Mass spectrometry (Analytical Technique)

CONTENTS
• INTRODUCTION
• HISTORY
• PRINCIPLE
• THEORY
• TERMINOLOGY
• INSTRUMENTATION
• IONISATION TECHNIQUES
• MASS ANALYSERS & TYPES
• MASS DETECTORS & TYPES
• TYPES OF IONS
• MASS FRAGMENTATION RULES
• MASS FRAGMENTATION PATTERNS
• MASS FRAGMENTATION TYPES
• TYPES OF PEAKS
• TANDEM MASS SPECTROMETRY
• ADVANTAGES & DISADVANTAGES OF
MS
• APPLICATIONS OF MS
• REFERENCES
21-12-2019V.K. VIKRAM VARMA 2

INTRODUCTION
•MASS SPECTROMETRY(MS) IS THE MOST ACCURATE
METHOD AMONGST OTHER SPECTROSCOPIC METHODS.
•IN WHICH SAMPLE IS CONVERTED TO RAPIDLY MOVING
POSITIVE IONS BY ELECTRON BOMBARDMENT &
CHARGED PARTICLES ARE SEPARATED ACCORDING TO
THEIR MASSES.
•MASS SPECTRA: IT IS A PLOT OF RELATIVE ABUNDANCE
AGAINST THE RATIO OF MASS/CHARGE.

HISTORY
1897
•J.J. Thomson. Discovered electrons by cathode rays experiment.
Nobel prize in 1906.
1919
•Francis Aston recognized 1st mass spectrometer and measure z/m
of ionic compounds.
1934
•First double focusing magnetic analyser was invented by Johnson
& Neil.
1966
•Munson & field described chemical ionisation.

CONTD.
1968
•Electrospray ionisation was invented by Dole, Mack & friends.
1975
•Atmospheric pressure chemical ionisation(APCI) was developed
by Caroll & others.
1985
•F.Hillenkamo, M.Karas & co-workers describe & coin the term
matrix assisted laser desorption ionisation(MALDI)
1989
•Paul discovered the ion trap technique.

FIVE NOBEL PRIZE PIONEERS

MASS SPECTROMETER

PRINCIPLE
Organic molecules
are bombarded with
electrons
Converted into highly
energetic positively
charged ions
(Molecular/Parent
ions )
Further breakup into
smaller ions
(Fragment/Daughter
ions)
Formed ions are
separated by
deflection of magnetic
field according to
their mass & charge.
Mass spectrum

CONTD.

CONTD.
Neopentane
(C5H12)
CH
+
3
+C4H9⦁
C2H
+
5
+
C3H7⦁
C3H
+
7
+
C2H5⦁
C4H
+
9
+ CH
⦁
3
[C5H12]⦁+
Fragmentation
example
Electron bombardment
⦁ Cations
⦁ Free radicals

THEORY
•IT IS AN INSTRUMENT WHICH HELP IN SEPARATING THE
INDIVIDUAL ATOMS OR MOLECULES BECAUSE OF THE
DIFFERENCE IN THE MASSES.
•THREE BASIC FUNCTIONS:
TO VAPORISE COMPOUNDS OF VARYING VOLATILITY.
PRODUCE IONS FROM THE NEUTRAL COMPOUNDS IN THE
VAPORISE PHASE.
TO SEPARATE IONS ACCORDING TO THEIR MASS/CHARGE
RATIO & TO RECORD THEM.

TERMINOLOGY
•MASS NUMBER (A): TOTAL NUMBER OF PROTONS AND
NEUTRONS IN AN ATOMIC NUCLEUS.
•MOLECULAR ION: IONS OBTAINED BY THE LOSS OF
AN ELECTRONS FROM THE MOLECULE.
•BASE PEAK: MOST INTENSE PEAK IN THE MASS
SPECTROMETRY, ASSIGNED 100% INTENSITY.

CONTD.
• 𝐌+
: SYMBOL OFTEN GIVEN TO THE MOLECULAR ION.
•RADICAL ION: POSITIVE CHARGED SPECIES WITH AN
ODD NUMBER OF ELECTRONS.
•FRAGMENT ION: LIGHTER CATIONS FORMED BY THE
DECOMPOSITION OF THE MOLECULAR ION. THESE
OFTEN CORRESPOND TO STABLE CARBOCATIONS.

INSTRUMENTATION

COMPONENTS OF MASS
SPECTROMETER
•INLET SYSTEM
•ION SOURCE
oIONISATION METHODS
•MASS ANALYSERS
•ION DETECTORS
•VACCUM SYSTEM

INLET SYSTEM
For Solids, Liquids, Gases
IONISATION SOURCE
EI, CI, FD, FAB, MALDI, ESI, APCI, APPI.
IONISATION MECHANISMS
Protonation, Deprotonation, Cationization,
Electron ejection, Electron capture
MASS ANALYSER
Magnetic field deflection, Double
focusing, Quadrupole, Time of
flight, Ion trap analyser, FT-ICR
DETECTOR
Electron multiplier, Faraday cup,
Photomultiplier conversion
dynode, Array
Mass
Spectrometer

 SAMPLE INLET•THREE TYPES OF SAMPLES, IF SOLID OR LIQUID SAMPLE IS
PRESENT CONVERTED INTO GAS UNDER SUITABLE ATMOSPHERIC
PRESSURE.
SAMPLE
SOLID
Solids with lower vapour
pressure directly inserted
into ionisation chamber.
Volatilisation is
controlled by heating the
probe.
LIQUID
Handled by hypodermic
needles.
Injected by silicon rubber
dam.
GAS
Directly introduced into
ionisation chamber by
mercury manometer.

 IONISATION CHAMBER
•IONISES THE MATERIAL UNDER ANALYSIS.
•MOLECULAR IONS ARE FORMED WHEN ENERGY OF
THE ELECTRON BEAM IS 10 − 15eV.
•FRAGMENT IONS ARE FORMED WHEN ENERGY OF
THE ELECTRON BEAM IS 70eV.
•ELECTRONS ARE RELEASED BY M ARE COLLECTED
BY ION COLLECTOR.

 ION ACCELERATION
CHAMBER
•ELECTRODE POTENTIAL OF ION ACCELERATION
CHAMBER IS 8 − 10KV.
•THE ACCELERATED IONS FURTHER GOES TO MAGNETIC
FIELD & DEFLECTION OF IONS.
•IONS HAVING HIGH MOLECULAR WEIGHT WILL REACH
DETECTOR FIRST.

CONTD.
ENERGY OF ACCELERATED IONS
𝟏
𝟐
𝑴𝒗 𝟐
= ⅇ𝑽 (EQUATION 1)
ENTERED INTO MAGNETIC FIELD
𝑯ⅇ𝑽 =
𝑴𝒗 𝟐
𝒓
(EQUATION 2)
SQUARING BOTH THE SIDES IN EQUATION 2
𝑯 𝟐ⅇ 𝟐 𝑽 𝟐 =
𝑴 𝟐 𝒗 𝟒
𝒓 𝟐 (EQUATION 3)
𝑯 𝟐ⅇ 𝟐 =
𝑴 𝟐 𝒗 𝟐
𝒓 𝟐 (EQUATION 4)
Where,
M= mass of the ion.
e= charge on the ion.
v= velocity of the ion.
V= potential difference.
Where,
H= Applied magnetic field.
r= radius of path.

CONTD.
REARRANGING EQUATION 1
𝑴𝒗 𝟐 = 𝟐ⅇ𝑽 (EQUATION 5)
PUT THE VALUE OF 𝑀𝑣2 FROM EQUATION 5 TO 4
𝑯 𝟐
ⅇ 𝟐
=
𝑴.𝟐ⅇ𝑽
𝒓 𝟐
𝑯 𝟐ⅇ =
𝑴𝑽
𝒓 𝟐
𝑴
ⅇ =
𝑯 𝟐 𝒓 𝟐
𝟐𝑽
(EQUATION 6)

 MASS ANALYSER
•IONS WILL BE SEPARATED ON
THE BASIS OF MOLECULAR
WEIGHT.
•TYPES OF ANALYSER
oMAGNETIC FIELD
DEFLECTION
oDOUBLE FOCUSING
oQUADRUPOLE
oTIME OF FLIGHT
oION TRAP ANALYSER
oFT-ICR
 AMPLIFIER•DIRECT ELECTRIC AMPLIFIER IS USED TO AMPLIFY THE
SIGNAL.

 RECORDER
•RECORDS THE SPECTRA.
 VACCUM SYSTEM
•ALL MASS SPECTROMETERS NEED A VACCUM TO ALLOW
IONS TO REACH THE DETECTOR WITHOUT COLLIDING
WITH OTHER GASEOUS MOLECULES OR ATOMS.
•IF SUCH COLLISION OCCUR, THE INSTRUMENT WOULD
SUFFER FROM REDUCED RESOLUTION & SENSITIVITY
10−2 𝑡𝑜10−5OR 10−4 𝑡𝑜10−7 𝑡𝑜𝑟𝑟.

IONISATION TECHNIQUES
•MASS SPECTRUM IS SIGNIFICANTLY DEPENDED UPON THE
IONISATION METHOD.
•VARIATION IN THE SPECTRUM IS INTRODUCED IN TERMS OF
NUMBER OF PEAKS.
INTENSITY OF PEAKS(SPECIALLY MOLECULAR ION).
•IONISATION TECHNIQUE CAN BE CATEGORIES INTO 2 PARTS:
HARD IONISATION TECHNIQUE:
–HIGH ENERGY, INCREASED FRAGMENTATION.
SOFT IONISATION TECHNIQUE:
–LOW ENERGY, DECREASED FRAGMENTATION.

TYPES or METHODS
GAS PHASE
Electron Ionisation (EI)
Chemical Ionisation
(CI)
DESORPTION
Field Desorption (FD)
Fast Atom
Bombardment (FAB)
Matrix Assisted Laser
Desorption Ionisation
(MALDI)
EVARPORATIVE
Electron Spray
Ionisation (ESI)
Atomic Pressure
Chemical Ionisation
(APCI)
Atomic Pressure Photo
Ionisation (APPI)

GAS PHASE IONISATION
•OLDEST & MOST POPULAR METHOD.
•SAMPLE IS VAPORISED BEFORE IONISATION.
•CLASSIFIED INTO:
– ELECTRON IONISATION (EI)
–CHEMICAL IONISATION (CI)
–FIELD IONISATION (FI)

 ELECTRON IONISATION (EI)

CONTD.
•DIRECT IONISATION THROUGH ELECTRON BEAM.
•IT IS THE MOST WIDELY USED & HIGHLY DEVELOPED METHOD. IT
IS ALSO KNOW AS ELECTRON IMPACT IONISATION OR ELECTRON
BOMBARDMENT.
•IONISATION TECHNIQUE IS USED TO CONVERT THE GASEOUS
SAMPLE INTO MOLECULAR IONS.
•HARD IONISATION TECHNIQUE (70𝑒𝑉).
𝐌 + ⅇ− → 𝐌⦁+ + 𝟐ⅇ−

CONTD.
•OPERATING PRESSURE 10−5 𝑡𝑜10−6 𝑡𝑜𝑟𝑟.
•POTENTIAL DIFFERENCE FROM 𝐺3 𝑡𝑜𝐺5 IS UP TO 8000V OR 8KV.
Gaseous sample
is entered from
slit 1.
Electrons emitted
from the filament
(tungsten) by
thermic emission.
Gaseous sample
& electrons
collides in the
ionisation
chamber.
𝐌⦁+Radical is
converted into
𝑴 𝟏
+
, 𝑴 𝟐
+
, 𝑴 𝟑
+
&
electrons
collected at
anode.

CONTD.
Positive ions will
move to
acceleration
chamber.
Potential difference
b/w the repller
plate(+vely charged
G3) & first
accelerating plate (-
vely charged G4)
Move to second
accelerating plate (-
vely charged G5)
Accelerated ions
are separated to
their masses to
their final
velocities.
The ions emerge from the final accelerating slit as a collimated
ribbon of ions. The energy and velocity of ions are given by :
𝒁𝑽 =
𝟏
𝟐
𝑴 𝟏 𝒗 𝟏 =
𝟏
𝟐
𝑴 𝟐 𝒗 𝟐 =
𝟏
𝟐
𝑴 𝟑 𝒗 𝟑
Where, z = charge of the ion V
= accelerating potential v =
velocity of ion.

CONTD.
ADVANTAGES
• CAN BE USED AS GC/MS INTERFACE.
• GIVES REPRODUCIBLE MASS
SPECTRA.
• GIVES MOLECULAR MASS & ALSO
THE FRAGMENTATION PATTERN OF
THE SAMPLE.
• EXTENSIVE FRAGMENTATION &
LARGE NUMBER OF PEAKS GIVES
STRUCTURAL INFORMATION.
DISADVANTAGES
• SAMPLE MUST BE THERMALLY
STABLE & VOLATILE.
• UNSTABLE MOLECULAR ION
FRAGMENTS ARE FORMED SO
READILY THAT ARE ABSENT FROM
MASS SPECTRUM.
• A SMALL AMOUNT OF SAMPLE IS
IONISED (1 IN 1000 MOLECULES).

 CHEMICAL IONISATION (CI)

CONTD.
•CARRIER GAS WILL BE IONISED → SECONDARY IONS WILL BE
PRODUCED & IONS WILL BE TRANSFERRED TO THE ANALYTES.
•IT IS VERY IMPORTANT SOFT IONISATION TECHNIQUE.
•FRAGMENTATION IS LESS & GIVES INTENSE PEAK OF
MOLECULAR ION.
•SOME MOLECULES LIKE ALCOHOLS, ETHERS, AMINES,
ESTERS, AMINO ACIDS ARE HIGHLY FRAGMENTED IN
ELECTRON IONISATION (EI), SO MOLECULAR ION PEAKS WILL
NOT BE DETECTED, TO GET PROPER ION PEAK WE ARE USING
THIS TECHNIQUE.

CONTD.
•STEPS OF CHEMICAL IONISATION:
A CARRIER / REAGENT GAS IS INTRODUCED INTO
IONISATION SOURCE AT SLIGHTLY HIGHER PRESSURE(1
TORR) (METHANE, AMMONIA & ISOBUTANE GASES ARE
USED)
CARRIER GAS WILL BE IONISED DUE TO ELECTRON
IMPACT FROM THE IONISATION SOURCE.
𝑪𝑯 𝟒 + ⅇ−
→ 𝑪𝑯 𝟒
+⦁ + 𝟐ⅇ−
𝑪𝑯 𝟒
+⦁ → 𝑪𝑯 𝟑
+
+ 𝑯∎
Where,
𝑪𝑯 𝟒
+⦁ &𝑪𝑯 𝟑
+
:Primary
ions

CONTD.PRIMARY IONS (𝑪𝑯 𝟒
+⦁& 𝑪𝑯 𝟑
+
) WILL REACT WITH EXCESS 𝑪𝑯 𝟒 & IT WILL
PRODUCE DIFFERENT TYPES OF SECONDARY IONS.
𝑪𝑯 𝟒
+⦁ + 𝑪𝑯 𝟒 → 𝑪𝑯 𝟓
+
+ 𝑪𝑯 𝟑
⦁
𝑪𝑯 𝟑
+
+ 𝑪𝑯 𝟒 → 𝑪 𝟐 𝑯 𝟓
+
+ 𝑯 𝟐
𝑪 𝟐 𝑯 𝟓
+
+ 𝑪𝑯 𝟒 → 𝑪 𝟑 𝑯 𝟓
+
+ 𝟐𝑯 𝟐
SECONDARY IONS WILL REACT WITH ANALYTE MOLECULE & FORM IONS
BY 3 WAYS:
PROTON TRANSFER.
HYDRIDE TRANSFER.
ELECTROPHILIC ADDITION.
Where,
𝑪𝑯 𝟓
+
, 𝑪 𝟐 𝑯 𝟓
+
& 𝑪 𝟑 𝑯 𝟓
+
:
Secondary ions

CONTD.
ADVANTAGES
•USED FOR SAMPLES WHICH
UNDERGO RAPID
FRAGMENTATION IN EI.
•USED FOR HIGH MOLECULAR
WEIGHT COMPOUNDS.
DISADVANTAGES
• RELATIVE LESS SENSITIVE THAN
EI IONISATION.
• NOT SUITABLE FOR THERMALLY
UNSTABLE & NON VOLATILE
SAMPLES.
• SAMPLES MUST BE DILUTED
WITH LARGE EXCESS OF
REAGENT GAS TO PREVENT
PRIMARY INTERACTION BETWEEN
THE ELECTRONS & SAMPLE
MOLECULES.

DESORPTION IONISATION
TECHNIQUE
•LIQUID/ SOLID SAMPLES WILL BE DIRECTLY
CONVERTED INTO GASEOUS IONS.
•CLASSIFIED INTO:
–FIELD DESORPTION (FD).
–FAST ATOM BOMBARDMENT (FAB).
–MATRIX ASSISTED LASER DESORPTION IONISATION
(MALDI).

 FIELD DESORPTION (FD)

CONTD.
•DESORPTION OF ELECTRONS FROM THE ANALYTE THROUGH
ANODE.
•LOW VOLATILE SAMPLES ARE USED TO PRODUCE STABLE
MOLECULAR IONS.
•WORKING:
SAMPLES ARE LOADED ON THE SURFACE OF THE CARBON
MICRONEEDLE BY DIPPING IN THE SAMPLE SOLUTION.
CARBON NEEDLES WILL PRODUCE HIGH GRADIENT VOLTAGE
ON THEIR TIPS, THAT IS WHY SHARP TIPS ARE USED.

CONTD.
ION FORMATION TAKES PLACE MAINLY BY 2 MECHANISMS:
FIELD IONISATION: ELECTRONS ARE REMOVED FROM THE SPECIES/
ANALYTE IN A HIGH ELECTRIC FIELD.
𝑴 → 𝑴+ + ⅇ−
CATIONS ATTACHED: CATIONS WILL BE ATTACHED WITH ANALYTE
MOLECULE 𝑖. 𝑒. 𝐻+
𝑜𝑟 𝑁𝑎+
𝑒𝑡𝑐.
𝑴 + 𝑯+
→ [𝑴 − 𝑯]+
POSITIVE IONS WILL BE REPELLED BY THE ANODE & THEY WILL
GO TOWARDS THE MASS ANALYSER.
IONS DOESN’T HAVE SUFFICIENT INTERNAL ENERGY FOR
FRAGMENTATION, DUE TO THIS STABLE IONS FORMED.

CONTD.
ADVANTAGES
•WORKS WELL FOR SMALL
ORGANIC MOLECULES, LOW
MOLECULAR WEIGHT
POLYMERS & PETROCHEMICAL
FUNCTIONS.
DISADVANTAGES
• SAMPLE MUST BE IN A SOLVENT.
• SENSITIVE TO ALKALI METAL
CONTAMINATION.
• NOT SUITABLE FOR THERMALLY
UNSTABLE & NON VOLATILE
SAMPLES.
• STRUCTURAL INFORMATION IS
NOT OBTAINED AS VERY LITTLE
FRAGMENTATION OCCURS.

 FAST ATOM BOMBARDMENT (FAB)

CONTD.
•SAMPLE IS MIXED WITH MATRIX & NEUTRAL ATOM BEAM
IS BOMBARDED.
•SOFT IONISATION METHOD.
•DETERMINE THE MOLECULAR WEIGHT OF THE
COMPOUNDS HAVING THE SIZE FROM 300-6OOODALTONS.
GENERALLY USED TO DETERMINE MOLECULAR WEIGHTS
OF PEPTIDES.

CONTD.
•METHODOLOGY:
CHARACTERISTICS OF THE MATRIX:
 NON VOLATILE
 LOW VAPOUR PRESSURE LIQUID
 EXAMPLES: GLYCEROL, THIOGLYCEROL, DIMETHANOLAMINE, TRIETHANOLAMINE.
 𝑋𝑒 OR 𝐴𝑟 (ACCELERATED NEUTRAL ATOMS)WILL BE BOMBARDED TO THE SAMPLE
MATRIX MIXTURE & IONISE THE SAMPLE DUE TO TRANSLATIONAL ENERGY. E.G.:
𝑿ⅇ + ⅇ− → 𝑿ⅇ⦁ + 𝟐ⅇ−
𝑿ⅇ + 𝑿ⅇ+⦁ → 𝑿ⅇ + 𝑿ⅇ+⦁
𝑮𝒍𝒚𝒄ⅇ𝒓𝒐𝒍 − 𝑯+
→ [𝑴𝑯]+
•IF 𝐶𝑠+
ION IS USED THAN IT IS KNOWN AS SIMS (SECONDARY
IONISATION MASS SPECTROMETRY).

CONTD.
ADVANTAGES
• FAB SPECTRA USUALLY PROVIDE
RELATIVELY ABUNDANT MOLECULAR
OR QUASIMOLECULAR IONS & SHOW
SOME STRUCTURALLY IMPORTANT
FRAGMENT IONS.
• USED FOR IONISATION OF HIGH
MOLECULAR WEIGHT SAMPLES OF
BIOLOGICAL ORIGIN.
• EXTENSIVELY USED FOR OBTAINING
MASS SPECTRA OF SALTS DEPENDING
UPON THE NATURE OF ITS CATION &
ANION.
DISADVANTAGES
• FAB SAMPLES THE SURFACE
RATHER THAN THE BULK
CONCENTRATION OF THE SOLUTE
PRESENT & HENCE LIMITS
QUANTITATIVE MEASUREMENTS.
• THE MATRIX ALSO FORMS IONS
ON BOMBARDMENT, IN ADDITION
TO THOSE FORMED BY THE
SAMPLE WHICH COMPLICATES
THE SPECTRUM.

CONTD.
•APPLICATIONS
THE SEPARATION & MS ANALYSIS OF PEPTIDES
ARISING FROM PROTEIN ENZYMATIC DIGESTION.
ELUCIDATION OF THE AMINO ACID SEQUENCE OF
THE OLIGOPEPTIDE EFRAPEPTIN D. THIS IS THE
POTENT INHIBITOR OF MITOCHONDRIAL 𝑨𝑻𝑷𝒂𝒔ⅇ
ACTIVITY.

 MATRIX ASSISTED LASER DESORPTION
IONISATION(MALDI)
• Matrix materials:
Nicotinic acid, Dihydroxy benzoic acid, Cinnamic acid derivatives, urea.
• Characteristics of Matrix:
Low molecular weight, Acidic nature, Strong laser beam absorption, Polar functional group.

CONTD.
Laser beams:
337nm- nitrogen laser of UV
range.
355nm- Frequency tripled
Nd:YAG
(Neodynium:Yterium:Alumini
um:Garnet)
326nm- Frequency
Quadrupole ND:YAG
294nm- IR laser
GENERALLY TIME OF FLIGHT(TOF) ANALYSER IS
USED.

CONTD.
• MIXED WITH POLAR MATRIX & LASER LIGHT IS USED FOR IONISATION.
• SOFT IONISATION METHOD, WHICH USES PULSED LASER BEAM.
• DETERMINE THE MOLECULAR WEIGHT OF PEPTIDES, ANTIBODIES,
PROTEIN, MOLECULES 𝑒𝑡𝑐 UP TO THE SIZE OF 300𝐾𝐷𝑎.
• LASER BEAM WILL HIT THE SAMPLE: MATRIX MIXTURE & ANALYTE/
SAMPLE WILL CONVERT INTO THE FORM OF GAS.
• ANALYTE/ SAMPLE & MATRIX WILL ALSO CONVERTS INTO THE IONS
DUE TO TRANSITIONAL ENERGY.
PROTONATION: 𝑴 + 𝑯+ → [𝑴𝑯]+
DEPROTONATION: 𝑴 → [𝑴 − 𝑯]−+𝑯+

CONTD.
ADVANTAGES
• HIGH MOLECULAR WEIGHT ANALYTE
CAN BE IONISED.
• GENTLE IONISATION TECHNIQUE.
• MOLECULE NEED TO BE VOLATILE.
• WIDE ARRAY OF MATRIXES.
• PRIOR SEPARATION BY
CHROMATOGRAPHY IS NOT REQUIRED.
• PRODUCES SINGLY CHARGED IONS
THUS INTERPRETATION BECOMES EASY.
DISADVANTAGES
• ANALYTE MUST HAVE VERY LOW
VAPOUR PRESSURE.
• MALDI MATRIX CLUSTER IONS NOT
DISCOVERED LOW 𝑚
𝑧 SPECIES (<6OO).
• PULSED NATURE OF SOURCE LIMITS
COMPATIBILITY WITH MANY MASS
ANALYSERS.
• ANALYTES THAT ABSORB THE LASER
CAN BE PROBLEMATIC.

CONTD.
•APPLICATIONS:
PHARMACEUTICAL ANALYSIS
a.DRUG METABOLISM STUDIES, PHARMACOKINETICS.
b.BIOAVAILABILITY STUDIES.
c. CHARACTERISATION OF POTENTIAL DRUGS.
d.IDENTIFYING DRUG TARGETS.
e. SCREENING OF DRUG CANDIDATES.
f. DRUG DEGRADATION PRODUCT ANALYSIS.

CONTD.
MICROBIOLOGY
a. IDENTIFICATION OF MICROORGANISMS.
b. SPECIES DIAGNOSIS BY THIS PROCEDURE IS MUCH FASTER, MORE
ACCURATE & CHEAPER THAN OTHER PROCEDURES BASED ON
BIOCHEMICAL TESTS.
FORENSIC & ENVIRONMENTAL ANALYSIS
a. PESTICIDES ON FOOD.
b. SOIL & GROUND WATER CONTAMINATION.
PROTEOMICS
a. TO IDENTIFY, VERIFY & QUANTITATE: METABOLITES, RECOMBINANT
PROTEINS, PROTEINS ISOLATED FROM NATURAL SOURCE, PEPTIDES &
THEIR AMINO ACIDS SEQUENCES.

EVARPORATIVE IONISATION
METHOD
•THESE TECHNIQUES ARE USED IN
CHROMATOGRAPHY.
•CLASSIFIED INTO:
–ELECTRON SPRAY IONISATION (ESI).
–ATMOSPHERIC PRESSURE CHEMICAL IONISATION
(APCI).
–ATMOSPHERIC PRESSURE PHOTO IONISATION
(APPI).

 ELECTRON SPRAY IONISATION (ESI)

CONTD.
•SOFT IONISATION TECHNIQUE.
•USED TO ANALYSE THE HIGH MOLECULAR WEIGHT
BIOMOLECULES, LIABLE & NON VOLATILE COMPOUNDS.
•USED TO IONISE PROTEINS, PEPTIDES, LIPIDS,
OLIGOSACCHARIDE, OLIGONUCLEOTIDE & SYNTHETIC
POLYMER.

CONTD.
Solution
containing the
sample through
the high voltage
potential
capillary by the
help of
Nebulisation gas.
Sprayed droplets
are ionised due to
high voltage
potential at
capillary.
Heated
desolvation gas
will evaporate the
solvent & it will
produce the
molecular ion.
Moves towards
ion accelerator
chamber.
Working:
• It can also produce multiply charged ions along with singly
charged.

CONTD.
ADVANTAGES
• GOOD SENSITIVITY & THEREFORE,
USEFUL IN ACCURATE QUANTITATIVE
& QUALITATIVE MEASUREMENTS.
• HAS THE ABILITY TO HANDLE
SAMPLES WITH LARGE MASSES.
• ONE OF THE SOFT IONISATION
TECHNIQUE AVAILABLE & HAS THE
ABILITY TO ANALYSE BIOLOGICAL
SAMPLES WITH NON COVALENT
INTERACTIONS.
DISADVANTAGES
• CANNOT ANALYSE MIXTURES VERY WELL &
WHEN FORCED TO DO SO, RESULTS ARE
UNRELIABLE.
• PRIOR SEPARATION OF CHROMATOGRAPHY
IS REQUIRED
• APPARATUS IS VERY DIFFICULT TO CLEAN &
HAS A TENDENCY TO BECOME OVERLY
CONTAMINATED WITH RESIDUES FROM
PREVIOUS EXPERIMENTS.
• MULTIPLE CHARGES THAT ARE ATTACHED TO
THE MOLECULAR IONS CAN MAKE FOR
CONFUSING SPECTRAL DATA.

CONTD.
•APPLICATIONS:
STUDYING NON COVALENT INTERACTION.
IDENTIFICATION & QUANTIFICATION OF HAEMOGLOBIN
VARIANTS.
PROTEIN IDENTIFICATION & CHARACTERISATION.
PROBING MOLECULAR DYNAMICS.
CHEMICAL IMAGING.
MONITORING CHEMICAL REACTIONS & STUDYING REACTIVE
INTERMEDIATES.
SCREENING FOR INBORN ERRORS OF METABOLISM.

 ATMOSPHERIC PRESSURE CHEMICAL
IONISATION (APCI)

CONTD.
•SOFT IONISATION TECHNIQUE, BASED ON THE
MECHANISM OF EVAPORATION & CARRIED OUT
ATMOSPHERIC PRESSURE.
•APCI IS A COMBINATION OF CI & ESI WITH
DEVIATION(ADVANCED VERSION OF CHEMICAL
IONISATION).
•GENERALLY APCI IS COUPLED WITH CHROMATOGRAPHIC
INSTRUMENT LIKE HPLC.

CONTD.
•WORKING:
SAMPLE WILL BE INJECTED THROUGH THE CAPILLARY THEN IT
WILL BE CONVERTED INTO SPRAYED DROPLET & FINALLY
ANALYTE.
SOLVENT VAPOUR DUE TO HEATING BY 𝑵 𝟐.
CORONA DISCHARGE ELECTRODE WILL IONISE THE SOLVENT
VAPOUR MOLECULE JUST LIKE PRODUCTION OF PRIMARY IONS
IN CHEMICAL IONISATION.
SOMETIMES ANALYTE VAPOURS MAY ALSO IONISED BY THE
ELECTRODE.

CONTD.
CORONA DISCHARGE ELECTRODE 𝒐𝒓 𝜷 −PARTICLE EMITTER IS
USED FOR IONISATION.
DUE TO COLLISION & ION MOLECULAR CHARGE TRANSFER
BETWEEN SOLVENT & ANALYTE TAKES PLACE & IT WILL
PRODUCE
𝑴𝑯+
𝒊𝒐𝒏 𝑨 + 𝑺+
→ 𝑴𝑯+
+ 𝑺−
𝒑𝒐𝒔𝒊𝒕𝒊𝒗ⅇ
[𝑴 − 𝑯]− 𝒊𝒐𝒏 𝑨+ + 𝑺 → [𝑴 − 𝑯]−+𝑺𝑯+ 𝒏ⅇ𝒈𝒊𝒕𝒊𝒗ⅇ
•APCI IS USED TO ANALYSE POLAR, THERMOSTABLE SUBSTANCE
WITH MOLECULAR WEIGHT LESS THAN 1500DALTONS.

CONTD.
ADVANTAGES
• MULTIPLE CHARGING IS TYPICALLY NOT
OBSERVED AS THE IONISATION PROCESS IS
MORE ENERGETIC THAN ESI.
• ELECTRON TRANSFER OR PROTON LOSS,
([𝑀 − 𝐻]−
) OCCURS IN THE NEGATIVE MODE.
• PROTON TRANSFER OCCURS IN THE POSITIVE
MODE.
• AT ATMOSPHERIC PRESSURE ANALYTE
MOLECULES COLLIDE WITH THE REAGENT
IONS FREQUENTLY & HENCE IONISATION IS
VERY DIFFICULT.
DISADVANTAGES
• RELATIVELY LOW ION CURRENTS.
• VERY SENSITIVE TO CONTAMINANTS
SUCH AS ALKALI METALS OR BASIC
COMPOUNDS.
• RELATIVELY COMPLEX HARDWARE
COMPARED TO OTHER ION SOURCES.

CONTD.
• APPLICATIONS:
DETERMINATION OF VITAMIN D3 IN POULTRY FEED SUPPLEMENTS.
CAN BE USED AS LC/MS INTERFACE.
ANALYSIS OF ORGANIC COMPOUNDS WITH MEDIUM-HIGH POLARITY.
ANALYSIS OF PESTICIDES.
SINCE POSITIVE IONISATION IS DEPENDENT ON PROTONATION, MOLECULES
CONTAINING BASIC FUNCTIONAL GROUPS SUCH AS AMINO, AMIDE ESTERS,
ALDEHYDE/ KETONE & HYDROXYL CAN BE ANALYSED.
NEGATIVE IONISATION DEPENDS UPON DEPROTONATION, MOLECULES
CONTAINING ACIDIC FUNCTIONAL GROUPS ARE ANALYSED BY THIS METHOD.
ANALYSIS OF TRIAZINES, PHENYLUREAS, CARBAMATES, &
ORGANOPHOSPHORUS COMPOUNDS.

 ATMOSPHERIC PRESSURE PHOTO
IONISATION (APPI)

CONTD.
•SOFT IONISATION TECHNIQUE.
•APPI SIMILAR TO APCI, BUT IONISATION IN APPI IS DUE TO
PHOTONS GENERATED BY UV LIGHT OF KRYPTON LAMP.
•SAMPLE SOLUTION WILL COME THROUGH THE HEATED
CAPILLARY & SPRAYED DROPLETS WILL BE FORMED DUE TO
NEBULISING GAS(𝑁2).
•DESOLVATION GAS(𝑁2) WILL BE SUPPLIED WHICH WILL
CONVERT THE SPRAYED DROPLETS INTO THE FORM OF
VAPOURS OF ANALYTE & SOLVENT.

CONTD.
•PHOTONS EMITTED BY THE KRYPTON LAMP HAVE A
SPECIFIC ENERGY 𝑖. 𝑒. 10𝑒𝑉; WHICH IS SUFFICIENT TO
IONISE THE TARGET MOLECULE 𝑖. 𝑒.ANALYTE & SOLVENT.
•PHOTONS OF 10𝑒𝑉 WILL NOT IONISE OTHER ATMOSPHERIC
GAS PRESENT, DUE TO LOW ENERGY.
•PHOTONS WILL IONISE THE ANALYTE BY 3 MECHANISMS
DIRECT APPI
𝑴 + 𝒉𝒗 → 𝑴⦁+ + ⅇ−

CONTD.
INDIRECT APPI
𝑺 + 𝒉𝒗 → 𝑺⦁+ + ⅇ−
𝑴 + 𝑺+
→ 𝑴+
+ 𝑺⦁
DOPANT ASSISTED APPI
TOLUENE IS USED AS DOPANT AGENT TO INCREASE THE
PERCENTAGE OF MOLECULAR ION.
𝑫 + 𝒉𝒗 → 𝑫⦁+ + ⅇ−
𝑺 + 𝑫⦁+ → 𝑴+ + 𝑫⦁

CONTD.
ADVANTAGES
•ADVANTAGE OVER APCI:
APPLICABLE TO HIGHLY NON
POLAR COMPOUNDS & LOW
FLOW RATED(<100𝜇𝑙/𝑚𝑖𝑛).
DISADVANTAGES
•IT CAN GENERATE
BACKGROUND IONS FROM
SOLVENTS.
•IT REQUIRES VAPORISATION
TEMPERATURES RANGING
FROM 350-500℃, WHICH CAN
CAUSE THERMAL
DEGRADATION.

CONTD.
•APPLICATIONS:
IT HAS THE CAPABILITY TO IONISE COMPOUNDS WITH A
WIDE RANGE OF POLARITIES WHILE BEING
REMARKABLY TOLERANT OF MATRIX COMPONENTS OF
HPLC ADDITIVES.
APPI HAS BEEN PROVED TO BE A VALUABLE TOOL FOR
ANALYTES WHICH ARE POORLY IONISED OR NOT
IONISED BY ESI & APCI, IN PARTICULAR.

CONTD.
APPI WAS SHOWN TO BE ABLE TO DETECT STEROID
HORMONES & HAD BEEN PROVEN TO HAVE MUCH
HIGHER SENSITIVITY THAN ESI.
RESULTS INDICATE THAT APPI USING TOLUENE AS
DOPANT PROVIDES EXCEPTIONAL IONISATION
CAPABILITIES FOR A BROAD RANGE OF COMPOUNDS,
IN PARTICULAR FOR HORMONES & STEROLS
COMPARED TO APCI & HESI.

MASS ANALYSER
•IONS AFTER LEAVING ION SOURCE, THE IONS ARE SEPARATED
ACCORDING TO THEIR 𝑚
𝑒 RATIO.
•IN THIS AREA, THE IONS ARE ACCELERATED BY BOTH
ELECTROSTATIC & MAGNETICALLY
•TYPES OF ANALYSERS:
MAGNETIC SECTOR ANALYSER.
DOUBLE FOCUSING ANALYSER.
QUADRUPOLE ANALYSER.
TIME OF FLIGHT ANALYSER(TOF).
ION TRAP ANALYSER.

 MAGNETIC SECTOR ANALYSER

CONTD.
•POSITIVELY CHARGED PARTICLES ARE SEPARATED BY
APPLICATION OF MAGNETIC FIELD, THEY TRAVEL I THE
CURVED PATH & MOLECULAR IONS ARE SEPARATED
ACCORDING TO THEIR MASSES & COLLECTED.
•GIVEN BY
𝒎
𝒛
=
𝑯 𝟐 𝒓 𝟐
𝟐𝑽
Where,
•
𝒎
𝒛
= mass
• H=magnetic
field
• r= Radius of
curvature
• V= Applied
voltage

 DOUBLE FOCUSING ANALYSER

CONTD.
•DOUBLE FOCUSING MAGNETIC SECTOR MASS ANALYSER
ARE THE CLASSICAL MODEL AGAINST WHICH OTHER MASS
ANALYSERS ARE COMPARED.
•THE LIMITATION IN THE SINGLE FOCUSING INSTRUMENT
IS THAT THE RESOLVING POWER IS LIMITED BY INITIAL
SPREAD OF TRANSLATIONAL ENERGY OF ION LEAVING
THE SOURCE.
•MAGNETIC SECTOR ANALYSER + ELECTROSTATIC
SECTOR→RESOLUTION INCREASED.

CONTD.
ADVANTAGES
•CLASSICAL MASS SPECTRA.
•HIGH RESOLUTION.
•HIGH SENSITIVITY.
•BEST QUANTITATIVE
PERFORMANCE OF ALL MS
ANALYSERS.
•VERY HIGH REPRODUCIBILITY.
DISADVANTAGES
•REQUIRED SKILLED OPERATOR
•DIFFICULT TO INTERFACE TO
ESI.
•USUALLY LARGER & HIGHER
COST THAN OTHER MASS
ANALYSERS.

CONTD.
•APPLICATIONS:
ACCURATE MASS MEASUREMENTS.
ALL ORGANIC MS ANALYSIS METHODS.
QUANTITATION.
ISOTOPE RATIO MEASUREMENTS.

 QUADRUPOLE ANALYSER

CONTD.•QUADRUPOLE ANALYSER CONSISTS OF TWO PAIRS OF
RODS WITH A HYPERBOLIC CROSS SECTION THAT ARE
ACCURATELY POSITIONED PARALLEL IN RADIAL ARRAY.
•APPLIED DC & RF VOLTAGE.
•IF RF>DC:- LARGER IONS WILL HIT THE DETECTOR FIRST.
•IF RF<DC:- SMALLER IONS WILL HIT THE DETECTOR
FIRST.
•UNSTABLE OR NON TRANSMITTED IONS WILL HIT THE
RODS & WILL NOT BE DETECTED.

CONTD.
ADVANTAGES
• RELATIVELY SMALL & COST
EFFECTIVE SYSTEMS.
• GOOD REPEATABILITY.
• CLASSICAL MASS SPECTRA.
• LOW ENERGY COLLISION INDUCED
DISSOCIATION (CID) MS/MS SPECTRA
LEADS TO EFFICIENT CONVERSION OF
PRECURSOR TO PRODUCT.
DISADVANTAGES
• LIMITED RESOLUTION.
• PEAK HEIGHT 𝑉𝑆 MASS RESPONSE
SHOULD BE TUNED.
• PEAK HEIGHTS ARE VARIABLE AS A
FUNCTION OF MASS DISCRIMINATION.
• LOW ENERGY COLLISION INDUCED
DISSOCIATION (CID) MS/MS SPECTRA
RELY MOST PROBABLY ON ENERGY,
COLLISION GAS, PRESSURE, &
ALTERNATIVE FACTORS.

CONTD.
•APPLICATIONS:
MAJORITY OF BENCH TOP GCMS & LCMS SYSTEMS.
SECTOR / QUADRUPOLE HYBRID MS/MS SYSTEMS.
TRIPLE QUADRUPOLE MS/MS SYSTEMS.

 TIME OF FLIGHT ANALYSER (TOF)

CONTD.
•TOF ANALYSER- 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.
•SORTING OF IONS IS DONE IN ABSENCE OF MAGNETIC FIELD.
•IONS PRODUCED HAVE DIFFERENT VELOCITIES DEPENDS ON THEIR
MASSES.
•LIGHTER IONS HAVE HIGHER VELOCITY COMPARED TO HEAVIER
IONS.
•LIGHTER IONS WILL STRIKE THE DETECTOR FIRST DUE TO HIGHER
VELOCITY.

CONTD.
ADVANTAGES
• FASTEST MS ANALYSER.
• HIGH ION TRANSMISSION.
• WELL SUITED FOR PULSED
IONISATION METHODS(METHOD OF
CHOICE FOR MAJORITY OF MALDI
MS SYSTEMS).
• HIGHEST PRACTICAL MASS RANGE
OF ALL MS ANALYSERS.
• MS/MS INFORMATION FROM POST
SOURCE DECAY.
DISADVANTAGES
•LOW RESOLUTION
•LIMITED PRECURSOR ION
SELECTIVITY FOR MOST MS/MS
EXPERIMENTS.
•REQUIRES PULSED IONISATION
METHOD OR ION BEAM
SWITCHING (DUTY CYCLE IS A
FACTOR).

CONTD.
•APPLICATIONS:
QUALITATIVE ANALYSIS
a.MOLECULAR WEIGHT DETERMINATION.
b.STRUCTURE DETERMINATION.
QUANTITATIVE ANALYSIS
a.PHARMACEUTICAL ANALYSIS
b.DRUG DISCOVERY
c. BIOTECHNOLOGY
d.ANALYSIS OF PROTEINS & PEPTIDES.

 ION-TRAP ANALYSER

CONTD.
•QUADRUPOLE ION TRAP CONSISTS OF A RING ELECTRODE & 2
HYPERBOLIC END CAP ELECTRODES.
•AS THE RF VOLTAGE IS INCREASED, THE ORBITS OF HEAVIER IONS
BECOME STABILISED, & PASSED INTO THE DETECTOR.
•IONS ARE INJECTED INTO THE TRAP & ALL IONS ARE TRAPPED.
•RF & DC ARE SCANNED TO SEQUENTIALLY EJECT IONS FOR
DETECTION.
•SPECIFIC IONS CAN BE TRAPPED WHILE OTHERS ARE EJECTED.
•ION VELOCITY CAN BE INCREASED TO INDUCED FRAGMENTATION.

CONTD.
ADVANTAGES
•INEXPENSIVE.
•EASILY INTERFACED TO
MANY IONISATION
METHODS.
•MS/MS IN ONE ANALYSER.
DISADVANTAGES
•LOW ACCURACY (>100𝑝𝑝𝑚).
•LOW RESOLUTION (<4000).
•SLOW SCANNING.
•SPACE CHARGING CAUSES
MASS SHIFTS.
•LOW MASS RANGE (<4000).

CONTD.
•APPLICATIONS:
TARGET COMPOUND SCREENING.
ION CHEMISTRY.
NON-DESTRUCTIVE ION DETECTION.
BENCHTOP GCMS, LCMS &MS/MS SYSTEMS.

MASS DETECTORS
•ONCE THE IONS ARE SEPARATED BY THE MASS ANALYSER,
THEY REACH THE ION DETECTOR, WHICH GENERATES A
CURRENT SIGNAL FROM THE INCIDENT IONS.
•TYPES OF DETECTORS:
FARADAY CUP DETECTOR.
ELECTRON MULTIPLIER DETECTOR.
PHOTOMULTIPLIER DYNODE DETECTOR.
ARRAY DETECTOR.

 FARADAY CUP MASS DETECTOR

CONTD.
• BASIC PRINCIPLE: THE INCIDENT ION STRIKES THE DYNODE SURFACE WHICH
EMITS ELECTRONS & INDUCES A CURRENT WHICH IS AMPLIFIED & RECORDED.
• THE DYNODE ELECTRODE IS MADE OF A SECONDARY EMITTING MATERIALS LIKE
𝐶𝑠𝑆𝑏, 𝐺𝑎𝑃 𝑜𝑟 𝐵𝑒𝑂.
• IT IS IDEALLY SUITED TO ISOTOPE ANALYSIS.
• ADVANTAGES:
GOOD FOR CHECKING ION TRANSMISSION & LOW SENSITIVITY
MEASUREMENTS.
• DISADVANTAGES:
LOW AMPLIFICATION.

 ELECTRON MULTIPLIER MASS
DETECTOR

CONTD.
• ELECTRON MULTIPLIER ARE THE MOST COMMON ESPECIALLY WHEN POSITIVE &
NEGATIVE IONS NEED TO BE DETECTED ON THE SAME INSTRUMENT.
• DYNODES MADE UP OF COPPER-BERYLLIUM WHICH TRANSDUCES THE INITIAL
ION CURRENT & ELECTRON EMITTED BY FIRST DYNODE ARE FOCUSED
MAGNETICALLY FROM DYNODE TO THE NEXT.
• FINAL CASCADE CURRENT IS AMPLIFIED MORE THAN MILLION TIMES.
• ADVANTAGES:
FAST RESPONSE
SENSITIVE
• DISADVANTAGES:
SHORTER LIFETIME THAN SCINTILLATION COUNTING(~3 𝑌𝑒𝑎𝑟𝑠).

 PHOTOMULTIPLIER DYNODE MASS
DETECTOR

CONTD.
• THE DYNODE CONSISTS OF A SUBSTANCE (A SCINTILLATOR) WHICH EMITS
PHOTONS.
• THE EMITTED LIGHT IS DETECTED BY PHOTO MULTIPLIER TUBE & IS
CONVERTED INTO ELECTRIC CURRENT.
• USEFUL IN STUDIES ON METASTABLE IONS.
• ADVANTAGES:
SENSITIVE
LONG LIFETIME(>5YEARS)
• DISADVANTAGES:
CANNOT BE EXPOSED TO LIGHT WHILE IN OPERATION.

 ARRAY DETECTOR

CONTD.
• AN ARRAY DETECTOR IS A GROUP OF INDIVIDUAL DETECTORS ALIGNED IN AN ARRAY
FORMAT.
• ARRAY DETECTOR, WHICH SPATIALLY DETECTS IONS ACCORDING TO THEIR DIFFERENT
𝑚
𝑧 , HAS BEEN TYPICALLY USED ON MAGNETIC SECTOR MASS ANALYSERS.
• SPATIALLY DIFFERENTIATED IONS CAN BE DETECTED SIMULTANEOUSLY BY AN ARRAY
DETECTOR.
• ADVANTAGES:
FAST & SENSITIVE.
• DISADVANTAGES:
REDUCES RESOLUTION
EXPENSIVE

TYPES OF IONS
•MOLECULAR IONS / PARENT IONS
•FRAGMENT IONS
•REARRANGEMENT IONS
•METASTABLE IONS
•MULTICHARGED IONS
•NEGATIVE IONS
•QUASI-MOLECULAR IONS

 MOLECULAR / PARENT
IONS:
•MOLECULE IS BOMBARDMENT WITH ELECTRONS IN
HIGH VACCUM IT IS CONVERTED TO POSITIVE IONS BY
LOSS OF ELECTRONS.
𝐌 + ⅇ−
→ 𝐌⦁+
+ 𝟐ⅇ−

•GENERATED BY THE FRAGMENTATION OF THE
MOLECULAR ION IN THE IONISATION CHAMBER.
𝐌⦁+
→ 𝐌 𝟏
+
+ 𝐌 𝟐
+
DUE TO UNSTABILITY OF 𝐌⦁+
& ENERGY OF
IONISATION POTENTIAL.
 FRAGMENT IONS / DAUGHTER
IONS:

 REARRANGEMENT IONS:
•IONS RESULTS FROM THE INTRAMOLECULAR
ATOMIC REARRANGEMENT DURING
FRAGMENTATION.
 METASTABLE IONS:
•FRAGMENT OF PARENT ION WILL GIVE RISE TO A NEW
ION(DAUGHTER ION) + NEUTRAL MOLECULE /
RADICAL.
𝐌 𝟏
+
→ 𝐌 𝟐
+
+ 𝐧𝐨𝐧 𝐜𝐡𝐚𝐫𝐠ⅇ𝐝 𝐩𝐚𝐫𝐭𝐢𝐜𝐥ⅇ𝐬

MULTICHARGED IONS:
•IONS MAY EXIST AS 2 OR 3 CHARGES INSTEAD OF
USUAL SINGLE CHARGE.
𝐌⦁+
+ ⅇ−
→ 𝐌++ + 𝟑ⅇ−
•RARELY FORMED UNDER NORMAL CONDITIONS.
•COMMON IN INORGANIC MASS SPECTRA.

NEGATIVE IONS:
•POSITIVE IONS PREDOMINATE IN ELECTRONIC IMPACT IONISATION
BECAUSE OF GREATER STABILITY.
•BUT NEGATIVE IONS ARE NOT VERY USEFUL IN STRUCTURE
DETERMINATION, FORMATION OF NEGATIVE IONS ARE VERY RARE.
𝐀𝐁 + ⅇ−
→ 𝐀𝐁−
𝐀𝐁 + ⅇ−
→ 𝐀+
+ 𝐁−
𝐀𝐁 + ⅇ−
→ 𝐀+
+ 𝐁−
+ ⅇ−

QUASI-MOLECULAR IONS:
•A PROTONATED MOLECULAR ION “OR” AN ION
FORMED BY REMOVAL OF ONE HYDROGEN ATOM
FROM MOLECULAR ION.
𝐌 + 𝐇+
→ 𝐌𝐇 +
(M+1)
𝐌+
→ 𝐌 − 𝐇 +
+ H (M+2)

 METASTABLE IONS
•DIFFUSED METASTABLE PEAKS.
•ARISE FROM FRAGMENTATION THAT TAKES PLACE DURING
FLIGHT DOWN THROUGH ION DEFLECTOR.
•DECOMPOSE RAPIDLY & RARELY REACH THE DETECTOR.
𝐌 =
𝐌 𝟐
𝟐
𝐌 𝟏
•E.G.: TOLUENE → 𝐂 𝟕 𝐇 𝟕
+
→ 𝐌 𝟏 → 𝟗𝟏 𝐌
𝐙
→ 𝐂 𝟓 𝐇 𝟕
+
→ 𝐌 𝟐 → 𝟔𝟓 𝐌
𝐙
𝐌 =
𝐌 𝟐
𝟐
𝐌 𝟏
=
𝟔𝟓 𝟐
𝟗𝟏
= 𝟒𝟔. 𝟖𝟒 𝐌
𝐙

MASS FRAGMENTATION RULES
۞HEIGHT OF M+⦁ PEAK DECREASES WITH INCREASING
DEGREE OF BRANCHING.
۞HEIGHT OF M+⦁ PEAK DECREASES WITH INCREASING
MOLECULAR WEIGHT.
۞CLEAVAGE IS FORMED AT ALKYL SUBSTITUTED
CARBONS WHICH LEADS TO FORMATION OF
CARBOCATION.

CONTD.
۞DOUBLE BONDS, CYCLIC STRUCTURES, AROMATIC RINGS
STABILISE M+⦁ & INCREASE THE PROBABILITY OF ITS
APPEARANCE.
۞DOUBLE BONDS FAVOUR ALLYLIC CLEAVAGE TO GIVE
THE RESONANCE STABILISED CATION.
CH
+
𝟐
− 𝐂𝐇 = 𝐂𝐇 𝟐 ↔ 𝐂𝐇 𝟐 = 𝐂𝐇 − 𝐂𝐇
+
𝟐

CONTD.
۞SATURATED RINGS TENDS TO LOOSE ALKYL SIDE
CHAINS AT THE 𝛼 – BOND & UNSATURATED RINGS
UNDERGO RETRO-DIELS ALDER REACTION.

CONTD.
۞ALKYL SUBSTITUTED AROMATIC COMPOUNDS ARE
CLEAVED PREFERABLY AT 𝛽- BOND TO THE RING, GIVING
RESONANCE STABILISED “BENZYL ION” OR “TROPYLIUM
ION”.

CONTD.
۞CLEAVGE IS OFTEN ASSOCIATED WITH ELIMINATION OF
SMALL STABLE, NEUTRAL MOLECULES, SUCH AS CO,𝐇 𝟐 𝐎,
N𝑯 𝟑, 𝑯 𝟐 𝐒, KETONES. E.G.: MCLAFFERTY REARRANGEMENT.
۞C-C BONDS NEXT TO HETEROATOM ARE FREQUENTLY
CLEAVED, LEAVING THE CHARGE ON THE HETEROATOM.

CONTD.
۞NITROGEN RULE:
•NONE/EVEN NUMBER OF N ATOMS: EVEN NOMINAL
MASS.
•ODD NUMBER OF N ATOMS: ODD NOMINAL MASS.
•IHD (INDEX OF HYDROGEN DEFICIENCY INDEX OR
DEGREE OF UNSATURATION)
FOR CXHY 𝐈𝐇𝐃 =
𝟐𝐱+𝟐𝐲
𝟐
DOUBLE BONDS/RING= IHD=1 ; TRIPLE BOND= IHD=2

CONTD.
۞STEVENSON RULE: WHEN AN ION FRAGMENTS, THE POSITIVE
CHARGE REMAIN ON THE FRAGMENT OF LOWEST IONISATION
POTENTIAL.
۞RING RULE: CAN CALCULATE THE NUMBER OF UNSATURATED
SITES IN THE COMPOUND FROM RING RULE. 𝐑 = 𝐂 + 𝟏 +
𝐍−𝐇
𝟐
FOR HALOGEN 𝐑 = 𝐂 + 𝟏 +
𝐗−𝐍
𝟐
−
𝐱
𝟐
Where, R= no. of unsaturated sites.
C= no. of carbons
N= no. of nitrogen X= halogen

FRAGMENTATION PATTERNS
•HOMOLYTIC CLEAVAGE: HERE
FRAGMENTATION IS DUE TO ELECTRONS
REDISTRIBUTION BETWEEN BONDS.

CONTD.
•HETEROLYTIC CLEAVAGE: CLEAVAGE OF C-X (X=O, N, S,
CHLORINE) BOND IS MORE DIFFICULT THAN C-C BOND.
IN SUCH CLEAVAGE, THE POSITIVE CHARGE IS CARRIED
BY THE CARBON ATOM & BY THE HETEROATOM.

CONTD.
•MCLAFFERTY REARRANGEMENT: OCCUR IN
KETONE, ALDEHYDES, CARBOXYLIC ACIDS &
ESTER.
SEPERATION OF 𝛾- HYDROGEN FOLLOWED BY 𝛽-
BOND CLEAVAGE TO FORM FRAGMENTS.

MASS FRAGMENTATION
•TYPES OF FRAGMENTATION:
COLLISION INDUCED DISSOCIATION (CID)
ELECTRON CAPTURE DISSOCIATION (ECD)
ELECTRON TRANSFER DISSOCIATION (ETD)
ELECTRON DETACHMENT DISSOCIATION (EDD)
PHOTO DISSOCIATION
SURFACE INDUCED DISSOCIATION (SID)
CHARGE REMOTE FRAGMENTATION
HIGH ENERGY C-TRAP DISSOCIATION (HCD)

 COLLISION INDUCED DISSOCIATION (CID):
•MOLECULAR IONS ARE ACCELERATED BY ELECTRICAL
POTENTIAL TO HIGH KINETIC ENERGY AND THEN ALLOWED
TO COLLIDE WITH NEUTRAL MOLECULES LIKE HELIUM,
NITROGEN OR ARGON.
•COLLISION BETWEEN THESE MOLECULES LEADS TO
FORMATION OF FRAGMENT IONS WHICH ARE ANALYZED BY
MASS SPECTROMETER.
•EXAMPLE:- TRIPLE QUADRUPOLE SPECTROMETER
PRODUCES CID FRAGMENTS

 ELECTRON CAPTURE
DISSOCIATION(ECD):
•IT IS A METHOD OF FRAGMENTING GAS PHASE IONS
FOR TANDEM MASS SPECTROMETRIC ANALYSIS
(STRUCTURAL ELUCIDATION).
•DIRECT INTRODUCTION OF LOW ENERGY
ELECTRONS TO TRAPPED GAS PHASE IONS.
•ECD TYPICALLY INVOLVES A MULTIPLY PROTONATED
MOLECULE M INTERACTING WITH A FREE
ELECTRON TO FORM AN ODD-ELECTRON ION.

•ETD INDUCES FRAGMENTATION OF CATIONS BY
TRANSFERRING ELECTRONS TO THEM.
• EXAMPLE:-PEPTIDES OR PROTEINS.
 ELECTRON TRANSFER DISSOCIATION(ECD):
 ELECTRON DETACHMENT DISSOCIATION(EDD):
• METHOD FOR FRAGMENTING ANIONIC SPECIES.

 PHOTO DISSOCIATION:
•PHOTODISSOCIATION IS A CHEMICAL REACTION IN
WHICH A CHEMICAL COMPOUND IS BROKEN DOWN BY
PHOTONS.
•IRMPD:- ABSORPTION OF MULTIPLE INFRA RED PHOTONS
BY A MOLECULE AND LEADS TO DISSOCIATION.
•BIRD:- LONG INTERACTION OF MOLECULE WITH
RADIATION FIELD LIKE CARBON DIOXIDE LASER.

 HIGH ENERGY C-TRAP
DISSOCIATION (HCD):
• IONS PASS THROUGH C-TRAP & INTO HCD CELL, WHERE
DISSOCIATION TAKES PLACE.
•IT IS A FRAGMENTATION TECHNIQUE, USED FOR PEPTIDE
MODIFICATION ANALYSIS.
• IMMONIUM IONS GENERATED VIA HCD PINPOINT MODIFICATIONS
SUCH AS PHOSPHO-TYROSINE.
•AN ADDED OCTOPOLE COLLISION CELL FACILITATES DE NOVO
SEQUENCING.

•IT IS A TYPE OF COVALENT BOND BREAKING THAT OCCURS IN A GAS
PHASE ION IN WHICH THE CLEAVED BOND IS NOT ADJACENT TO THE
LOCATION OF THE CHARGE. THIS FRAGMENTATION CAN BE OBSERVED
USING TANDEM MASS SPECTROMETRY.
 SURFACE INDUCED DISSOCIATION(SID):
•IT IS A TECHNIQUE USED IN MS TO FRAGMENT MOLECULAR IONS IN
THE GAS PHASE BY COLLISION OF AN ION WITH A SURFACE UNDER
HIGH VACCUM
 CHARGE REMOTE FRAGMENTATION:

FACTORS INFLUENCING
FRAGMENTATION
• THERMAL DECOMPOSITION: UNDERGO THERMAL DECOMPOSITION IN THE ION
SOURCE BEFORE IONISATION.
DUE TO THIS FRAGMENTATION OF COMPOUND WILL BE AFFECTED & PROBLEM
WILL OCCUR DURING INTERPRETATION OF SPECTRA.
• BOMBARDMENT ENERGIES: MORE NUMBER OF FRAGMENTS –HIGH
BOMBARDMENT ENERGY REQUIRED.
LESS FRAGMENTATION-LESS BOMBARDMENT ENERGY REQUIRED.
• FUNCTIONAL GROUPS.

TYPES OF PEAKS
•MOLECULAR ION PEAK
•FRAGMENT ION PEAK
•REARRANGEMENT ION PEAK
•METASTABLE ION PEAK
•MULTICHARGED ION PEAK
•BASE PEAK
•NEGATIVE ION PEAK

CONTD.
•MOLECULAR ION PEAK: SAMPLE IS BOMBARDED WITH
ELECTRONS OF 9-15EV THE MOLECULAR ION IS
PRODUCED BY THE LOSS OF SIMPLE ELECTRON.
•FRAGMENT ION PEAK: ENERGY IS GIVEN FURTHER
MORE UP TO 70EV. FRAGMENTS WHICH HAS LOWER
MASS NUMBER.
•REARRANGEMENT ION PEAK: RECOMBINATION OF
FRAGMENT ION.

CONTD.
•METASTABLE ION PEAK: IONS RESULTING FROM
DECOMPOSITION BETWEEN THE SOURCE & MAGNETIC
ANALYSER.(BROAD PEAKS)
•MULTICHARGED ION PEAK: IONS EXIST AS 2 OR 3
CHARGES INSTEAD OF SINGLE CHARGE.
•BASE PEAK: MOST INTENSE PEAK IN THE MS, ASSIGNED
100% INTENSITY.
•NEGATIVE ION PEAK: NEGATIVE IONS FORMED FROM
ELECTRON BOMBARDMENT OF SAMPLE.

ISOTOPIC PEAK•EACH ISOTOPE WILL SHOW UP AS A SEPARATE LINE IN MS.
•THE PRESENCE OF ISOTOPES READILY PRODUCE THE ISOTOPE
IONS IN THE SPECTRUM ACCOMPANIED BY A MAIN MOLECULAR
& FRAGMENT ION PEAK.
•.12
C – 98.9% NATURAL ABUNDANCE- VERY HIGH PEAK- M+
PEAK
(BASE PEAK).
•.13 C – 1.1% NATURAL ABUNDANCE- VERY LOW PEAK-
[M + 1]+PEAK.
•BASE PEAK IS THE LARGEST PEAK IN THE SPECTRUM &
INTENSITY OF EVERY OTHER PEAK IS REPORTED IN
COMPARISON TO BASE PEAK.

TANDEM MASS
SPECTROMETRY
• PURPOSE IS TO FRAGMENT IONS FROM PARENT ION TO PROVIDE STRUCTURAL
INFORMATION ABOUT A MOLECULE
• ALSO ALLOWS MASS SEPARATION AND AA IDENTIFICATION OF COMPOUNDS IN
COMPLEX MIXTURES
• USES TWO OR MORE MASS ANALYZERS/FILTERS SEPARATED BY A COLLISION CELL
FILLED WITH ARGON OR XENON
• COLLISION CELL IS WHERE SELECTED IONS ARE SENT FOR FURTHER
FRAGMENTATION

CONTD.
• IN TANDEM MASS SPECTROMETRY (MS/MS), DISTINCT IONS OF INTEREST ARE
SELECTED BASED ON THEIR M/Z FROM THE FIRST ROUND OF MS AND ARE
FRAGMENTED BY A NUMBER OF METHODS OF DISSOCIATION.
• ONE SUCH METHOD INVOLVES COLLIDING THE IONS WITH A STREAM OF INERT
GAS, WHICH IS KNOWN AS COLLISION-INDUCED DISSOCIATION (CID) OR HIGHER
ENERGY COLLISION DISSOCIATION (HCD). OTHER METHODS OF ION
FRAGMENTATION INCLUDE ELECTRON-TRANSFER DISSOCIATION (ETD) AND
ELECTRON-CAPTURE DISSOCIATION (ECD).

 DIAGRAM OF TANDEM MASS
SPECTROMETRY (MS/MS)

CONTD.
• THESE FRAGMENTS ARE THEN SEPARATED BASED ON THEIR INDIVIDUAL M/Z RATIOS IN A
SECOND ROUND OF MS. MS/MS (I.E., TANDEM MASS SPECTROMETRY) IS COMMONLY USED TO
SEQUENCE PROTEINS & OLIGONUCLEOTIDES AND THESE CAN BE MATCH WITH DATABASES
SUCH AS IPI, REFSEQ & UNIPROTKB/SWISS-PROT.
• THESE SEQUENCE FRAGMENTS CAN THEN BE ORGANIZED IN SILICO INTO FULL- LENGTH
SEQUENCE PREDICTIONS.
• A SAMPLE IS INJECTED INTO THE MASS SPECTROMETER, IONIZED, ACCELERATED &
ANALYZED BY MASS SPECTROMETRY (MS1).
• IONS FROM THE MS1 SPECTRA ARE THEN SELECTIVELY FRAGMENTED & ANALYZED BY A
SECOND STAGE OF MASS SPECTROMETRY (MS2) TO GENERATE THE SPECTRA FOR THE ION
FRAGMENTS.

 HOW TANDEM MS SEQUENCING
WORKS• USE TANDEM MS: TWO MASS ANALYZERS IN SERIES WITH A COLLISION
CELL IN BETWEEN
• COLLISION CELL: A REGION WHERE THE IONS COLLIDE WITH A GAS (HE,
NE, AR) RESULTING IN FRAGMENTATION OF THE ION
• FRAGMENTATION OF THE PEPTIDES OCCUR IN A PREDICTABLE FASHION,
MAINLY AT THE PEPTIDE BONDS.
• THE RESULTING DAUGHTER IONS HAVE MASSES THAT ARE CONSISTENT
WITH KNOWN MOLECULAR WEIGHTS OF DIPEPTIDES, TRIPEPTIDES,
TETRAPEPTIDES… SER-GLU-LEU-ILE-ARG-TRP COLLISION CELL SER-GLU-
LEU-ILE-ARG SER-GLU-LEU SER-GLU-LEU-ILE ETC.…

CONTD.
ADVANTAGES
• FAST
• NO GELS
• DETERMINES MW AND AA SEQUENCE
• CAN BE USED ON COMPLEX
MIXTURES-INCLUDING LOW COPY
• CAN DETECT POST-TRANSLATIONAL
MODIFICATION.
DISADVANTAGES
• VERY EXPENSIVE-CAMPUS
• REQUIRES SEQUENCE DATABASES
FOR ANALYSIS

ADVANTAGES & DISADVANTAGES
OF MASS SPECTROMETRY:
ADVANTAGES
• MOLECULAR WEIGHT & FORMULA
DETERMINATION.
• QUANTITATIVE & QUALITATIVE
ANALYSIS.
• LESS AMOUNT OF SAMPLE REQUIRED.
• LESS THAN 1 MINUTE REQUIRED FOR
ANALYSIS.
DISADVANTAGES
• SAMPLE DESTRUCTION.
• SAMPLE SHOULD BE IN GASEOUS
FORM.
• COMPLEX & HIGH COST.
• SHOULD MAINTAIN VACCUM
THROUGHOUT THE PROCESS

APPLICATIONS OF MASS
SPECTROMETRY
•STRUCTURE ELUCIDATION.
•PHARMACEUTICAL ANALYSIS:
oBIOAVAILABILITY STUDIES.
oDRUG METABOLISM STUDIES, PHARMACOKINETICS.
oCHARACTERISATION OF POTENTIAL DRUGS.
oDRUG DEGRADATION OF PRODUCT ANALYSIS.
oSCREENING OF DRUG CANDIDATES.
oIDENTIFYING DRUG TARGETS.

CONTD.
•BIOMOLECULE CHARACTERIZATION:
oPROTEINS & PEPTIDES.
oOLIGO NUCLEOTIDES.
•ENVIRONMENTAL STUDIES:
oPESTICIDES IN FOOD.
oSOIL & GROUND WATER CONTAMINATION.
•FORENSIC ANALYSIS/ CLINICAL STUDIES:
oINVESTIGATE USE OF ILLEGAL DRUGS THROUGH ANALYZING BODY FLUIDS &
TISSUES. THE SAMPLE FOR FORENSICS IN THE CASE OF DRUG ABUSE IS
MAINLY URINE, HAIR & BLOOD.

REFERENCE
• INTRODUCTION TO SPECTROSCOPY BY PAVIA.
• A TEXTBOOK OF ORGANIC CHEMISTRY BY BAHL ARUN & BAHL B.S.
• HTTP://WWW.CHEM.UCALGARY.CA/COURSES/350/CAREY5TH/CH13/CH13-0.HTML
• HTTP://PREMIERBIOSOFT.COM/TECH_NOTES/MASS-SPECTROMETRY.HTML
• HTTPS://EN.WIKIPEDIA.ORG/WIKI/MASS_SPECTROMETRY
• WWW.YOUTUBE.COM
• WWW.SLIDESHARE.COM
• WWW.GOOGLE.COM

Mass spectrometry (Analytical Technique)

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Mass spectrometry (Analytical Technique)