MASS SPECTROMETRY

MASS SPECTROMETRY: 

MASS SPECTROMETRY PRESENTED BY, S.SUNDARARAMAN. FIRST M.PHARM (DEPARTMENT OF PHARMACEUTICAL ANALYSIS) S.B .COLLEGE OF PHARMACY 1

MASS SPECTROMETRY: 

MASS SPECTROMETRY INTRODUCTION PRINCIPLE INSTRUMENTATION TYPES OF MASS SPECROMETRY TYPES OF PEAK APPLICATION S.B .COLLEGE OF PHARMACY 2

INTRODUCTION: 

INTRODUCTION Mass spectrometry, one of most generally applicable tools, provides both qualitative and quantitative information about the atomic and molecular composition of inorganic and organic materials. The first mass spectrometer dates back to the work in England of J.J.Thompson in1912 and of F.W. Aston in 1919. S.B .COLLEGE OF PHARMACY 3

MS HISTORY: 

MS HISTORY JJ Thomson built MS prototype to measure m/z of electron, awarded Nobel Prize in 1906 MS concept first put into practice by Francis Aston, a physicist working in Cambridge England in 1919 Designed to measure mass of elements Aston Awarded Nobel Prize in 1922 S.B .COLLEGE OF PHARMACY 4

MS HISTORY: 

MS HISTORY 1948-52 - Time of Flight (TOF) mass analyzers introduced 1955 - Quadrupole ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins 1989 Nobel Prize) 1968 - Tandem mass spectrometer appears Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY S.B .COLLEGE OF PHARMACY 5

MASS SPECTROMETRY: 

MASS SPECTROMETRY Analytical method to measure the molecular or atomic weight of samples S.B .COLLEGE OF PHARMACY 6

PRINCIPLE: 

PRINCIPLE Organic molecules are bombarded with electrons and are converted to highly energetic positively charged ions ( molecular ions or parent ions ). Which can break up into smaller ions ( fragment ions or daughter ions ). This loss of electron from a molecule leads to a radical cation. m 2 M M + . S.B .COLLEGE OF PHARMACY 7

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S.B .COLLEGE OF PHARMACY 8 This molecular ion commonly M + . decomposes to a pair of fragments which may be either radical + anion or a small molecule + radical cat ion. molecular ions, the fragment ions and the fragment radical ions are separated by the deflection in a variable magnetic field according to their mass and charge and generate current (the ion current) at the collector in proportion to their relative abundances.

MASS SPECTROMETRY: 

MASS SPECTROMETRY Molecular weight can be obtained from a very small sample. 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. S.B .COLLEGE OF PHARMACY 9

MASS SPECTROMETRY : 

MASS SPECTROMETRY . DETERMINE THE MOLECULAR MASS OF ORGANIC COMPOUNDS DETERMINE THE MOLECULAR FORMULA OF ORGANIC COMPOUNDS S.B .COLLEGE OF PHARMACY 10 THE MAIN USE OF MS IN ORG CHEM IS:

How do we achieve this?: 

How do we achieve this? PERSUADE THE MOLE CULE TO ENTER THE VAPOR PHASE (CAN BE DIFFICULT) PRODUCE IONS FROM THE MOLECULES THAT ENTER THE GAS PHASE. SEPARATE THE IONS ACCORDING TO THEIR MASS-TO-CHARGE RATIOS(m/z). S.B .COLLEGE OF PHARMACY 11 MEASURE AND RECORD THESE IONS

MASS SPECROMETY NEEDS: 

MASS SPECROMETY NEEDS IONIZATION : how a sample is injected into MS machine SEPARATION : mass and charge is determined. ACTIVATION : sample is broken into small fragments. MASS DETERMINATION : m/z for the ionized sample is determined. S.B .COLLEGE OF PHARMACY 12

Mass Spec Principles: 

Mass Spec Principles S.B .COLLEGE OF PHARMACY 13 Ionizer Sample + _ Mass Analyzer Detector

HOW DOES A MASS SPECTROMETER WORKS?: 

HOW DOES A MASS SPECTROMETER WORKS? Ionization method MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF MW Triple Quadrapole AA seq MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif. S.B .COLLEGE OF PHARMACY 14 Create ions Separate ions Detect ions Mass spectrum Database analysis

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INSTRUMENTATION: 

INSTRUMENTATION SAMPLE INLET SYSTEM HIGH VACCUN SYSTEM. ION SOURCE MASS ANALYSERS DETECTORS IN MS DATA HANDLING SYSTEM S.B .COLLEGE OF PHARMACY 17

instrumentation: 

instrumentation S.B .COLLEGE OF PHARMACY 18

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MASS SPECROMETER SCHEMATIC: 

MASS SPECROMETER SCHEMATIC S.B .COLLEGE OF PHARMACY 20 Inlet Ion Source Mass Filter Detector Data System High Vacuum System Turbo pumps Diffusion pumps Rough pumps Rotary pumps Sample Plate Target HPLC GC Solids probe MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. Hybrid Detec. PC’s UNIX Mac

Inlet Systems: 

Inlet Systems Purpose is to introduce a very small amount of sample into the mass spectrometer so that its components may be converted into gas ions Sample may be gas,liquid and solids. S.B .COLLEGE OF PHARMACY 21 Want minimal loss of vacuum

Direct Probe Inlet: 

Direct Probe Inlet Good for solid and non-volatile liquids Sample is on surface, probe positioned near ionization source and slit leading to spectrometer Good for low concentrations or thermally unstable compounds S.B .COLLEGE OF PHARMACY 22

Different Ionization Methods: 

Different Ionization Methods 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

Mass analysers: 

Mass analysers The function of mass analysers is to separate the ions produced in the ion source according to their difference in m/z ratio After entering the ion source,the ions are accelerated by a system of electrostatic slits and then they enter the particular analysers S.B .COLLEGE OF PHARMACY 24

TYPES OF MASS ANALYSERS: 

TYPES OF MASS ANALYSERS Magnetic Sector Analyzer ( MSA ) High resolution, exact mass, original MA Quadrupole Analyzer ( Q ) Low (1 amu) resolution, fast, cheap Time-of-Flight Analyzer ( TOF ) No upper m/z limit, high throughput Ion Trap Mass Analyzer ( QSTAR ) Good resolution, all-in-one mass analyzer Ion Cyclotron Resonance ( FT-ICR ) Highest resolution, exact mass, costly S.B .COLLEGE OF PHARMACY 25

MAGNETIC SECTOR ANALYSERS: 

MAGNETIC SECTOR ANALYSERS S.B .COLLEGE OF PHARMACY 26

MAGNETIC SECTOR: Single focusing: 

MAGNETIC SECTOR: Single focusing 1.Magnetic Sector: Single Focusing : A magnetic field is used to focus ions based on their momentum as they are ejected from an ion source at high energy. Resolution <2000 S.B .COLLEGE OF PHARMACY 27 Focuses based on momentum

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2.Magnetic sector double focusing : An electrostatic analyzer is used serially with a magnet to select monoenergetic ions. High resolution >10,000 mass up to 100,000Da S.B .COLLEGE OF PHARMACY 28

Magnetic sector mass analyser: 

Magnetic sector mass analyser The ions move at different speed, but have the same kinetic energy (ideally The paths of the ions is curved in the sector where the magnetic field is present The selection of ions is achieved by the fact that only ions traveling on a path with a narrowly defined radius will go through the exit slit to the detector. S.B .COLLEGE OF PHARMACY 29

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S.B .COLLEGE OF PHARMACY 30 To consecutively detect ions with different m/z, we can: fixed magnetic field, kinetic energy sweep larger B : larger m/z fixed kinetic energy, magnetic field sweep larger V : smaller Ions that exit the analyzer through the slit will become neutralized due to collisions with the walls of the analyzer and pumped away by the vacuum pump.

QUADRUPOLE MASS ANALYSERS: 

Q UADRUPOLE M ASS A NALYSERS 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 S.B .COLLEGE OF PHARMACY 31

Quadrupole Mass Analyzer: 

Quadrupole Mass Analyzer S.B .COLLEGE OF PHARMACY 32

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Time of flight analysers: 

Time of flight analysers The Time of flight (TOF) method of measuring partic le mass-to-charge ratio is done as follows. An ion of known electrical charge and unknown mass enters a mass spectrometer and is accelerated by an electrical field of known strength. This acceleration results in any given ion having the same kinetic energy as any other ion given that they all have the same charge. The velocity of the ion will depend however on the mass-to-charge ratio . The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will depend on the mass-to-charge ratio of the particle (heavier particles reach lower speeds). From this time and the known experimental parameters one can find the mass-to-charge ratio of the particle. This method of analysis is a powerful tool for finding the mass-to-charge ratio of charged particles, atoms and molecules. S.B .COLLEGE OF PHARMACY 35

Time of flight analyser: 

Time of flight analyser t = time of flight m/z = mass to charge ratio k = constant depending on instrumental settings and characteristics. S.B .COLLEGE OF PHARMACY 36

TIME OF FLIGHT: 

TIME OF FLIGHT S.B .COLLEGE OF PHARMACY 37

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ION TRAP MASS ANALYSERS: 

ION TRAP MASS ANALYSERS 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 S.B .COLLEGE OF PHARMACY 39

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FT-ICR (Fourier Transform Ion Cyclotron Resonance): 

FT-ICR (Fourier Transform Ion Cyclotron Resonance) Advantages of FT: Improved S/N Greater speed per spectrum Higher sensitivity Greater resolution S.B .COLLEGE OF PHARMACY 41

FT-ICR: 

FT-ICR S.B .COLLEGE OF PHARMACY 42

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ANALYSERS AND THEIR SEPARATIONS: 

ANALYSERS AND THEIR SEPARATIONS TYPES OF ANALYSERS ELECTRIC SECTOR MAGNETIC SECTOR QUADRAPOLE,ION TRAP TOF FT ION CYCLOTRON RESONANCE BASIS OF SEPARATION KINETIC ENERGY/Z MOMENTUM/Z m/z FLIGHT TIME M/Z (resonance frequencies) S.B .COLLEGE OF PHARMACY 44

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S.B .COLLEGE OF PHARMACY 45 • High voltage (keV energy range) – magnet (B) – electrostatic (E) – time-of-flight (TOF) • Low voltage (eV energy range) – quadrupole (Q) – ion trap (IT, LT) – ion cyclotron resonance (ICR) Analyzers

Comparison of Quads and Traps : 

Comparison of Quads and Traps Ion traps Mass Separation in time High sensitivity Full Scan Lower sensitivity SRM Offer multiple stages of MS Offer multiple stages of MSn Quads Mass Separation in space Lower sensitivity Full Scan High sensitivity SIM and SRM No mutiple stages of MS Lower resolution S.B .COLLEGE OF PHARMACY 46

DETECTORS USED IN MS: 

DETECTORS USED IN MS Resolved ion beams ,after passage through a mass analysers,sequentially strikes a detector Several types of detectors are available. They are, Faraday cup collector Electron multiplier tubes Channel electron multiplier arrays S.B .COLLEGE OF PHARMACY 47

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S.B .COLLEGE OF PHARMACY 48 FARADAY CUP COLLECTOR: It is simple and effective means of monitoring ion current in the focal plane of the ms. It consists of cup with suitable suppressor electrodes

Electron multiplier tubes: 

Electron multiplier tubes When the ion beams strike the conversion dynodes, metal plates convert impinging ions to electrons. The current generated are further multiplied, the secondary electrons ,emitted by the dynodes are constrained in mag.field to follow circular paths Need to be very sensitive Typical gains are 10^5 up to 10^8! Provides high current with nanosecond response time S.B .COLLEGE OF PHARMACY 49

Mass Detectors: 

Mass Detectors S.B .COLLEGE OF PHARMACY 50 Electron Multiplier (Dynode)

CHANNEL ELECTRON MULTIPLIER ARRAY: 

CHANNEL ELECTRON MULTIPLIER ARRAY It composed of a regular close packed array of channels in a plate of semi conducting material Inside each pore or channel is coated with secondary electron emissive material . Thus each channel constitutes an multiplier individually. S.B .COLLEGE OF PHARMACY 51

DATA HANDLING: 

DATA HANDLING On modern mass spectrometry,the data are digitized and collected on magnetic tape or stored in the memory of computer for subsequent processing. Scanning and peak switching . S.B .COLLEGE OF PHARMACY 52

MASCOT: 

MASCOT

PEAKS IN MS: 

PEAKS IN MS Peak with max intensity and high m/z value are important. The peak with maximum intensity iS called “base peak”. It is most stable fragment. The peak with lower m/z value is called molecular ion peak.It gives direct evidence on mol.wt. S.B .COLLEGE OF PHARMACY 54

Typical Mass Spectrum: 

Typical Mass Spectrum aspirin Relative Abundance 120 m/z-for singly charged ion this is the mass

Different Types of MS: 

Different Types of MS GC-MS - Gas Chromatography MS separates volatile compounds in gas column and ID’s by mass LC-MS - Liquid Chromatography MS separates delicate compounds in HPLC column and ID’s by mass MS-MS - Tandem Mass Spectrometry separates compound fragments by magnetic field and ID’s by mass LC/LC-MS/MS-Tandem LC and Tandem MS Separates by HPLC, ID’s by mass and AA sequence S.B .COLLEGE OF PHARMACY 56

Different Types of MS: 

Different Types of MS ESI-QTOF Electrospray ionization source + quadrupole mass filter + time-of-flight mass analyzer MALDI-QTOF Matrix-assisted laser desorption ionization + quadrupole + time-of-flight mass analyzer Both separate by MW and AA seq S.B .COLLEGE OF PHARMACY 57

APPLICATIONS: 

APPLICATIONS Structure elucidation : using nitrogenrule , peak matching, fragmentation patterns of compounds and %abundance of isotopes,stucture elucidation of compounds can be done. Detection of impurities: impurities present in the sample can be dedected by the additional peaks,highest value of mass peaks than the compound it self and from the fragmentation pattern. S.B .COLLEGE OF PHARMACY 58

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S.B .COLLEGE OF PHARMACY 59 Quantitative analysis: the intensity of the peaks corresponds to the proportion of fragments.by comparing with the standard drug, the quantity of sample can be estimated. Drug metabolism studies: by recording the mass spectrum of the metabolite and that of the pure drug can be known. Clinical, toxicological and forensic applications: very minute quantities of the drug can be used to find out the mass and hence the substance can be identified from the mass spectral pattern. GC MS is used in volatile oil identification and separation.

REFERENCES: 

REFERENCES 1. organic spectroscopy by william Kemp,Third edition page no 285-290 2.spectrometric identification of organic compounds by silverstein. Sixth edition page no-2-5 3.Instrumental methods of analysis by willard,seventh edition page no-466 S.B .COLLEGE OF PHARMACY 60

THANK YOU: 

THANK YOU S.B .COLLEGE OF PHARMACY 61