Mass spectrometry

chromatogarphy,mass spectrometry

1. IN 504 Analytical Instruments Module 5 1 Presented by; Anju Sunny CUSAT Reference Text: R S Khandpur “Handbook of Analytical Instrumentation”

2. Mass Spectrometer 2

3. Introduction Mass spectrometers is an analytical instrument which can be used for all kind of chemical analysis like; To measure the elemental composition of matter To analyze the structures of inorganic, organic and biological molecules To find the isotopic ratios of atoms in the sample For the detection of poisons in a sample

4. General Block Diagram – Mass Spectrometer

5. Analogy between Optical Spectrophotometer & Mass Spectrometer

6. Components of a Mass Spectrometer Ionisation Ion Detection Ion Separation Ion Source Mass Analyser Detector Electron Ionisation (EI) Chemical Ionisation (CI) Fast Atom Bombardment (FAB) Electrospray Ionisation (ESI) Matrix-Assisted Laser Desorption Ionisation (MALDI) Quadrupole Magnetic deflection type Electrostatic type Time-Of-Flight (TOF) Ion Trap Electron Multiplier Multichannel plate Faraday Cup

7. IONIZATION SOURCES 1) Electron Ionization (EI) - Commonly used for analysis of organic samples - Electrons are emitted from a heated tungsten filament cathode. - Electrons are accelerated towards the anode with a potential of about 50 – 100 V - Electrons meet at right angles with the sample molecules - Interaction with the high energy electrons causes ionization of sample molecules and fragmentation into smaller ions.

8. IONIZATION SOURCES 2) Chemical Ionization (CI) - A large excess of reagent gas (1000 – 10000 times) is introduced into the ionization region - Pressures in source are typically higher than EI - Electrons are allowed to bombard the gas-sample mixture Examples of reagent gas - Methane, ammonia, isobutane

9. IONIZATION SOURCES 3) Atmospheric Pressure Ionization (API) Sources Two major types Electrospray Ionization (ESI) - Atmospheric Pressure Chemical Ionization (APCI) - Operate at atmospheric pressure - Modified version of ESI is the Ion Spray Source - Used for mixtures of nonvolatile high molecular weight compounds

10. IONIZATION SOURCES 4) Desorption Ionization - For direct ionization of solids - Excellent tool for analysis of large molecules - Solid samples are placed on a support and then bombarded with ions or photons - Different types are available

11. IONIZATION SOURCES 4) Desorption Ionization a) Laser Desorption Ionization - Uses pulsed laser - Provides selective ionization by choosing appropriate λ - Laser is focused on a solid surface to ionize material Examples of Lasers - IR laser: CO 2 laser - UV laser: Nd:YAG (yttrium aluminum garnet)

12. IONIZATION SOURCES 4) Desorption Ionization b) Matrix-Assisted Laser Desorption Ionization (MALDI) - Matrix disperses large amounts of energy absorbed by the laser - Minimizes fragmentation of the molecule - Used for study of polymers, proteins, peptides

13. IONIZATION SOURCES 4) Desorption Ionization c) Fast Atom Bombardment (FAB) - Employs fast moving neutral inert gas atoms ( Ar ) to ionize large molecules - Sample is dissolved in glycerol and spread in a thin layer on a metal probe - Probe is then inserted into the mass spectrometer and a beam of fast moving atoms probe the surface

14. Types of Mass Spectrometers Various types of Spectrometers are available in the means of separating the ions according to their mass-to-charge ratio or simply, on the basis of Mass Analyzers used . They are; 1) Magnetic Deflection Mass Spectrometer 2) Electrostatic Mass Spectrometer 3) Time-of-Flight Mass Spectrometer 4) Quadrupole Mass Spectrometer

15. 1) Magnetic Deflection Mass Spectrometer

16. Magnetic Deflection Mass Spectrometer In a mass spectrometer, the sample to be analyzed is first bombarded with an electron beam to produce ionic fragments of the original molecule. These ions are accelerated in a high vacuum into a magnetic field , which deflects them into circular paths. Since the deflection for light ions is greater than that for heavy ions, the ion stream separates into beams of different molecular weight. A suitably placed slit allows a beam of selected molecular weight to pass through to a collection electrode or detector.

17. 2) Electrostatic Mass Spectrometer Electrostatic sector analyzer consists of 2 curved plates of equal and opposite potential. As the ion travels through the electric field, it is deflected and the force on the ion due to the electric field is equal to the centripetal force on the ion. Here the ions of the same kinetic energy are focused, and ions of different kinetic energies are dispersed. Electrostatic and magnetic sector analyzers when employed individually are single focusing instruments . When both techniques are used together, it is called a double focusing instrument , because in this instrument both the energies and the angular dispersions are focused.

18. 3)Time of Flight ( ToF ) Mass Spectrometer In ToF Mass Spectrometer, ions of different mass/charge ratio are separated by the difference in time they take to travel over an identical path from the ion source to the collector at the detector . Here ions generated by bombardment of the sample with a brief pulse of electrons, secondary ions or laser-generated photons. Ions accelerated by electric field and they enter into field-free drift tube. Ions enter tube with same kinetic energy and ion velocity vary inversely with mass. Lighter particles arrive at detector before heavier particles and ions are separated in the drift tube according to their velocities (v)

19. 3)Time of Flight ( ToF ) Mass Spectrometer

20. 3)Time of Flight ( ToF ) Mass Spectrometer  V = accelerating voltage  If L is the length of tube (typically 1-2 m) and t is the flight time of ion, then v = L/t  Implies mass-to-charge ratio and flight time can be found from

21. Advantages of ToF High speed operation Ability to record entire mass spectrum at one time Accuracy only depend on electronic circuits 21 Disad vantages of ToF Poor resolution due to display on an Oscilloscope screen

22. Applications of ToF Qualitative Analysis Molecular Weight Determination Structure Determination Quantitative Analysis Biotechnology Analysis of proteins & peptides Pharmaceutical drug discovery 22

23. 4) Quadrupole Mass Spectrometer 23

24. 4) Quadrupole Mass Spectrometer Four parallel cylindrical rods serve as electrodes. Opposite rods are connected electrically One pair attached to positive side of variable dc source One pair attached to negative side of variable dc source Variable radio-frequency ac potential (180 o out of phase) applied to each pair of rods. The result is an ac potential superimposed on a dc potential and this creates an electric field. Ions accelerated through the electric field between rods. ac and dc voltages increased simultaneously with ratio being constant All ions without specific m/e strike rods and become neutral Only ions having a limited range of m / e reach detector. 24

25. Features of Quadrupole Mass Spectrometer The quadrupole acts as a filter so is often called the mass filter. Sample must be ionized and it should be in gas phase. Has smaller range and lower resolution than magnetic sector but faster. More compact less expensive High scan rate 25

26. DETECTORS 1) Multichannel plate - Detector which measures one m/e value at a time called single channel detectors. - Multichannel plate detectors are used for the detection of multiple ion beams with different m/e value. - High resolution magnetic sector instruments use multichannel detectors (called multicollectors )

27. DETECTORS 2) Electron Multiplier (EM) - The most common detector in Mass spectrometer for ions. - Similar to PMT. - Very sensitive and has fast response.

28. DETECTORS 3) Faraday Cup - A metal or carbon cup serves to capture ions and store the charge. - Cup shape decreases loss of electrons. - Least expensive detector for ions. - Has long response time.

29. DETECTORS 4) Array Detectors - Used in ToF Mass spectometers . - Employs a focal plane camera (FPC) consisting of an array of 31 Faraday Cup - Up to 15 m/e values can be measured simultaneously. - Exhibits improved precision compared with single channel detectors.

30. Molecular weight can be obtained from a very small sample. It does not involve the absorption or emission of light. Detection limits better by 3 orders of magnitude of other techniques. Remarkably simple spectra that are unique and easily interpreted. Ability to measure isotopic ratios. Mass Spectrometry : Advantages Mass Spectrometry : Disadvantages Instrument costs are 2 to 3 times higher than others. Instrument drift that can be as high as 5-10% per hour. Interference effects may occur.

31. Chromatography 31

32. Principle It is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium. Components: Mobile phase : a solvent that flows through the supporting medium. Stationary phase : a layer or coating on the supporting medium that interacts with the analyte . Supporting medium : a solid surface on which the stationary phase is bound or coated. 32

33. Types of Chromatography The primary division of chromatographic techniques is based on the type of mobile phase used in the system. Type of Chromatography Type of Mobile Phase Gas chromatography (GC) Gas Liquid chromatography (LC) Liquid 33

34. Types of Chromatography Further divisions can be made based on the type of stationary phase used in the system: Gas Chromatography Name of GC Method Type of Stationary Phase Gas-solid chromatography(GSC) solid support Gas-liquid chromatography(GLC) liquid-coated support Bonded-phase gas chromatography chemical support 34

35. Types of Chromatography Liquid Chromatography Name of LC Method Type of Stationary Phase Adsorption chromatography - solid support Partition chromatography - liquid-coated support Ion-exchange chromatography - support containing fixed charges Size exclusion chromatography - porous support Affinity chromatography - support with immobilized material 35

36. Gas Chromatography (GC) 36

37. Gas Chromatography- Equipments 37 Carrier source with pressure and flow regulators. ( Eg : Ar , He, N 2 , H 2 ) Injector or sample application system. Chromatographic column with oven for temperature control. ( Eg : 2-50 m, coiled stainless steel/glass/Teflon tube) Detector & computer or recorder. Eg : Thermal Conductivity Detector (TCD), Flame Ionization Detector (FID), Electron Capture Detector (ECD)

38. Gas Chromatography- Equipments 38 Mobile Phase or Carrier gas Main purpose of carrier gas in GC is to move the solutes along the column. Common carrier gas include He, Ar , H 2 , N 2 etc. Carrier Gas or Mobile phase does not affect solute retention time . (Time required for the sample to travel from the injection port through the column to the detector). But does affect: Desired efficiency for the GC System Stability of column and solutes Response of the detector

39. Gas Chromatography- Equipments 39 Stationary Phase It is the main factor determining the selectivity and retention of solutes. There are three types of stationary phases used in GC: Solid adsorbents (GSC) Liquids coated on solid supports (GLC) Bonded-phase supports

40. How Does Chromatography Work? In all chromatographic separations , the sample is transported in a mobile phase. The mobile phase can be a gas or a liquid. The mobile phase is then forced through a stationary phase held in a column or on a solid surface. The stationary phase needs to be something that does not react with the mobile phase or the sample. The sample then has the opportunity to interact with the stationary phase as it moves past it. Samples that interact greatly, then appear to move more slowly. Samples that interact weakly, then appear to move more quickly. Because of this difference in rates, the samples can then be separated into their components. 40

41. Chromatography is based on a physical equilibrium that results when a solute is transferred between the mobile and a stationary phase. K = distribution coefficient or partition ratio K = C S C M Where C S is the molar concentration of the solute in the stationary phase and C M is the molar concentration in the mobile phase.

42. Gas-Solid Chromatography (GSC) Here same material is used as both the stationary phase and support material Common adsorbents include: Alumina Molecular sieves (crystalline aluminosilicates [ zeolites ] and clay) Silica Active carbon 42

43. Gas-Liquid Chromatography (GLC) Here stationary phase is some liquid coated on a solid support. Over 400 liquid stationary phases available for GLC. Material range from polymers ( polysiloxanes , polyesters, polyethylene glycols) to fluorocarbons, molten salts and liquid crystals . Disadvantage is the liquid may slowly bleed off with time. 43

44. Bonded Phase Gas Chromatography Stationary phase is covalently attach to the solid support. So it avoids column bleeding in GLC. Bonded phases are prepared by reacting the desired phase with the surface of a silica-based support. Commonly recommended bonded-phases: Dimethylpolysiloxane Methyl(phenyl) polysiloxane Polyethylene glycol ( Carbowax 20M) 44

45. Liquid Chromatography (LC) Introduction: A chromatographic technique in which the mobile phase is a liquid. LC is a much older technique than GC, but was overshadowed by the rapid development of GC in the 1950’s and 1960’s. LC is currently the dominate type of chromatography and is even replacing GC in its more traditional applications. 45

46. Advantages of LC compared to GC Separation of any compound that is soluble in a liquid phase is possible. ( Eg : biological compounds, synthetic or natural polymers, inorganic compounds etc.) Can be used at lower temperatures than GC. Retention of solutes in LC depend on their interaction with both the mobile phase and stationary phase, where GC retention based on volatility and interaction with stationary phase. Most LC detectors are non-destructive and most GC detectors are destructive. 46

47. Classification of LC based on overall efficiency or performance Low-performance Liquid Chromatography (LPLC) High-performance Liquid Chromatography (HPLC) 47

48. Low-performance liquid chromatography (LPLC) LC methods that use large, non-rigid support material. Poor system efficiency. Such systems have the following characteristics: broad peaks poor limits of detection long separation times columns can only tolerate low operating pressures 48

49. Low-performance liquid chromatography (LPLC) Column chromatography – an example of the equipment used in low-performance liquid chromatography . 49 Solvent reservoir Column head Column Column packing Porous glass plate Sample is usually applied directly to the top of the column. Detection is by fraction collection with later analysis of each fraction

50. High-performance liquid chromatography (HPLC) LC methods that use small, uniform, rigid support material. Good system efficiencies Such systems have the following characteristics: narrow peaks good limits of detection short separation times columns can tolerate high operating pressures and faster flow-rates 50

51. High-performance liquid chromatography (HPLC) It requires higher operating pressures for mobile phase delivery and uses special pumps and other system components. Sample applied using closed system (i.e., injection valve) Detection uses a flow through detector. 51

52. High-performance liquid chromatography (HPLC) Advantages: fast analysis time ease of automation good limits of detection preferred choice for analytical applications popular for purification work Disadvantages: greater expense lower sample capacities 52

53. HPLC Vs LPLC

54. Types of Liquid Chromatography Techniques in LC are classified according to the method of solute separation. 1) Adsorption chromatography: Separates solutes based on their adsorption to the stationary phase. 2) Partition chromatography: Separates solutes based on their partitioning between a liquid mobile phase and a liquid stationary phase coated on a solid support. 3) Ion-exchange chromatography: Separates solutes by their adsorption onto a support containing fixed charges on its surface. 4) Affinity chromatography: Separates based on the use of immobilized biological molecules as the stationary phase. 5) Size-exclusion chromatography: separates molecules according to differences in their size.

55. Types of Liquid Chromatography

56. Chromatographic Detectors After the components of a mixture are separated using chromatography, they must be detected as they exit the chromatographic column. Most commonly used detectors are: Thermal Conductivity Detector (TCD) F lame I onization D etectors (FID) Electron C apture D etector (ECD) Atomic E mis s ion D etector (AED) Mass spectrometer (MS) 56

57. Thermal Conductivity Detector (TCD) A TCD detector consists of an electrically-heated wire or thermistor . The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it. Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance. The TCD is not as sensitive as other detectors but it is non-specific and non-destructive. 57

58. Thermal Conductivity Detector (TCD) 58

59. Flame Ionization Detectors (FID) The effluent from the column is mixed with hydrogen and air and then ignited electrically. Most organic compounds, when pyrolyzed at the temperature of a hydrogen/air flame, produce ions and electrons that can conduct electricity through the flame. The ions are collected on a biased electrode and the resulting current (~10-12 A) is then measured. Extremely sensitive and have large dynamic range. Disadvantage of FID is that it is destructive of sample . 59

60. Flame Ionization Detectors (FID) 60

61. Electron Capture Detectors(ECD) The ECD uses a radioactive source such as Ni 63 which produces Beta particles, which react with the carrier gas producing free electrons. These electrons flow to the anode & producing an electrical signal . When electrophillic molecules are present, they capture the free electrons, lowering the signal. The amount of lowering is proportional to the amount of analyte present. It is widely used detectors for environmental samples, because it detects halogen containing compounds, such as pesticides and polychlorinated biphenyls. 61

62. Electron Capture Detectors(ECD) 62 63

63. Atomic Emission Detector (AED) One of the newest gas chromatography detector & it is quite expensive compared to other chromatographic detectors. The strength of the AED lies in the detector's ability to simultaneously determine elements. It uses microwave energy to excite helium molecules (carrier gas) which emit radiation & which breaks down molecules to atoms such as S, N, P, Hg, As, etc. These excited molecules emit distinctive wavelengths which can be separated by a grating and sent to the detector (typically a photodiode array) which produces the electrical signal. 63

64. Atomic Emission Detector (AED) 64

65. Mass Spectrometer (MS) Uses the difference in mass-to-charge ratio (m/e) of ionized atoms or molecules to separate them from each other. Molecules have distinctive fragmentation patterns that provide structural information to identify structural components. The general operation of a mass spectrometer is: C reate gas-phase ions S eparate the ions in space or time based on their mass to charge ratio Measure the quantity of ions of each mass-to-charge ratio. 65

66. Mass Spectrometer (MS) 66

67. Centrifugal force  is the apparent force that draws a rotating body away from the centre of rotation. Centripetal force is a force that makes a body follow a curved path. 67 Extra reading…