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Mass Spectroscopy

Mass Spectroscopy. Instrumental Analysis Chemistry 3590 Laboratory. Different MS Systems in MCAL. GC-MS, Gas Chromatography Triple Quadrupole MS separates volatile compounds in GC column and identifies by mass and fragmentation pattern LC-MS, Liquid Chromatography ESI Ion Trap MS

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Mass Spectroscopy

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  1. Mass Spectroscopy Instrumental Analysis Chemistry 3590 Laboratory

  2. Different MS Systems in MCAL • GC-MS, Gas Chromatography Triple Quadrupole MS • separates volatile compounds in GC column and identifies by mass and fragmentation pattern • LC-MS, Liquid Chromatography ESI Ion Trap MS • separates dissolved compounds by HPLC column chromatography or direct injection and identifies by mass • LC-MS, Quadrupole Time of Flight (qTOF) • separates dissolved compounds by HPLC column or direct injection and identifies by mass • MS-MS,Tandem Mass Spectrometry • identifies compound fragments by mass

  3. GC-MS Spectrometry in MCAL GC-MS, GC separates small, volatile, non-polar material • MS is detection devise (Agilent 320-MS TQ Mass Spectrometer).Three modes of monitoring ions produced: • Full scan monitoring • SIM single ion monitoring • MSMS monitoring • Sample can be injected as a liquid, a gas or even a solid

  4. GC-MS Injection port MS 320 Probe GC 3800

  5. Headspace vials Liquid sampling syringe Heating block with stirrer

  6. GC Column Oven

  7. The GC-MS A mixture of compounds is separated by gas chromatography, then identified by mass spectrometry. =>

  8. How Does Gas Chromatography Separate compounds • Chromatographic technique that separates volatile organic compounds. • A gas chromatograph consists of a 1) flowing mobile phase, 2)injection port, 3) a separation column containing the stationary phase, 4) controller (computer) detector and a (integrator) and data collection and storage (computer). • Organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column.

  9. GC-MS C

  10. Mass Spectrometry (MS) • Uses the interaction of electric and/or magnetic fields with matter to determine weight or mass • Measures mass, not absorption or emission of electromagnetic radiation • Measures molecular weight • Sample vaporized, separated by GC column, and then subjected to bombardment by electrons that remove an electron • Creates a cation-radical • Bonds in cation radicals begin to break (fragment) • Charge to mass ratio is measured

  11. Seperation of Alcohols C1 to C8 on GC column Chromographic separation by retention time Signal (uV) Time (minutes)

  12. Caffeine Fragmentation Pattern 109 55 67 194 82 137 44 165

  13. Caffeine fragmentation pattern GC-MS 70eV

  14. Ion Source sample separated by GC column Electons from filament are energized by a potential of 70 Ev. M + e- = M+ + 2e-

  15. Single Quadrupole Triple Quadrupole

  16. Quadrupole

  17. General Principle of Operation of the Mass Filter Light ions (low mass to charge ratio) are able to follow the alternating component of the field. For the X-direction, those ions will stay in phase with the RF drive, gain energy from the field and oscillate with increasingly large amplitude until they encounter one of the rods and are discharged. Therefore the X-direction is a high-pass mass filter: only high masses will be transmitted to the other end of the quadrupole without striking the X-electrodes. In the Y-direction, heavy ions will be unstable because of the defocusing effect of the DC component, but some lighter ions will be stabilized by the AC component if its magnitude is such as to correct the trajectory whenever its amplitude tends to increase. Thus the Y-direction is a low-pass mass filter: only low masses will be transmitted to the other end of the quadrupole without striking the Y electrodes. By a suitable choice of RF/DC ratio, the two directions together give a mass filter which is capable of resolving individual atomic masses.

  18. Quadrupole

  19. THE MOLECULAR ION In the spectrum of octane, a signal occurs at 114 due to the species C8H18+ The species due to the final signal is known as the molecular ion and is usually corresponds to the molecular mass of the compound. Mass spectrum Base peak 20 40 60 80 100 Abundance % molecular ion 114 . m/z 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 Mass/charge

  20. The Mass Spectrum • Plot mass of ions (m/z) (x-axis) versus the intensity of the signal (roughly corresponding to the number of ions) (y-axis) • Tallest peak is base peak (100%) • Other peaks listed as the % of that peak • Peak that corresponds to the unfragmented radical cation is parent peak or molecular ion (M+)

  21. The Mass Spectrum 1) Spectra (full scan) obtained for organic molecules have many peaks. 2) Each peak is due to a particular fragment with a certain m/z value. 3) Highest m/z value usually corresponds to the molecular ion 4) The tallest peaks come from the most stable species

  22. N -CH2- OH COOH HO -CH2CH-NH2 HO HO MS Principles • Different compounds can be identified by their mass Butorphanol L-dopa Ethanol CH3CH2OH MW = 327.1 MW = 197.2 MW = 46.1

  23. Electron Impact MS of CH3OH Molecular ion EI Breaks up Molecules in Predictable Ways

  24. EI Fragmentation of CH3OH CH3OH CH3OH+ CH3OH CH2O=H+ + H CH3OH + CH3 + OH CH2O=H+ CHO=H+ + H

  25. MOLECULAR MASS DETERMINATION USING MASS SPECTROMETRY Mass spectrometry is used to identify unknown or new compounds. When a molecule is ionized it forms a MOLECULAR ION which can also undergo FRAGMENTATION or RE-ARRANGEMENT to produce particles of smaller mass. Only particles with a positive charge will be deflected and detected. The resulting spectrum has many peaks. The final peak (M+) shows the molecular ion (highest m/z value) and indicates the molecular mass. The rest of the spectrum provides information about the structure. IONIZATION MOLECULAR ION FRAGMENTION RE-ARRANGEMENT FRAGMENTION

  26. Hexane (C6H14) with MW = 86.18

  27. GC-MS in MCAL Essential Oils Experiment

  28. Essential oils • Two essential oils will be analysed: peppermint oil and lavender oil to compare the molecular substances in the oils. • The identity of the molecules will be determined using the NIST library of the GC-MS. • Two oil components will be quantified using purchased standards.

  29. Essential oils • The MS is equipped with a triple quadrupole analyzer and allows several types of mass detection to be performed. • Full scan (direct MS) mode is used. • SIM will be used • MS-MS will be used

  30. GC Seperation • The essential oils are separated by gas chromatography. The separation is based on the particianing of the sample between the stationary phase and the gas phase. • A temperature gradient separates the sample based on boiling point.

  31. Capillary column: thin fused-silica capillary. 50 m in length and 250 µm inner diameter. The stationary phase is CP-sil 8, with 5% phenyl 95% dimethyl polysiloxane coated on the inner surface. Varian (Agilent) Factor Four Capillary GC Columns.

  32. GC Settings

  33. GC Parameters • You can set the flow rate of gas through column. There is an ideal flow rate which gives the best separation. • The split ratio determines how mush of your sample in injected onto column and how much goes to waste.

  34. MS The gas containing the separated analytes from the GC are ionized (as they elute from the GC) in the ion volume and the ions focused into the first quadrapole (Q1). The first quadrapole can measure the ions (full scan or selected ions [SIM]) or select certain ions to be transported to the collission quad (Q2). In Q2 the ions can be broken into smaller fragments by interaction with energized argon collission gas (CID). These ions are transported to the Q3 where they are analysed usually as single or small groups of ions (MSMS). These ions are directed to the detector (photomultiplier) where the produce a enhance signal.

  35. Triple Quodrapole From GC Collect full scan or SIM Ion volume CID in this quod ms-ms Collect ms-ms fragment ions in this quod Detector

  36. Mass Spectrometer (Q3) Q2 Q1 Q3 Diffusion pump Sample from GC Detector photomultiplier Ion Source tungsten filament

  37. Key Components of MS • Ionization – gas phase ions created in source • High vacum – creates free path • Mass Analyser- sorts ions by M/Z ratio - electric field • Detector – creates signal multiplication

  38. Electron Ionization • It uses a heated filament to produce electrons. The filament is usually made of rhenium or tungsten. • Once the electrons are produced they are accelerated through a potential difference of 70V this gives electrons with 70eV of energy.

  39. Electron Impact • Electrons produced by the source will then collide with the sample and remove an electron to give ions   e- + M  M+. + 2e-   • Fragmentation is a result of an excited molecular ion, which in attempting to gain stability • Fragmentation is not random and occurs in a repeatable chemical reaction.

  40. Q1 Window full scan Ion Range

  41. Various constituents

  42. Quantitation of Ingredients • You quantitate the ingredient that you are interested in by: - Full scan: measure the full peak area - SIM: measures one of the ions in your sample - MSMS: measures one of the ions produced in the collision cell (Quad 2)

  43. Mass 294 Q1 only Single ion monitoring SIM or SIR

  44. Q1 Window

  45. Fractionating the 293 ion in Quad 2 will produce product ions which can be scanned at 100 to 300 to identify the product ions. MW 293 - molecular ion Product ions

  46. MSMS product ions 183-185

  47. Sample Probe (solid samples)

  48. Probe

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