Mass spectrometry(MS)

It is an analytical technique useful for the determination of molecular mass, molecular formula and fragmentation pattern of particular molecule and compounds. It has greater application in pharmaceutical and medicinal fields.

1. Mass Spectrometry PREPARED BY:-AJAY KUMAR

2. History of Mass Spectrometry 2 Year Scientist Discovery/Award 1886 E. Goldstein Discovers anode rays (positive gas ions) in gas discharge 1897 J.J. Thomson Discovers the electron and determines its m/z ratio. Nobel Prize in 1906 1898 W. Wien Analyzes the anode rays by magnetic deflection, and establishes that they carry a positive charge. Nobel Prize in 1911 1909 R.A. Millikan & H. Fletcher Determine the elementary unit of charge

3. Mass Spectrometry 3 Year Scientist Discovery/Award 1912 J.J. Thomson First Mass Spectrometer. In 1913 J. J. Thomson separated the isotopes 20 Ne and 22 Ne 1919 A.J. Dempster Electron ionization and magnetic sector MS 1942 Atlantic Refining Company First commercial use This technique resolves ionic species by their m/e ratio 1953 W. Paul and H.S. Steinwedel Quadrupole and the ion trap. Nobel Prize to Paul in 1989.

4. Mass Spectrometry 4 Year Scientist Discovery/Award 1956 First GC-MS 1968 First commercial quadrupole 1975 First commercial GC-MS 1990s Explosive growth in biological MS, due to ESI & MALDI 2002 Fenn & Tanaka Nobel Prize to Fenn & Tanaka for ESI & MALDI 2005 Commercialization of Orbitrap MS

5. Mass Spec - Introduction Very different from IR and NMR Absorption of electromagnetic energy Sample can be recovered and reused Mass spectrometry Records what happens when an organic molecule is hit by a beam of high-energy electrons Sample is completely destroyed

6. Mass Spec - Introduction What does a mass spectrum tell us? Molecular weight Molecular formula Either directly or in conjunction with other kinds of spectra such as IR or NMR Fragmentation pattern Key pieces of what the molecule looks like (such as methyl, ethyl, phenyl, or benzyl groups

7. Ms spectrometry gives composition of sample. Structure of inorganic, organic & biological sample Qualitative & quantitative composition of solid surfaces Isotopic ratios of atoms in samples Atomic or Molecular weight expressed in terms of atomic mass unit (amu) or daltons (Da). Introduction to Mass Spectrometry

8. The amu is based upon the relative scale in which the reference is carbon isotope C-12. Thus amu is defined as 1/12 the mass of the one neutral C-12 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. 8

9. Separation of Ions Only the cations are deflected by the magnetic field. Amount of deflection depends on m/z . The detector signal is proportional to the number of ions hitting it. By varying the magnetic field, ions of all masses are collected and counted. 9

10. Atomic MS Acronym Atomic ion source Typical Ms analyzer Inductive coupled plasma ICPMS High temp. argon plasma Quadruple Direct current plasma DCPMS High temp. argon plasma Quadruple Microwave induced plasma MIPMS High temp. argon plasma Quadruple Spark source SSMS Radio frequency electric spark Double focusing Thermal ionization TIMS Electrically heated plasma Double focusing Glow discharge GDMS Glow discharge plasma Double focusing Laser microprobe LMMS Focused laser beam Time of flight Secondary ion SIMS Accelerated ion bombardment Double focusing

11. - Used quantitatively and qualitatively (identification) Useful for both organic and inorganic compounds Can measure ~ 75 elements Rapidly evolving technology Expensive and complex 11 General Characteristics and Features

12. 12 Mass Spectrometry… Sample is ionized (an electron is removed)  M . + Ionization frequently fragments molecules  bonds most likely to break are the weakest -> form cations & radicals Modern techniques can be used to study non-volatile molecules such as proteins and nucleotides

13. MS perform three functions: Creation of ions – the sample molecules are subjected to a high energy beam of electrons (70 eV ), converting some of them to ions Separation of ions – as they are accelerated in an electric field, the ions are separated according to mass-to-charge ratio (m/z) Detection of ions – as each separated population of ions is generated, the spectrometer needs to qualify and quantify them All type of MS need very high vacuum (~ 10 -6 torr ),

14. source analyzer ion detection data system Vacuum pumps Sample Introduction Ion Formation Ion Sorting Ion Detection Data Handling Data Output Mass spectrum Basic Components of a MS

15. Mass Spectrometer 15

16. Components of MS Sample Introduction System Volatilizes the sample and introduces it to the ionization chamber under high vacuum Ion Source Ionizes the sample (fragmentation may occur) and accelerates the particles into the mass analyzer Mass Analyzer (or Mass Separator) Separates ionized particles based on their mass-to-charge ratio (m/e - ) 16

17. Components of MS Detector - Ion Collector Monitors the number of ions reaching detector per unit time as a current flow Signal Processor Amplifies the current signal and converts it to a DC Voltage Vacuum Pump System A very high vacuum (10 -4 to 10 -7 torr) is required so that the generated ions are not deflected by collisions with internal gases 17

18. Mass Spectrometry The Mass Spectrometer Single Focusing Mass Spectrometer A small quantity of sample is injected and vaporized under high vacuum The sample is then bombarded with electrons having 70-80 eV of energy A valence electron is “ punched ” off of the molecule, and an ion is formed

19. Mass Spectrometry The Mass Spectrometer The Single Focusing Mass Spectrometer Ions (+) are accelerated using a (-) anode towards the focusing magnet At a given potential (1 – 10 kV) each ion will have a kinetic energy: ½ mv 2 = eV As the ions enter a magnetic field, their path is curved; the radius of the curvature is given by: r = mv eH If the two equations are combined to factor out velocity: m/e = H 2 r 2 2V m = mass of ion v = velocity V = potential difference e = charge on ion H = strength of magnetic field r = radius of ion path

20. Mass Spectrometry The Mass Spectrometer Single Focusing Mass Spectrometer At a given potential, only one mass would have the correct radius path to pass through the magnet towards the detector “ Incorrect ” mass particles would strike the magnet

21. Ion Sources Purpose: create gaseous ions out of the sample components Two types: Molecular sources gas phase desorption sources Elemental sources 21

22. Ion Sources MS (cont.) Type S.No Name and Acronym Ionizing Process Gas Phase 1 Electron Impact (EI) Exposure to electron stream 2 Chemical Ionization (CI) Reagent gaseous ions 3 Field Ionization (FI) High potential electrode Desorption 1 Field Desorption (FD) High potential electrode 2 Electrospray Ionization (ESI) High electric field 3 Matrix-assisted desorption ionization (MALDI) Laser beam 4 Plasma Desorption (PD) Fission fragments from 252 Cf 5 Fast Atom Bombardment (FAB) Energetic atomic beam 6 Secondary Ion Mass Spectrometry (SIMS) Energetic beam of ions 7 Thermospray Ionization (TS) High temperature 22

23. Electron Impact Ionization Ionization methods required for gaseous sample . This method is not useful for non volatile or thermally unstable molecule. In desorption technique sample directly converted in to gaseous ions. We hit an organic molecule with a beam of electrons (usually 70-75 eV ) M + e –  M + + e – + e – ionization M +  A + + B fragmentation That removes an electron from the molecule resulting in the molecular ion (a radical cation ) The molecular ion then fragments in smaller radicals and cations The cations are detected by the MS instrumentation

24. Electron Impact Ion Source 24

25. Chemical Ionization 25 Gaseous sample atoms are ionized by collision with positively charged “reagent” gases (e.g. CH 4 + ). The reagent ions are produced by electron bombardment A (g) + CH 4 + (g) -------> A + (g) + CH 4 (g)

26. Chemical Ionization (CI) A modified form of EI Higher gas pressure in ioniation cavity ( 1 torr ) Reagent gas (1000 to 10000-fold excess) added; usual choice is methane, CH 4 A “soft ionization” technique Reagent gases are ionized methanol, methane, ammonia, others Sample molecules collide with the ionized reagent gas usually results in a proton transfer from the reagent gas to the sample compound so M+1 ions are common

27. Chemical Ionization Reactions Reagent gas ionization: CH 4  CH 4 + +e – (also CH 3 + , CH 2 + ) Secondary reactions: CH 4 + + CH 4  CH 5 + + CH 3 CH 3 + + CH 4  H 2 + C 2 H 5 + (M+29) Tertiary reactions CH 5 + + MH  CH 4 + MH 2 + (M+1) proton exchange CH 3 + + MH  CH 4 + M + (M–1) hydride exchange CH 4 + + MH  CH 4 + MH + (M) charge exchange 27

28. Comparison of EI and CI 28

29. Field Ionization and Desorption Intense electric field (10 7 -10 8 V/cm) Electrons “tunnel” into pointed electrode, yielding positive ions with little excess energy Very gentle; little fragmentation In Field Desorption , anode coated with analyte Not as efficient as EI sources by an order of magnitude Waller 1972, Mc Fadden 1973, Beckey 1969 29

30. Electrospray Ionization Source 30 How it Works Small, electrically charged droplets are formed from a solution flowing out of a hollow needle into a chamber under low vacuum The charged droplets are attracted to an electrode across an open volume by the application of an electrical field in the open chamber

31. Electrospray Ionization Source Some of the solvent is evaporated (and concentration occurs) during transit across the chamber As the droplets shrink, ions are forced closer together. At some point the repulsive forces between the ions is greater than surface tension and small droplets break off the larger drops. This process continues several times as the droplets transit across the chamber Eventually the solvent disappears and ions are generated, a process called ion evaporation& analysed by quadrupole Mass analyser 31

32. Matrix-Assisted Laser Desorption/Ionization (MALDI) Analyte mixed with radiation-absorbing material such as Nicotinic acid, Benzoic acid deriv., Pyrazine –carboxylic acid, cinnamic acid deriv., Nitrobenzyl alcohol The resulting solution was evaporated on the metallic probe surface and dried Sample mixture was exposed to pulsed laser beam, which result in the sublimation of analyte ion and were drawn into time-of-flight (TOF) analyser for analysis Excellent for larger molecules, e.g. peptides, polymers 32

33. Inductively Coupled Plasma (ion source) Plasma An electrically conducting gaseous mixture containing cations and anions ∑ cation(s) charge = ∑ electron charge Argon Plasma Ar is the principal conducting species Temperatures of 10,000 K possible Powered by radiofrequency energy (2 kW @ 27 Mhz) 33

34. Inductively Coupled Plasma (ion source) An ICP “torch” consists of: Three concentric quartz tubes through which a stream of argon flows at a rate of 5-20 L/min The three concentric rings are constructed to eliminate atmospheric gases from contacting the sample stream in the inner-most ring 34

35. Inductively Coupled Plasma (ion source) An argon plasma is generated by a water-cooled induction coil through which a radio-frequency energy (0.5 to 2 kW of power at 27-41 MHz) is transmitted Ionization of the flowing argon must be “initiated” by a Tesla coil 35 Radiofrequency Induction Coil Argon Plasma

36. Inductively Coupled Plasma (ion source) Sample is introduced through the inner-most ring in the torch as a “mist” carried by the argon stream The “mist” is generated by a nebulizer 36 Sample Inlet Tube Cetac Ultrasonic Nebulizer

37. Inductively Coupled Plasma (ion source) Analytes are ionized in the argon plasma and the ionized gas (i.e. plasma) is positioned on the entrance to the mass spectrometer. The interface consists of a series of metal (Pt, or Ni) cones with a small hole permitting the ions to be drawn in by the large vacuum on the inside. Can measure 90% of the elements in the periodic table can be simultaneously measured 37 MS Interface

38. Fast Atom Bombardment Ion source for biological molecules Ar ions passed through low pressure Ar or Xe gas to produce beam of neutral ions Atom-sample collisions produce ions as large as 25,000 Daltons 38

39. Fast Atom Bombardment Ionization Source 39 Ar + * + Ar ----------------> Ar + + Ar * Production of “fast atoms” Charge transfer Accelerated argon ion from “ion gun” Ground state argon atom “slow ion” “fast atom” The atom gun The atom beam Metal sample holder The end of the probe arm used to insert the sample into the chamber (e) The sample in the low volatility solvent (f) The sample ion driven from the surface (g) Ion extraction plate-select positive ions for mass analysis (h) Ion lens system leading to mass analyser

40. FAB Characteristics Used with high molecular weight organic molecules The fast atom interacts with analyte on a “target” to produce ions by “sputtering” (i.e. transfer of energy from argon to analyte ) Analyte ions are accelerated into the MS by application of an electric field (ion extraction plate and lenses) 40

41. Thermal Ionization (Ion) Source 41 How it Works It employ two wire filaments (usually tungsten or rhenium) closely spaced (~ 1 mm) situated in a chamber under high vacuum The sample is coated (usually in a silica gel matrix with phosphoric acid coated on top) on one wire filament that is heated gently The second filament, the ionizing filament is heated to incandescenc e The analyte desorbs from the filament and become ionized by the second filament. Sample ions are accelerated into the MS by application of an electric field Characteristics Old ionization method (70+ years) Used primarily for very high precision isotopic ratio studies of the elements

42. Example Resolution Calculation What is the resolution required to separate particles having masses of 999 and 1001? 42 (1 part in 500) For Masses of 28.0061 (N 2 + ) and 27.9949 (CO + ) (1 part in 2500)

43. LC-MS Inlets Direct inlet Moving-belt inlet Thermo spray inlet 43

44. Sample Introduction -Direct MS Inlet Four Basic Types : Batch Inlet Sample is volatilized externally and allowed to “leak” into the ion source Good for gas and liquid samples with boiling points < 500 °C Major interface problem – carrier gas dilution 44 Purpose: Introduce the sample (as a gas) into the ion source under high vacuum- GC MS

45. Direct Probe Good for non-volatile liquids, thermally unstable compounds and solids Sample is held on a glass capillary tube, fine wire or small cup 45 A mixture of compounds is separated by gas chromatography, then identified by mass spectrometry (GC-MS Inlets )

46. Moving-Belt LC-MS Inlet 46

47. Thermospray LC-MS Inlet 47

48. Thermospray - Inductively Coupled Plasma (ICP) Operates somewhat like a nebulizer in an AAS Also ionizes the sample in argon stream (at very high temperatures, >6000 °C) Only a small amount of analyte is utilized (< 1%) 48

49. Mass Analyzer The function of the MS analyzer like monochromator in an optical spectrometer. Transducer converts the beam of ions to an electrical signal that can be then Processed, stored in memory. MS require an elaborate vacuum system to maintain a low pressure in all of the components except signal processor

50. Mass Analyzers Type Mass Range Resolution Sensitivity Advantage Disadvantage Magnetic Sector 1-15,000 m/z 0.0001 Low High resolution Expensive Quadrupole 1-5000 m/z Unit High Easy to use; inexpensive Low res; low mass Ion trap 1-5000 m/z Unit High Easy to use; inexpensive Low res; low mass Time of Flight Unlimited 0.0001 High High mass; simple design Fourier Transform Up to 70 kDa 0.0001 High Very high res and mass Very expensive Silverstein, et. al., Spectrometric Identification of Organic Compounds, 7 th Ed, p 13. Single Focus Double Focus

51. Mass Analyzers There are several methods for separating different masses Elemental analysis - Want to distinguish between individual mass units 51

52. Single Focus Determine m/e by varying H , r , or V R = 500-5000 52

53. Magnetic Sector Mass Spectrometer Carey, Chapter 13

54. Single Focusing Magnetic Sector Mass Analyzer 54 Masses are separated in a (single) magnetic field Ions are deflected 60-180° Varying the magnetic field separates the masses

55. Double Focus Ion source produces ions with a certain spread of Kinetic Energy (K.E.). Electrostatic field and exit slit select only ions with same K.E. Net effect is to increase R to 2500-150000 Can distinguish very similar ions, e.g., C 2 H 4 + (28.0313) and CO + (27.9949) 55

56. Double Focusing Magnetic Sector Mass Analyzer 56 A “double focusing” analyzer has two regions of mass separation Magnetic Field preceded by an electrostatic field The electrostatic field helps to isolate particles of a specific kinetic energy Ion sources which produce particles of variable kinetic energy have low resolution

57. Quadrupole Mass Analyzers 57 Mass separation is achieved using 4 electrically connected rods (two “+” and two “-”) Both DC and AC signals are passed through the rods to achieve separation Scans are achieved by varying the frequency of the (AC) radio-frequency or by varying potentials of the two sources while keeping their ratio and frequency constant

58. resonant ion nonresonant ion detector source focusing lens quadrupole rods Quadrupole Mass Analyzers Mass filters

59. Quadrupole Analyzer Ions forced to wiggle between four rods whose polarity is rapidly switched Small masses pass through at high frequency or low voltage; large masses at low frequency or high voltage Very compact, rapid (10 ms) R = 700-800 59

60. Merit and Demerit Classical mass spectra Good reproducibility Relatively small/ compact, Relatively low-cost systems Limited resolution Peak heights variable as a function of mass (mass discrimination). Peak height vs. mass response must be 'tuned'. Not well suited for pulsed ionization methods 60

61. Quadrupole Ion Trap Ions follow complex trajectories between two pairs of electrodes that switch polarity rapidly Ions can be ejected from trap by m/z value by varying the frequency of end cap electrodes 61

62. Time of Flight Mass Analyzer 62 Operation Characteristics Separates ions based on flight time in drift tube Positive ions are produced in pulses and accelerated in an electric field (at the same frequency) All particles have the same kinetic energy but the velacities vary with mass of the ions Lighter ions reach the detector first Typical flight times are 1-30 µsec

63. Time-of-Flight Analyzer Pulsed ion source Arrival of ions at detector is timed,typically 1- 30 m s 63

64. Time of Flight Mass Analyzer Separation Principle All particles have the same kinetic energy In terms of mass separation principles: V particle = Her/m Hold H,e, and r constant V particle = 1/m (constant) V particle is inversely proportional to mass 64

65. Inductively Coupled Plasma Mass Spectrometer 65

66. Detectors for MS Two Basic Types Electron Multipliers Faraday Cup Time of Flight (TOF) and Fourier Transform Ion-Cyclotron Resonance (FTICR) instruments can separate more than one m/e - ratio simultaneously Multiple detectors are required in this case 66

67. Discrete Dynode Electron Multiplier Operates somewhat like a PMT tube Each dynode is at successively higher potential Produces a cascade of electrons 67

68. Channel (or Continuous) Dynode Electron Multiplier A glass tube that is coated with lead or a conductive material A potential of ~ 2000 V is applied between the opening and the end of the tube The curved shape helps to reduce electrical noise by preventing positive ions returning upstream. 68

69. Dynode-Based Detectors A disadvantage of dynode-based detectors is that the number of secondary electrons released in a detector depends on the type of primary particle, its energy and its incident angle, Mass discrimination occurs when ions enter the detector with different velocities. 69

70. Electron Multiplier Detector Tilted so that ions do not pass through undetected 70

71. Faraday Cup 71 How it Works Ions exiting the analyzer strike the collector electrode The faraday cage prevents escape of reflected ions and ejected secondary electrons The inclined collector reflects ions away from the entrance The collector is connected to ground via a large resistor Positive ions are neutralized on the surface of the collector by a flow of electrons (from ground) through the resistor The potential energy difference across the resistor is amplified

72. Faraday Cup Characteristics Inexpensive Low sensitivity Slow response A metal or carbon cup Produces a few micro amps of current (that is then amplified) Current is directly proportional to number of ions entering Detector response is independent of ion Kinetic energy Mass Chemical nature Does not exhibit mass discrimination Used in isotope ratio MS 72

73. Application of MS Drug discovery, combinatorial chemistry, Drug testing/Pharmacokinetics Antiterror /Security (e.g. bomb molecule ‘sniffers’) Environmental Analysis (e.g. water quality testing) Quality Control (food, pharmaceuticals) Medical Testing (various blood illnesses and… cancer?) Validation of art/History/Anthropology etc. Validation during chemical synthesis Biochemical research (proteomics, interact… omics ) Tissue imaging (with MALDI) Analysis of Proteins, peptides, olegonucleotides Clinical testing etc 73 -

74. 74 Thank You