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Presented by:

OBSERVATION OF ISOTOPE USING MASS SPECTROMETRY

oan.sahito@gmail.com

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OUTLINES

INTRODUCTION BASIS OF SEPARARION IONIZER, ANALYZER AND DETECTOR TYPES ISOTOPES AND OBSERVATION OF ISOTOPES CLASSIFICATION OF ISOTOPES SPECTRAS ISOTOPIC ABUNDANCE CONCLUSION REFERENCES ACKNOWLEDGEMENTS

It subjects vaporized molecules to bombardment by a stream of high-energy electrons, converting these

molecules to ions

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INTRODUCTION

These ions are then accelerated in an electric fieldThe accelerated ions are then separated according to their mass-to-charge ratio in a magnetic or electric

field

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BASIS OF SEPARATION

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INSTRUMENTAL DIAGRAM

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IonizerSample introduction/ionization method:

Ionizationmethod

Typical Analytes

Sample Introduction

Mass Range

Method Highlights

Electron Impact (EI)

Relatively small volatile

GC or liquid/solid probe

to1,000

Daltons

Hard method versatile provides structure

info

Chemical Ionization (CI)

Relatively smallvolatile

GC orliquid/solid

probe

to1,000

Daltons

Soft method molecular ion peak

[M+H]+

Electrospray (ESI)PeptidesProteins

non-volatile

LiquidChromatography 

or syringe

to200,000  Daltons

Soft method ions often multiply charged

Fast Atom Bombardment (FAB)

Carbohydrates Organometallics

Peptides nonvolatile

Sample mixed in viscous

matrix

to6,000

Daltons

Soft method but harder than ESI or

MALDI

Matrix Assisted Laser Desorption(MALDI)

Peptides  Proteins 

Nucleotides

Sample mixed  in solid  matrix

to  500,000  Daltons

Soft method  very high 

mass

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Ion Analyzer

Analyzer System Highlights

QuadrupoleUnit mass resolution, fast scan, low cost

Sector (Magnetic and/or Electrostatic) High resolution, exact mass

Time-of-Flight (TOF)Theoretically, no limitation for m/z maximum, high throughput

Ion Cyclotron Resonance (ICR)Very high resolution, exact mass, perform ion chemistry

Tandem Mass Spectrometry (MS/MS) Molecular structure determination

Detector, Vacuum

DetectorConvert the beam of ions in an electrical signal that can be processed, stored, displayed and recorded in many ways.

VacuumSystem

MS require the high vacuum is maintained in all spectrometer components (except signal processing)

Electron Multiplier (most commonly used)

Faraday cupPhotographic platesScintillation type

Other:

Mass spectrometers analyze gas-phase ions, not

neutral moleculesNeutrals don’t respond to electric and

magnetic fields If a molecule cannot ionize, MS cannot help

MS is not a “magic bullet” techniqueMS can describe atomic composition of an ionConnectivity of the atoms is much more

challenging

Although MS requires a vacuum, it cannot be performed in a vacuum of informationDeriving useful information from MS data

often requires some knowledge of the system under investigation

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IMPORTANT POINTS TO REMEMBER

Francis William Aston

"For his discovery, by means of his mass spectrograph, of isotopes, in a large number of non-radioactive elements, and for his

enunciation of the whole-number rule04/12/2023 11

1922 Nobel Prize

Isotopes can be classified asMono-isotopeDi-isotopePoly-isotope

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Isotopic Classification of the Elements

Monoisotopic Elements.

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Among 82 naturally occurring stable elements, 20 elements do exist in the form of only one single naturally occurring stable isotope.

Among the monoisotopic elements,Fluorine (19F), Sodium (23Na), Phosphorus (31P), and Iodine (127I)belong to the more prominent examples in organic mass spectrometry.

Methyl Floride

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Nonetheless,there are many more such as9Be, 27Al, 55Mn, 59Co, 75As, 93Nb, 103Rh, 133Cs, and 197Au

The monoisotopic elements are also referred to as

M, A or X elements.If radioactive isotopes were also taken into account, not a single monoisotopic element

would remain.

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Di-isotopic Elements

Several elements exist naturally in two isotopes.These elements can even be sub-classified into those havingONE isotope that is 1 u heavier than the most abundant isotope. The first group has been termed M+1 elements.

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Prominent examples of M+1 elements areHydrogen (1H, 2H = D),Carbon (12C, 13C),andNitrogen (14N, 15N).

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Those having one isotope that is 2 u heavier than the most abundant isotope

They have been termed M+2 elements, respectively

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Elements such as chlorine, bromine, oxygen, sulfur, and silicon, can be dealt with as X+2 elements

Silicon

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Among the X+2 elements,Chlorine (35Cl, 37Cl),Bromine (79Br, 81Br) are relatively common but Copper (63Cu, 65Cu),Gallium (69Ga, 71Ga),Silver (107Ag, 109Ag),Indium (113In, 115In),and antimony (121Sb, 123Sb) also belong to this group.

84-49=35 49-14=35

A+2 peak with fragment of 35 indicates Cl presence.

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Chlorine

35Cl (P= .75)37Cl (P= .25) Ratio 3:1

Probability : [M+] / [M + 2] = 0.75 / 0.25 = 3 / 1

100 : 33

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Methyl Bromide:  An example of A+2 isotopes

The ratio of peaks containing 79Br and its isotope 81Br (100/98) confirms the presence of bromine in the compound.

A-1 Peak

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If we do not restrict our view to the elements, one should add the class of A–1 elements with one minor isotope of 1 u lower mass than the most abundant one

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The elementsLithium (6Li, 7Li),Boron (10B, 11B),and Vanadium (50V, 51V)come along with a lighter isotope of lower abundance than the heavier one and thus, they can be grouped together as X–1 elements.

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BORON

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Poly-isotopic Elements

The majority of elements are grouped as poly-isotopic elements because they consist

of three or more isotopes showing a wide variety of isotopic distributions.

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Tungsten

Index MS-fragmentation04/12/2023 35

Isotopic abundances are listed either as their sum being 100% or with the abundance of the most abundant isotope normalized to 100%. The custom of reporting mass spectra normalized to the base peak.

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Representation of Isotopic Abundance

Isotopic abundances for Carbon containing compounds

Relative ratio: [M+1]+ / [M+ ] = n(0.989)n-1 (0.011) / (0.989)n

= n (0.011) / (0.989)

= n (0.0111)

In percentage: n x 1.1 %

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Isotopic abundances for other common nuclei

For 15N: [M+1]+ / [M]+ => n x 0.36%

For 33S: [M+1]+ / [M]+ => n x 0.80%

For 18O: [M+2]+ / [M]+ => n x 0.20%

For 34S: [M+2]+ / [M]+ => n x 4.42%

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Have seen that for Cl and Br, having two common

isotopes, two radical cation peaks produced. What about other elements having more than one isotope?

We know what the isotopes are and their natural occurrence.

For the M+1 peak, one atom must be using an isotope heavier by one.

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Molecular Peaks, M+1

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We will use isotopic occurrence data for H, C, O for the M + 1 peak.

Technique to obtain molecular formula using intensities of M, M+1, M+2 peaks.

Consider the M+1 peak, nominal mass + 1. If we know the formula we should be able to calculate the relative intensity of that peak due to the contributions from each of the atoms present. Here are the major contributors to M+1.

Example. Given the data.

Peak Intensity

150 (M) 100

151 (M+1)

10.2

152 (M+2)

0.88Looking at M+2 there is no Br, Cl or S. There could be oxygen.Even mass for M means there could only be even number of Nitrogen

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Technique to obtain molecular formula using intensities of M, M+1, M+2 peaks.

Example. Given the data.

Peak Intensity

150 (M) 100

151 (M+1)

10.2

152 (M+2)

0.88EquationsM+1: (1.11% x # of C) + (0.38 x # of N+ small contributions from OM+2: (0.20 x # of O) + (1.1 x # of C)2/200

We can have 0 or 2 nitrogens. Even number.

We can have 0,1,2,3,4 oxygens. 0.88/0.2 < 5Can have 0,1,2,3,4,5,6,7,8,9 carbons. 10.2/1.11 <10

Find molecular formulas having reasonable M+1 peaks

M+1 M+2

C7H10N4 9.25 0.38

C8H10N2O 9.61 0.61

C9H10O2 9.96 0.84

C9H14N2 10.71

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Examine reasonable formulae. Calculate M+1, M+2 peaks

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Example. Identify this molecule

m/e Abundance

1 <0.1

16 1.0

17 21

18 100

19 0.15

20 0.22Due to heavier isotopes

Molecular radical ionEjection of an H

H2O

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Example 2

m/e Abundance

12 3.3

13 4.3

14 4.4

15 0.07

16 1.7

28 31

29303132

100891.30.21

Heavier isotopes

parent

H ejection

Oxygen

carbon

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The “exact mass” feature in ChemDraw will give

you a monoisotopic mass Not always correct for complex isotope patterns

Two freeware apps are available from MSF website “Links” page These can be used to predict the entire isotopic pattern

as an exportable image

MS-Search program on GC-MS computer can be used to retrieve mass spectra from NIST’02 library

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Some useful software tools

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Mass spectrometry is very useful technique for identification of ISOTOPES, through different ionizaion sources for different molecules

It is observed through study that isotope of some elements are 1unit heavier while others are 2unit heavier than most abundant isotope hence shown as M+1 and M+2 peaks respectively in Mass Spectra.

Isotopes 1unit lighter observed as M-1 peak in Mass Spectra. Observation of poly-isotopes as M+3 and so on.

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REFERENCES

1. MASS SPECTROMETRY BY JURGEN H GROSS, SPRINGER INTERNATIONAL EDITION

2. NIST WEB BOOK http://webbook.nist.gov3. LECTURE SLIDES OF DR SHAHABUDDIN MEMON4. WIKIPEDIA5. SPECTROMETRIC IDENTIFICATION OF OGANIC COMPOUNDS

BY ROBERT M.SILVERSTEIN 7th EDITION 20056. http//www.chemistry.ccsu/glagovich/teaching/316/ms/7. GOOGLE IMAGES FORM www.google.com

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