Mass Spectrometry by ANITHA SRI

8/3/2019 Mass Spectrometry by ANITHA SRI

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OVERVIEW OFMASS SPECTROMETRY

M. Anitha Sri (Y11MPH448)

I/II M.Pharmacy, Industrial pharmacy

CHALAPATHI INSTITUTE OF

PHARMACEUTICAL SCIENCES.


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CONTENTS Introduction

Instrumentation

Mass Spectrum

Resolution Determination of molecular formula

Data analysis and interpretation

Applications

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INTRODUCTION A mass spectrometer is an instrument that measures the masses

of individual molecules. Three Basic functions:

1. creating gaseous ion fragments from the samples

2. separating them according to their mass-to-charge ratio

3. records the relative abundance of each ionic species

present

Also known as positive ion spectra or line spectra.

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Block diagram of Components of Mass Spectrometer


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INSTRUMENTATION

Inlet system

Ion source

Electrostatic accelerating system

Magnetic field Ion separator

Ion collector and Detector

Vacuum system

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INLETSYSTEM

Direct vapor inlet

Direct insertion probe

Gas chromatography(GC-MS)

Liquid chromatography(LC-MS)Particle Beam Interface

Thermospray Interface

Electrospray Interface

Desorption techniques(FAB and LSIMS)

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DIRECT VAPOUR INLET

Gases or volatile liquidsMethod is Molecular leak or Molecular pumping

The sample can be introduced through a septum port or

through a valve port.

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DIRECT INSERTION PROBE

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Solids and liquid samples.

Autoprobe.


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GC-MS

Most common technique for

introducing samples.

Several different interface

designs are used to connect

these two instruments.

The MS coupled to the GC

should be capable of high

resolution.

Highly specific.

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LC-MS

Used for Thermo labile compounds.

Several interfaces are used to connect LC and MS.

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PARTICLE BEAM INTERFACE

Solvent is removed from an

aerosol of LC effluent

The resulting analyte is

analysed in the ion source

Known as MAGIC

(Monodisperse Aerosol

Generator Interface for

Chromatography)

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THERMOSPRAY

Involves simply heating the

tip of the entry tube to

promote vaporisation.

Through the centre of the

stainless steel tube, passes asmall diameter tube which

carries the column eluent.

The tube projects slightly

beyond the end of the heater

cap which is situated in acartridge heater together with

a thermocouple.

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ELECTRO SPRAY

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Sample is dissolved in

a solvent and pumped

through a narrow

capillary.Voltage is applied to

the capillary tip and

the sample is dispersed

into an aerosol, aidedby a coaxially

introduced nebulising

gas.


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ESI

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The charged droplets diminish

in size by solvent evaporation

assisted by a flow of drying

gas.

Eventually charged sample

ions, free from solvent, are

released from the droplets,

which pass through the orificeinto an intermediate vacuum

region and from these through

a small aperture into the

analyser of the MS.


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IONISATION TECHNIQUES

Name Ionising agent

Electron Impact(EI) Energetic electronsChemical Ionisation (CI) Reagent gaseous ions

Field Ionisation (FI) High potential electrode

Field Desorption (FD) High potential electrode

Electro Spray Ionisation (ESI) High electrical field

Matrix assisted Laser Desorption

Ionisation (MALDI)

Laser beam

Fast Atom Bombardment (FAB) Energetic atomic beamSecondary ion Mass

Spectrometry (SIMS)

Energetic beam of ions

Thermospray Ionisation (TS) High temperature

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ELECTRON IMPACT

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CHEMICAL IONISATION

Chemical interaction between reagent gas ions and analytemolecule.

Two-step process.

CH4 + e- = CH4

+ + 2e-

Secondary ions of reagent gas are produced, which react with

the analyte molecules. The mechanism may be proton transfer, hydride abstraction or

charge transfer.

CH4+ + MH = CH4 + MH

+

CH5+ + MH = CH4 + MH2

+

CH3+ + MH = CH4 + M

+

Reagent gases: Argon, Helium, Nitrogen.18


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FIELD IONISATION

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Ions are formed under the

influence of high electric field

produced by applying high

voltages.

On the surface of fine tube,

many hundreds of projecting

carbon microtips are present.

These extract the electron from

the sample and ionise the

sample molecules.


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FAST ATOM BOMBARDMENT

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High energy primary

beam is directed at a

target surface to obtain

high yield of secondaryions.

Primary beam may be

ions, electrons, photons

or neutral atoms. SIMS may be

dynamic or static.


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MALDI

Two step process.

Desorption is triggered

by a laser beam.

The second step isionization.

Nitrogen laser of 337 nm

wavelength is used.

Sinapinic acid is used asmatrix for proteins and -

cyano-4-

hydroxycinnamic acid for

peptides.21


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CHOOSINGAN IONISATIONTECHNIQUE

Information desired Ionization techniqueDepth profiling Fast atom bombardment/secondary ion

mass spectroscopyChemical speciation/component

analysis (fragmentation desired) Electron impactMolecular species identification of

compounds soluble in common

solventsElectrospray ionization

Molecular species identification of

hydrocarbon compounds Field ionizationMolecular species identification of

high molecular weight compoundsMatrix assisted laser desorption

ionizationMolecular species identification of

halogen containing compoundsChemical ionization (negative mode)

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ELECTROSTATIC ACCELERATINGSYSTEM

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The positive ions formed in the ionisation chamber

are accelerated by pairs of accelerator plates to

impart velocities to the ions.

Ions are sorted acc. to m/e ratio based on 3

properties: energy, velocity and momentum.

The beam from the slits of these plates consists of

a collimated ribbon of ions having equal energies.

K.E = eV = m1v12 = m2v2

2.


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MAGNETIC FIELD

eV = mv2

F = HeV

HeV = mv2/r

m/e = H2r2/2V

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e = charge

m= mass

v = velocity

V = voltage

F = Magnetic force

H = Magnetic field strength

r = radius


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ION SEPARATOR

Single Focussing

Double Focussing

Cycloidal

Quadrupole TOF

MS/MS

Radio Frequency

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SINGLE FOCUSSING

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SINGLE FOCUSSING

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DOUBLE FOCUSSING

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CYCLOIDAL

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QUADRUPOLE

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TIMEOF FLIGHT

The time-of-flight (TOF) mass analyzer separates ions in time as they travel downa flight/drift tube.

This is a very simple mass spectrometer that uses fixed voltages and does not

require a magnetic field. The greatest drawback is that TOF instruments have

poor mass resolution, usually less than 500.

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MS/MS

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Hybrid MS.

The two analysers are separated by a field free collision chamber,

which contains an inert gas.


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RADIO FREQUENCY

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An assembly of grids is

employed to select ions acc. to

their velocities.

Alternative grids are connected

to a radiofrequency source and

the other grids are connected to

a steady potential.

It is simple in construction and

doesnt require a magnet.


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IONCOLLECTORAND DETECTOR

Detection of ions is based on their charge

Detectors monitors the ion current, amplifies it and

the signal is transmitted to the data system where it

is recorded in the form of mass spectra.

Types of Detectors:

Faraday Cup Collector.

Electron Multiplier

Channel Electron Multiplier ArrayThe detection is either by pulse counting or analog

measurement.

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FARADAY-CUP

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Ions enter the cup and transfer

their charge to the cup.

Secondary electrons are

generated.

No. of secondary electrons

generated depends on several

factors:

mass of ionsenergy of ions

charge on the ions

Angle of incidence

material of cup

nature of the ion.


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ELECTRON MULTIPLIER

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A metal plate called

conversion dynode that

converts the impinging ionsto electrons is present.

Ion beams strikes the

conversion dynode.

Secondary electrons are

produced by the electron

multiplier.


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VACUUMSYSTEM

All mass spectrometers operate at very low pressure (high vacuum).

This reduces the chance of ions colliding with other molecules in the

mass analyzer. Any collision can cause the ions to react, neutralize,

scatter, or fragment. All these processes will interfere with the mass

spectrum.

To minimize collisions, experiments are conducted under high

vacuum conditions, typically 10-2 to 10-5 Pa (10-4 to 10-7 torr)

depending upon the geometry of the instrument.

This high vacuum requires two pumping stages. The first stage is a

mechanical pump that provides rough vacuum down to 0.1 Pa (10-3

torr). The second stage uses diffusion pumps or turbo molecular

pumps to provide high vacuum.

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GENERAL FRAGMENTATION PATTERNS

Simple Direct cleavage

Retro-Diels Alder Reaction

Hydrogen Transfer Rearrangement

Mc lafferty rearrangement

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MC-LAFFERTYREARRANGEMENT

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Involves intramolecular migration of -

hydrogen from electron rich center to electron

deficit center followed by cleavage at position

resulting in the formation of neutral alkene.

Common in ketones, esters and carboxylic acids


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TYPESOFIONS

Molecular ion

Fragment ions

Rearrangement ions

Multiply charged ions

Negative ions

Metastable ions

Pseudomolecular or Quasi ions

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METASTABLEIONS

Ions formed in the analyser after moving away from

the ionisation chamber.

Gives broad bands.

Formed at non-integral mass numbers.

Mass of metastable ion is calculated by:

m* = m22/m1

m* = mass of metastable ionm1 = mass of molecular ion

m2 = mass of daughter ion42


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DERIVITISATION

For some ionisation techniques, the compound should bederivitised before being analysed.

Derivitisation is the use of chemicals to modify theanalyte, usually to reduce its polarity. Often OH and NHgroups are reacted with silylating reagents, or acetic

anhydride, to form compounds with O-Si, N-Si, O-C orN-C bonds instead.

The derivative then lacks the ability to form hydrogenbonds and is more volatile than the analyte was.

Mass spectrometry is a gas phase technique; irrespective

of the nature of the sample the analysis is on gaseousions, hence the need for volatility.

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MASS SPECTRUM

The mass spectrum is presented in terms of ion abundance vs. m/eratio (mass)

The most abundant ion formed in ionization gives rise to the tallest

peak on the mass spectrumthis is the base peak

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base peak, m/e

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All other peak intensities are relative to the base peak as a


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All other peak intensities are relative to the base peak as a

percentage

If a molecule loses only one electron in the ionization process,

a molecular ion is observed that gives its molecular weightthis is designated as M+ on the spectrum

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M+,m/e 114

In most cases, when a molecule loses a valence electron, bonds


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, ,

are broken, or the ion formed quickly fragment to lower energy

ions.

The masses of charged ions are recorded as fragment ions by

the spectrometerneutral fragments are not recorded !

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fragment ions


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RESOLUTION

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Adjacent peaks must be clearly separated.

The valley between the two adjacent peaks should not be more

than 10% of the height of the larger peak.

R = Mn/Mn - Mm


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DETERMINATIONOF MOLECULAR FORMULA

Nitrogen Rule

Rule Of Thirteen

When a molecular mass, M+, is known, a base formula can be

generated from the following equation:

M/13 = n + r/13

the base formula being: CnHn+r

Index of Hydrogen Deficiency:

HDI = n-r+2 / 2 Ring rule:

For the molecule CwHxNyOz, R = w + 1 + y-x/248


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DATA ANALYSISFROMMASSSPECTRUM

The molecular ion peak in aromatic compounds is

relatively much intense.

Conjugated olefins show more intense molecular ion

peak as compared to the corresponding non-

conjugated olefins with same no. of unsaturation.

The relative abundance of the saturated hydrocarbon

is more than the corresponding branched chain

compound. In aromatic compounds, the substituent groups like -

OH, -OR, -NH2 increase the relative abundance and

NO2, -CN decrease the relative abundance.49


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Absence of molecular ion peak in the mass

spectrum means that the compound under

examination is highly branched or tertiary alcohol.

In case of Chloro or Bromo compounds, isotope

peaks(M+ + 2) are also formed along with the

molecular ion peak.

Isotope peak is not observed when Fluorine or

Iodine atom is present in the compound.

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COMPUTERISEDMATCHINGOFSPECTRA

WITHSPECTRALLIBRARIES

The computer can compare a mass spectrum it has

determined with the spectra in the databases of the

libraries.

The output is a table called HIT LIST.

Hit list includes the name of each compound that the

computer has used for matching, its molecular weight,

molecular formula, probability that the spectrum of the

test compound matches the spectrum in the data base.

The probability is determined by the no. of peaks andtheir intensities that can be matched.

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APPLICATIONS

Determination of molecular mass and structure.

Determination of Isotopic abundance.

Distinction between isomers.

Determination of Ionisation potential and BondDissociation energies.

Detection of presence of impurities.

Identification of unknown compound.

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REFERENCES

U.S.P. Y.R. SHARMA. ELEMENTARY ORGANIC SPECTROSCOPY,

PRINCIPLES AND CHEMICAL APPLICATIONS. 4th ed. S.CHAND.

2007.

D.A.SKOOG, F.J. HOLLER, S.R.CROUCH. PRINCIPLES OF

INSTRUMENTAL ANALYSIS. 6th

ed. THOMPSON BROOKS. 2007. D.L.PAVIA, G.M.LAMPMAN, G.S.KRIZ. INTRODUCTION TO

SPECTROSCOPY. 3rd ed. THOMPSON BROOKS. 2001.

G.R.CHATWAL, S.K.ANAND. INSTRUMENTAL METHODS OF

CHEMICAL ANALYSIS. 5th ed. HIMALAYA PUBLISHING HOUSE.

2002.

H.HWILLARD, L.L.MERRITT, J.A.DEAN, F.A.SETTLE.

INSTRUMENTAL METHODS OF ANALYSIS. 7th ed. CBS

PUBLISHERS.


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Mass Spectrometry by ANITHA SRI

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