PREPARED BY:

-Unique among the various spectroscopy techniques in both theory and

instrumentation.
- involves interaction of electromagnetic radiation or some form of
energy with molecules.
-ionization, separation, and detection (mass spectrum)
 -Ms with gas chromatography confirm the identity of unknown
compounds. The use of high performance liquid chromatography (HPLC) with
ms also has recently become more routine due to advances made in the
interconnecting interfaces.
● three basic functions
- Ionize the molecules, which occurs inthe ion source by a variety of
techniques such as electron impact, matrix-assisted-laser-
desorption, or atmospheric pressure ionization.
- Separated according to their m/z, and this occurs in the mass
analyzer section (e.g., quadrupoles, ion traps, time-of-flight
(TOF),Fourier transform).
- Monitored by a detector.
 A block diagram of the major components of a mass spectrometer

- Sample introduction can be static, direct insertion probes, or dynamic

which involves interfacing chromatographic equipment such as gas or liquid
chromatography (LC). Interface methods include Heated capillary transfer lines (GC–MS),
and LC–MS techniques such as Electrospray And atmospheric pressure ionization
interfaces.
Schematic of a typical mass spectrometer. The sample inlets (interfaces) at the top and
bottom can be used for direct injection or interfacing to a GC
 -The region between ion generation and detection is maintained by
different vacuum pumps. Each successive region from the source is kept at
lower vacuum than the preceding region , with the mass analyzer/detector
being in the region of the strongest vacuum (≈10−6 Torr). A vacuum is
necessary to avoid ion–molecule reactions between the charged ions and
other gaseous molecules before they reach the detector, thereby increasing
both sensitivity and resolution.
Sample introduction

- The initial step in operating the MS is to get the sample into the ion source chamber.
Pure compounds that are gases or volatile liquids are injected directly into the source
region
- Prior to sample introduction, 2 things must be achieved
- Sample must be introduced into vacuum and must be vaporized
- The sample is introduced by placing it on the probe, which is then inserted through a
vacuum lock into ionisation region of mass spectrometer
Ionization

- As the electrons pass through the source region, they come in close proximity to the
sample molecule and extract an electron, forming an ionized molecule.
- Ionization means placing a charge on a neutral molecule
Methods
- Electron ionization
- Electrospray
- Matrix-assisted laser desorption/ionization(MALDI)
Electron Ionization

- Also known as electron bombardment / electron impact method

- The sample is heated to vaporize it
- The sample in the gas phase is now delivered into electron ionization region
- Here a beam of electrons with energy of 70EV is made to interact with the sample
- This interaction causes electron ejection in the sample molecules leading to
ionization
Electrospray
Generates ions directly from aqueous or aqueous/organic solutions
The solution is forced through a narrow needle which is kept at a high potential (3.5 kV)
The voltage on the needle causes the spray to be charged as it is nebulized
Thus, very small droplets are created and they are charged on their surfaces
The electric charge density on the surface of the droplet is a function of its
size- smaller the droplet, larger is the electric charge density
Thus, as the droplets decrease in size, there is repulsion between mutually
charged droplets
At this point, ions begin to leave the droplet
Ions are led into mass analyzer
It nondestructively vaporizes ionizes both big and small molecules
The analyte is first co-crystallized with an excess of a matrix compound
Matrix compounds are organic acids, which absorb in the UV range
After the co-crystallization, a pulse UV laser beam is focused on the surface of the crystal
The matrix absorbs the radiation is vaporized
The analyte is also vaporized and carried along with the matrix
The matrix doubles up as a proton acceptor or donor thus also ionizes the analyte
Different matrices
2,5 dihydroxy benzoic acid-proteins,peptides oligonucleotides
Sinapinic acid proteins peptides
Mass Analyzers (heart of mass spectrometer)
- separating the charged fragments based on their m/z, and dictates the
mass range, accuracy, resolution, and sensitivity. There are seven basic
types of mass analyzers: quadrupoles (Q), ion traps (IT), time-of-
flight (TOF), magnetic sectors, isotope ratio MS, Fourier-transform-
based ion cyclotrons (FT-ICR) and Orbitraps (OT), and accelerator mass
spectrometers (AMS).
Magnetic sector mass analyzer
- J.J Thompson,who built the 1st mass spectrometer, used a magnet to
measure the m/z value of Electrons
- It separates ions in a magnetic field according to the momentum
charge of ion
- A 1 to 10kV electric field accelerates ions from the source region into
the magnetic sector
- The radius of the arc(r) depends on Momentum of the ion
- Charge of the ion Magnetic field strength
- The greater the momentum of the ions, the larger is their arc radius
- The separation of ions is thus based upon their momentum
- Hence, magnetic analyzers are also called momentum analyzers
Diagram of a magnetic sector mass analyzer Diagram of an ion trap mass analyzer
- In the mass spectrometer, an electric field accelerates ions out of the
source region and into the quadrupole analyzer
- The quadrupole analyzer consists of 4 rods/electrodes arranged across
from each other The ions are made to travel through the
Quadrupole Here, they get filtered according to their m/z
ratio
- Only one of the separated ion beams is allowed to strike the detector
- The separation according to m/z ratio is based upon the radio
frequency direct current
- voltages applied to these electrodes These voltages produce an
oscillating electric field that transmits ions according to their m/z
value by alternatively focusing them in different
planes
- Used mostly with MALDi
- The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions
through the same potential, and then measures the time
- they take to reach the detector
- The smaller ions will reach the detector first because they will achieve great
velocities
- The larger ions will have lesser velocities reach the detector late
The final element of the mass spectrometer is the detector
The detector generates a signal current from incident ions by generating secondary
electrons which are further amplified
Types
1) Faradey Cup
2) Electron Multiplier
3) Photomultiplier Conversion Dynode
Concept A change in charge on a metal plate results in a flow of electrons
The flow creates a current When a single ion strikes the surface of a dynode
in faradey cup, it results in ejection of several electrons
This ejection induces a current in the cup
Uses a series of dynodes maintained at successively higher potentials
Thus,electrons released by the 1st dynode (when ion impinges on it) are
dragged to 2nd dynode because it has a higher potential
Highly sensitive
- Ions strike a dynode resulting in emission of
Electrons
- These electrons are made to strike a phosphorous
Screen
- The screen releases photons
- Photons detected by a photomultipier
- Complex mixtures are now analyzed without prior purification by
tandem Ms
- It employs the equivalent of 2 mass spectrometers linked in series
- The 1st spectrometer separates individual peptides upon their differences
in mass By adjusting the field strength of 1st magnet, a
single peptide can be directed into 2nd mass spectrometer ,where
fragments are generated and their mass determined
1) Identification quantification of proteins
2) Drug screening
3) Pesticides pollutants screening
4) Used to screen blood samples from newborn for the presence conc of proteins,F.A,other
metabolites
5) Screening of inborn errors of metabolism (phenyl ketonuria, ethylmalonic
encephalopathy,glutaric acidemia type 1)
- In this technique, a gas chromatograph is used to separate different compounds.
- This stream of separated compounds is fed online into the ion source, a metallic
filament to which voltage is applied.
- This filament emits electrons which ionize
the compounds.
- The ions can then further fragment, yielding
predictable patterns.
- Intact ions and fragments pass into the mass spectrometer's analyzer and are
eventually detected
- Separates compounds chromatographically before they are introduced to
the ion source and mass spectrometer.
- It differs from GC/MS in that the mobile phase is liquid, usually a
mixture of water and organic solvents
- Most commonly, an electrospray ionization source is used in LC/MS.
- There are also some newly developed ionization techniques like laser
spray
- PROTEIN CHARACTERIZATION
- Proteins are 1st digested into smaller peptides using different proteases
- A collection of these smaller peptides is then introduced into the mass analyzer
- ANALYSIS OF BIOLOGICAL NONCOVALENT COMPLEXES
- Electrospray ionization gets these noncovalent complexes into gaseous phase MS can be
used to observe these complexes
- Eg Hb complex , DNA duplex , cell surface carbohydrates, whole viruses
- MS successfully discovered that cis-9,10-octadecenoamide was present in the sleep state
was absent during the wake state
- APPLICATIONS IN VIROLOGY
- Identification of a virus in a given sample by analyzing the mass of the capsid proteins
or DNA/RNA through MS
- SEQUENCING PEPTIDES OLIGONUCLEOTIDES
- MALDI has been used recently to sequence proteins
oligonucleotides
- selected ion flow tube (SIFT-MS), and proton transfer reaction (PTR-
MS), are variants of chemical ionization dedicated for trace gas
analysis of air, breath or liquid headspace
- Use well defined reaction time allowing calculations of analyte
concentrations from the known reaction kinetics without the need
for internal standard or calibration.
- Mass spectrometry (MS), with its low sample requirement and high
sensitivity, has been the predominantly used in glycobiology for
characterization and elucidation of glycan structures.
- Mass spectrometry provides a complementary method to HPLC for the
analysis of glycans
- As a standard method for analysis, mass spectrometers have reached other planets and
moons.Two were taken to Mars by the Viking program.
- In early 2005 the Cassini-Huygens mission delivered a specialized GC-MS instrument
aboard
- the Huygens probe through the atmosphere of Titan, the largest moon of the planet
Saturn.
- This instrument analyzed atmospheric samples and was able to vaporize and analyze
samples of Titan's frozen, hydrocarbon covered surface once the probe had landed.
Provides molecular weights of peptides and proteins with high accuracy (0.1-0.01)
Highly sensitive requires fmol-pmol quantities of sample
Sample purity not important Can be coupled with on-line separation methods such as HPLC
and capillary electrophoresis for the analysis of mixtures

Noncovalent complexes are often disrupted

Cannot distinguish stereoisomers
Expensive instrumentation