different m/z travel in arc with different
radii.

MAGNETIC SECTOR MASS ANALYZER

QUADRUPOLE MASS ANALYZER

Separates ions in an electric field (the
quadrupole field)
Oscillating radio frequency (RF) voltage and a
constant DC voltage are used to create the
field
These are applied to four precisely machined
parallel metal rods
The result is an AC potential superimposed on
a DC potential
Ion beam is directed axially between the four
rods

QUADRUPOLE MASS ANALYZER

Opposite pairs of rods are connected to
opposite ends of a DC source
Ions follow an oscillating (corkscrew) path
through the quadrupole to the detector
For a given ratio of DC to RF at a fixed
frequency, only ions of a given m/z value will
pass through the quadrupole

The Quadrupole Analyzers

Quadrupole analyzers ions are

filtered or trapped in a
device consisting of several
metal rods using specifically
tailored electromagnetic fields

DETECTORS

Measure one m/z value at a time (single

channel detectors)

Multiple detectors are used for multiple ion

detection
High resolution magnetic sector instruments
use multiple detectors called
multicollectors.

DETECTORS

Electron
Multiplier (EM)

Faraday Cup

Photomultiplier
Conversion
Dynode

MASS SPECTROMETRY APPLICATIONS

Field of
Study
Proteomics

Applications
Determine protein structure, function, folding
and interactions
Identify a protein from the mass of its peptide
fragments
Monitor enzyme reactions, chemical
modifications and protein digestion

DrugDiscove Determine structures of drugs and metabolites

ry
Screen for metabolites in biological systems
ClinicalTestin Perform forensic analyses such as confirmation
g
of drug abuse
Detect disease biomarkers (e.g., newborns
Source: https://www.thermofisher.com
screened for metabolic diseases)

MASS SPECTROMETRY APPLICATIONS

Field of Study

Applications

Genomics

Sequence oligonucleotides

Environment

Test water quality or food

contamination

Geology

Measure petroleum
composition
Perform carbon dating

Source: https://www.thermofisher.com

Some Pictures
MALDI-R

FT-ICR

Q-Tof Micro

LTQ-Orbitrap

1. Biophysical chemistry by Upadhyay,

Upadhyay and Nath
2. Sharma Y.R. Elementary organic
spectroscopy principles and chemical
applications. 1st ed. S. Chand and Company
ltd; New Delhi :2008.
3. http://www.chm.bris.ac.uk
4. http://chemwiki.ucdavis.edu/Analytical_Che
mistry/Instrumental_Analysis/Mass_Spectro
metry

Reference: