INTRODUCTION TO MASS SPECTROMETRY BY NISHANTH

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An Introduction To Mass Spectrometry PRESENTED BY P.NISHANTH OSMANIA UNIVERSITY 1

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CONTENTS : Introduction of mass spectrometry Principle Instrumentation Interpretation of mass spectrum Types of ions Isotopic abundance 2 Applications

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What is mass spectrometry ??? Mass spectrometry is a powerful technique for chemical analysis that is used to identify unknown compounds, to quantify known compounds, and to elucidate molecular structure It does not involve the absorption or emission of light. A beam of high-energy electrons breaks the molecule apart. The masses of the fragments and their relative abundance reveal information about the structure of the molecule. Molecular weight can be obtained from a very small sample. INTRODUCTION 3

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MASS SPECTROMETRY PRINCIPLE Vaporization Ionization Fragmentation Separation Detection 4

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INSTRUMENTATION Inlet Ion Source Mass Analyzer Detector Data System High Vacuum System EI CI MALDI ESI FAB SIMS TOF Quadrupole Ion Trap Magnetic Sector FTMS 5

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Pictorial diagram of mass spectrometry 6

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ION SOURCES : 1.HARD SOURCES: Electron Impact (EI) Chemical Ionization (CI) 2.SOFT SOURCES Field Ionization Field Desorption Method Matrix Assisted Laser Desorption-ionization (MALDI) Electron Spray Ionization (ESI) Fast Atom Bombardment (FAB) Secondary Ion Mass Spectrometry (SIMS) 7

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1. Electron Impact (EI) Beam of energetic electrons Tungsten or rhenium filaments : 70V Electrostatic repulsion 8

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ELECTRON IMPACT RADICAL CATION ONLY CATIONS ARE CARRIED TO DETECTOR CATION RADICAL BOND-BREAKING 9

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Limitations of EI : 1.THERMAL DEGRADATION 2. NON VOLATILE COMPD 3. EXTENSIVE FRAGMENTATION Overcoming problems : Derivatize sample Volatile and thermally stabile Other ionisation techniques lower ionisation energies Other means introducing sample 10

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2. CHEMICAL IONIZATION : Ionization- by collision with reagent ions . Conc. ratio of reagent to sample = 10 3 to 10 4 : 1 bath gas (CH 3 /NH 4 /CH 3 (CH 2 ) 2 CH 3 ) to protonate sample often forms MH + Still only applicable to volatile or Thermally stable samples The reagent gas -strong enough Bronsted acid to transfer a proton to the analyte . LIMITATION : 11

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Comparison of EI and CI spectra Mass spectra of 1-decanol: 12

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Desorption sources : Non volatile thermally unstable samples Biochemical species (mol wt > 100,000Da) Energy in various forms is used – solid or liquid gaseous ions Spectra – simplified Field desorption MALDI ESI FAB SIMS 13

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1.Matrix Assisted Laser Desorption-ionization (MALDI) Laser beam Low conc. Of analyte- dispersed in matrix deposited on steel probe Matrix properties : Strongly absorb laser radiation Preferably dissolves in same solvent as the sample Typically, the matrices are acidic . EXAMPLES 1. NITROGEN (337nm) 2. EXCIMER (308nm) 3. CARBON DIOXIDE (10.6 μ m) XeBr,XeCl,etc Analyte : matrix = 1:10 3 t0 1: 10 5 14

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MALDI 15

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Matrix Application α-Cyano-4-hydroxycinnamic acid (CCA) peptides 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid) proteins 2,5 Dihydroxybenzoic acid (DHB) peptides, proteins, polymers, sugars 3-Hydroxypicolinic acid (HPA) oligonucleotides Dithranol (anthralin) polymers MALDI- ToF Mass Spectrometry - Matrices 16

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ADVANTAGES OF MALDI Easier to interpret spectra (less multiple charges) Quick and easy Higher mass detection Higher Throughput (>1000 samples per hour) 17

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2.Electrospray Ionization (ESI) Sample solution-stainless steel capillary needle (RATE : μ l/ml) Capillary needle – high voltage Rayleigh limit- charge density becomes greater Columbic explosion – surface tension no longer support the charge Very sensitive technique, requires less than a picomole of material Contd… 18

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Positive ion mode measures (M + H) + (add formic acid to solvent) Contd… Negative ion mode measures (M - H) - (add ammonia to solvent) If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNA Strongly affected by salts & detergents LIMITATION 19

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3. Fast atom bombardment 4.Secondary ion mass spectrometry. 1. a beam of rare gas neutrals (FAB) 1. a beam of rare ions (SIMS ) 2. Analyte + liquid matrix (glycerol) end of insertion probe 3. Monolayer( sample in matrix) Limitation : Matrix interference at low masses Slow n skillful 2. No matrix is used 3. Very sensitive- material analysis - Surface chemistry Limitation : Difficult to quantitative analysis Large samples are required Formation of ion clusters 20

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FAB-Mass Spectrometry 21

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Electron Impact (EI - Hard method) small molecules, 1-1000 Daltons, structure Fast Atom Bombardment (FAB – Semi-hard) peptides, sugars, up to 6000 Daltons Electrospray Ionization (ESI - Soft) peptides, proteins, up to 200,000 Daltons Matrix Assisted Laser Desorption ( MALDI -Soft) peptides, proteins, DNA, up to 500 kD Different Ionization Methods

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MASS ANALYZERS What is analyzer? Analyzer is the section of instrument that separates ions of different m/z Separation based on angular momentum Magnetic sector Time of flight Quadrupole Ion trap magnetic sector : Separate electromagnetically Internal pressure : 10 -7 torr 22

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Large (Heavy!!), Expensive to operate Comparatively slow scan rates High Skill level required to operate and maintain Limitations of magnetic sector : 23

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Quadrupole Mass Analyzer A quadrupole mass filter consists of four parallel metal rods with different charges Two opposite rods have an applied + potential and the other two rods have a - potential The applied voltages affect the trajectory of ions traveling down the flight path For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path Can scan through all masses or sit at one fixed mass . 24

QUADRUPOLE: 

QUADRUPOLE 25

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single mass transmission mode m2 m2 m2 m2 m3 m1 m4 m2 Quadrupole Have Variable Ion Transmission Modes

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mass scanning mode m1 m3 m4 m2 m3 m1 m4 m2 single mass transmission mode m2 m2 m2 m2 m3 m1 m4 m2 Quadrupole have variable ion transmission modes TIME OF FLIGHT Measures the time for ions to reach the detector. Small ions reach the detector before large ones . The drift region is field free. ADVANTAGES Typical flight times : 1-50 μ s Simplicity, ease of accessibility, virtually unlimited mass range. LIMITATIONS Limited resolution and sensitivity 26

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Ion traps are ion trapping devices that make use of a three-dimensional quadrupole field to trap and mass-analyze ions invented by Wolfgang Paul (Nobel Prize1989) Offer good mass resolving power Ion trap mass analyzer 27

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Detectors: ion beam intensity Faraday Cups Faraday cups are simply a metal receptacle that is connected to an extremely sensitive current meter The positive charges associated with ions colliding with the “cup” must be balanced by a flow of electrons from “ground” through the electrometer and into the cup Measurement limits for electrometers are currently in the range of 10 -16 -10 -15 Amperes with some averaging. 28

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Vacuum system : Low pressure is maintained 10-2 to 10-5 Pa (10 -4 to 10 -7 torr) depending upon the geometry of the instrument First : Mechanical pumps (10 -3 torr) Second : Diffusion pumps 2 pumping stages - - reduce chance of collision of ions in analyzer

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Electrospray Mass Spectrometry (ESI-MS) MALDI- ToF Mass Spectrometry 30

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Masses are graphed or tabulated according to their relative abundance. Interpretation Of Mass Spectrum 31

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More stable carbocations will be more abundant Mass Spectrum of 2-methyl pentane 32

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Types of ions in mass spectrometry : Molecular ion Fragment ion Metastable ion Isotopic ions Rearrangement ions Multiple charged ion Negatively charged ion (M + ) STABILTY ORDER (M + ) : AR>-C=C-C=C->ALIC YC > -C0->-NH 2 >-C00>-CONH 2 >B. H.C>-OH weak diffused broad peaks different mass number aromatic compounds (0.1%) 33

Isotopic Abundance: 

Isotopic Abundance => 81 Br 34

Mass Spectrum with Sulfur: 

Mass Spectrum with Sulfur => 35

Mass Spectrum with Chlorine: 

Mass Spectrum with Chlorine => 36

Mass Spectrum with Bromine: 

Mass Spectrum with Bromine => 37

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Applications of mass spectrometry : To identify the pure compound – its mass, molecular formula , chemical structure . isotopic ratio Quantitative determination – area under the peaks ᾳ conc. of component MALDI -Protein Identifications Protein modification Bacteria identification Contd… 38

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Environmental analysis : - detection and quantitative determination of perfluororganics, pesticides, pharmaceuticals, algal toxins, arsenic, etc Forensic sciences - Detecting explosives, analyzing body fluids and hair, testing athletes for drugs ,etc Trace gas analysis Glycan analysis - used in glycobiology for characterization and elucidation of glycan structure Space exploration - Mass spectrometers are also widely used in space missions to measure the composition of plasmas. 39

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Analysis of Caffeine by Isotopic Dilution Using Mass Spectroscopy the concentration of an analyte is determined by spiking the analyte containing matrix with a known quantity of an isotopically labelled analogue of the analyte In this experiment caffeine is the analyte and tri- deuterated caffeine molecule would be the analogue mixed with it in the isotope dilution analysis protocol. In the SIM mode a small number of distinct ions are monitored many times per second by the detector. + CD 3 I + HI caffeine-d3 theophylline

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Synthesis of caffeine-d 3 2g theophylline Dimethyl formamide(50ml) 10M NaOH (1.1ml) 2.2 mL of iodomethane-d3 precipitate 3hrs stirring mixture overnight in the freezer

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Prepare three calibration standard solutions to determine R. Typically two stock solutions are made; 50mg of caffeine in 50.00mL methanol and 25.00 mg caffeine-d3 in25.00mL methanol. Make the three standards by mixing 500uL of caffeine stock with 500uL , 250uL and 125uL of caffeine-d3 stock in 1.00 mL volumetric flasks. R = area@194 / mass caffeine area@197 / mass caffeine-d 3 Standards Caffeine-d3 ( uL ) Caffeine ( uL ) Methanol ( uL ) area194 /area197 mass caffeine-d 3 / mass caffeine s1 100 100 -------- s2 50 100 50 s3 25 100 75 Examination of caffeine and caffeine-d3 show prominent peaks at 194 (197), 109 ( 112) and 82 (85) – deuterated compound peak in parenthesis. Analysis is based on the areas of the most prominent peak from each compound. Concentrations: caffeine stock 1mg/mL and caffeine-d3 stock 1mg/ml. Each standard and the test trials are run in SIM at m/z 194 and 197 separately

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test Caffeine-d3 ( uL ) unkown ( uL ) Methanol ( uL ) area194 /area197 mass caffeine-d 3 / mass caffeine t1 100 100 -------- t2 100 100 -------- t3 100 100 --------

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References : Principles Of Instrumental Analysis -SKOOG Spectroscopy - PAVIA Instrumental Method Of Chemical Analysis- CHATWAL An Introduction to Mass Spectrometry – SCOTT E. BRAMER Chemical Analysis By Mass Spectrometry - DR PHIL MORTIMER 40

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