Mass spectrometry

Dr. Amany M. Elshamy

Lecturer of Biochemistry and Molecular Diagnostics
Outline
Introduction
• The mass spectrometer was
invented by JJ THOMSON. He
performed a series of experiments
in 1897.
 Nobel Laureates:

Mass Spectrometry Joseph John Thomson Francis William Aston

Physics 1906 Chemistry 1922
first mass spectrometer mass spectrometry of isotopes

John B. Fenn
Wolfgang Paul
Chemistry 2002
Physics 1989
electrospray ionization of
quadrupole and
biomolecules
quadrupole ion trap MS

Koichi Tanaka
Chemistry 2002
Matrix-assisted laser
Desoprtion/ionization (MALDI)
A Long and Continuing History of Achievements
Mass spectrometry
• It is an analytic technique in
which a sample is first
volatilized and then ionized to
form charged molecular ions
and fragments.
• The separation is according to
their mass-to-charge (m/z)
ratio.
• The sample is then measured
by a detector, which gives the
intensity of the ion current for
each species.
 Mass Spectrometry - principle
• In mass spectrometry, a small sample of a compound is
introduced into an instrument called a mass spectrometer,
where it is vaporized and then ionized as a result of an
electron’s being removed from each molecule.
• Ionization can be accomplished in several ways.
 Mass Spectrometry - principle
• The most common method bombards the vaporized molecules
with a beam of high-energy electrons. The energy of the
electron beam can be varied, but a beam of about 70 electron
volts (eV) is commonly used.
• When the electron beam hits a molecule, it knocks out an
electron, producing a molecular ion, which is a radical
cation—a species with an unpaired electron and a positive
charge.
 • Mass spectrometry allows us to
Mass determine the molecular mass and
the molecular formula of a
Spectrometry - compound, as well as certain
principle structural features of the
compound.
 Definitive identification of
samples eluting from GC or
HPLC columns is possible when
an MS is used as a detector.
Mass
spectrometry
Mass spectrometry is
commonly used or screening
and confirmation.
 Mass spectrometer has powerful
analytic capabilities with widespread
clinical applications.

Determining the elemental

Mass composition and structure of organic
spectrometry and non organic compounds.

Performing quantitative analysis.

Steps These steps take
place in the four basic
components that are
standard in all MSs:

the
ionization mass ion
sample
source, analyzer, detector
inlet,
 The sample in an MS is first volatilized
and then ionized to form charged
molecular ions and fragments that are
separated according to their mass-to-
charge (m/z) ratio.
The sample is then measured by a
Sample detector, which gives the intensity of the
ion current for each species.
introduction

Fragment ions are formed when a

molecular ion breaks into smaller pieces.
 A mass spectrometer consists of an

(1) ion source

(2) vacuum system

Components (3) mass analyzer

(4) detector

(5) computer
General Block Diagram – Mass Spectrometer
Analogy between Optical Spectrophotometer
& Mass Spectrometer
Sample Introduction and Ionization

Types of ionization:
• Electron Ionization
• Chemical Ionization
• Electrospray Ionization
• Atmospheric pressure chemical ionization
(APCI)
Ion Source

• Electron ionization (EI) and chemical ionization (CI) are

ionization techniques that are used when gas-phase
molecules are introduced directly into the analyzer from a
gas chromatograph.
• The ionization methods used most frequently when a high-
performance liquid chromatograph is inter aced with a
mass spectrometer (HPLCMS) include (1) electrospray
ionization (ESI) and (2) atmospheric pressure chemical
ionization (APCI).
 • In EI, gas phase molecules are bombed
by electrons emitted from a heated
Electron filament and attracted to a collector
electrode.
ionization • The ions formed and their relative
(EI) proportions are reproducible and can
be used for qualitative identification of
the compound.
 IONIZATION SOURCES
1) Electron Ionization (EI)

- Commonly used for analysis of organic samples

- 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

- Interaction with the high energy electrons causes ionization of

sample molecules and fragmentation into smaller ions.
 • Chemical ionization (CI) is a “soft”
ionization technique, meaning that
Chemical relatively little fragmentation is
produced during the ionization process.
ionization • An electron beam ionizes the reagent
(CI) gas and produces reactive species,
often as a result of a cascade of ion
molecule reactions
 IONIZATION SOURCES
2) Chemical Ionization (CI)

-A large excess of reagent gas (1000 – 10000 times) is introduced

into the ionization region

- Pressures in source are typically higher than EI

-Electrons are allowed to bombard the gas-sample mixture

Examples of reagent gas

- Methane, ammonia, isobutane
 • Electrospray ionization (ESI) is a
Electrospray technique in which a sample is ionized
ionization at atmospheric pressure before it is
introduced into the mass analyzer
(ESI) (HPLC).
 IONIZATION SOURCES
3) Atmospheric Pressure Ionization (API) Sources

- Two major types

- Electrospray Ionization (ESI)
- Atmospheric Pressure Chemical Ionization (APCI)

- Operate at atmospheric pressure

- Modified version of ESI is the Ion Spray Source

- Used for mixtures of nonvolatile high molecular weight compounds

Atmospheric
Pressure • Another important ionization source is
APCI, which is similar to ESI in that the
Chemical liquid from LC is introduced directly
Ionization into the ionization source.
 Components of a Mass Spectrometer

Ionisation Ion Separation Ion Detection

Ion Source Mass Analyser Detector
Electron Ionisation (EI) Quadrupole Electron Multiplier

Chemical Ionisation (CI) Magnetic deflection type Multichannel plate

Fast Atom Bombardment (FAB) Electrostatic type Faraday Cup

Electrospray Ionisation (ESI) Time-Of-Flight (TOF)

Matrix-Assisted Laser Desorption Ion Trap

Ionisation (MALDI)
Types of Mass Spectrometers
 Various types of Spectrometers are available in the means of separating the ions
according to their mass-to-charge ratio or simply, on the basis of Mass Analyzers used.
They are;

 1) Magnetic Deflection Mass Spectrometer

 2) Electrostatic Mass Spectrometer

 3) Time-of-Flight Mass Spectrometer (TOF)

 4) Quadrupole Mass Spectrometer

1) Magnetic Deflection Mass Spectrometer
  In ToF Mass Spectrometer, ions of different mass/charge
ratio are separated by the difference in time they take to
travel over an identical path from the ion source to the
collector at the detector.
 Here ions generated by bombardment of the sample
with a brief pulse of electrons, secondary ions or laser-
Time of Flight generated photons.
(ToF) Mass  Ions accelerated by electric field and they enter into field-
free drift tube.
Spectrometer  Ions enter tube with same kinetic energy and ion
velocity vary inversely with mass.
 Lighter particles arrive at detector before heavier particles
and ions are separated in the drift tube according to their
velocities (v)
Time of Flight (ToF) Mass Spectrometer
 • Advantages of ToF
 High speed operation
 Ability to record entire mass spectrum at
one time
 Accuracy only depend on electronic
TOF circuits
 Disadvantages of ToF
 Poor resolution due to display on an Oscilloscope
screen
Spectrum
MS
• The spectrometer sorts out all the cations
(including the radical cations) according to their
mass/charge (m/z or m/e) values and records
them as line signals along the abscissa.
• At the same time, the instrument records their
relative abundances as signal heights plotted as
intensities along the ordinate. The mass
spectrometer does not detect the radicals.
 Spectrum
1. Molecular ion peak: Molecular weight of the compound
(at the end ).
2. Molecular ion isotope peak (M+2).
3. Base peak (the tallest one) (the ion exists most
abundantly in the ion source and reperesent the most
stable ions which are useful to identify the compound
MS of NAPHTHALENE C10H8
 • Qualitative Analysis
Mass Spectrometry - Molecular Weight Determination
Mass Spectrometry Data
- Structure Determination
• Quantitative Analysis
Quantitative Information

- Biotechnology
 analysis of proteins & peptides
Abundance

 analysis of oligonucleotides

- Pharmaceutical
 drug discovery, combinatorial chemistry

 pharmokinetics, drug metabolism

Qualitative Information - Clinical

neonatal screening, hemoglobin analysis
drug testing

- Environmental
 water, food, air quality (PCDs etcs)

- Geological
 oil composition
-Toxicology
 Mass Spectrometry
Advantages Over Atomic Optical Spectrometric
• Detection limits three orders of magnitude
better
• Remarkably simple spectra that are unique and
easily interpreted
• Ability to measure isotopic ratios

Disadvantages
• Instrument costs are two to three times higher
• Interference effects
 Mass spectrometers coupled to GC or LC can be
used not only for the identification and
quantitation of compounds but also for structural
information and molecular weight determination.
Applications of
Mass Spectrometry
GC/MS systems are widely used for measuring
in the drugs of abuse in urine toxicology confirmations.
Clinical Laboratory

Drugs and metabolites must be extracted from

body fluids and typically reacted with
derivatizing reagents to form compounds that are
more volatile for the GC process.
 LC offers a number of advantages over GC.
Typically, LC requires less extensive extraction
procedures and derivatization is rarely used,
saving time and Expense. However, it need more
Applications of frequent maintenance. Another disadvantage of
Mass Spectrometry LC/MS is the less reproducible mass spectra.
in the
Clinical Laboratory
LC/MS also has great potential for measuring low-
level and mixed-polarity analytes such as vitamin
D, testosterone, and immunosuppressant drugs
due to its superior sensitivity and specificity over
immunoassays.
 LC/MS has the advantage of being
able to detect multiple analytes (such
as a panel of drugs or a series of
Applications of metabolites) in one run.
Mass Spectrometry
in the
Clinical Laboratory LC/MS is free from the antibody
interferences seen in immunoassays,
although LC/MS has its own type of
interference in the form of ion
suppression.
Summary
Mass Spectrometry
• Mass Spectrometry (MS) measures the atomic or
molecular weight of a ion from the separation based on
its mass to charge ratio (m/z)
• elemental composition of matter
• structures of inorganic, organic and biological
molecules
• qualitative and quantitative composition of complex
mixtures
• isotopic ratios of atoms in the sample
• Which type of ionization technique uses an electron beam to
ionize the reagent gas and produce a reactive species as a
result of ion-molecule reactions?
• a. Electron
• b. Chemical
• c. Electrospray
• d. Inductively coupled plasma
• Which of the following main component of mass
spectroscopy deal with resolving the ions into their
characteristics mass components according to their mass-to-
charge ratio?
a) Ion Source
b) Analyzer
c) Detector System
d) Analyzer tube
• Who discovered the mass spectrometer?
a) Francis Aston
b) J. J Thomson
c) Ernest O. Lawrence
d) Walter Kaufmann
• In which state of matter mass spectroscopy is being
performed?
a) solid
b) liquid
c) gaseous
d) plasma
• What are the main criteria on which mass spectrometer used
for?
a) Composition in sample
b) Relative mass of atoms
c) Concentration of elements in the sample
d) Properties of sample
• Which species of the following is used to bombard with the
sample for which mass spectroscopy has been performed?
a) Alpha particles
b) Neutrons
c) Electrons
d) Protons
• Separation of ions in mass spectrometer take place on the
basis of which of the following?
a) Mass
b) Charge
c) Molecular weight
d) Mass to charge ratio
• Which type of ionic species are allowed to pass through the
slit and reach the collecting plate?
a) Negative ions of all masses
b) positive ions of the specific mass
c) Negative ions of the specific mass
d) Positive ions of all masses
• The mass analyser is similar to which of the following in
optical spectrometer?
1. Monochromator
2.Detector
3.Sample
4.Source
• What are the main criteria for which a mass
spectrometer is used?
1. To find the composition of the sample
2.To find the relative mass of atoms
3.To find the concentration of elements in the sample
4.To find the properties of the sample
• mass spectrometer separates ions based on which of
the following factors?
1. Mass
2.Charge
3.Molecular Weight
4.Mass to charge ratio
What is the primary purpose of ionization in a mass spectrometer?
A. To facilitate fragmentation of the analyte in the ion source
B. To ensure a radical is formed from the analyte
C. To ensure the analyte is volatile enough to enter the gas phase.
D. To be able to manipulate the analyte inside the mass spectrometer and facilitate
mass measurement and detection.
What is the typical energy of the electrons used to ionize
analytes by Electron Ionization (EI)?
A. 50eV
B. 70eV
C. 1eV
D. 1000eV