Download to read offline
![Isotope Peaks: [M+1]
[M+2]](https://image.slidesharecdn.com/doc-20240320-wa0004-250714042152-f91e6012/85/DOC-20240320-WA0004-pptnuclear-magnetic-resonance-spectroscopy-presentation-9-320.jpg)
![Isotope Peaks: [M+1]
[M+2]](https://image.slidesharecdn.com/doc-20240320-wa0004-250714042152-f91e6012/75/DOC-20240320-WA0004-pptnuclear-magnetic-resonance-spectroscopy-presentation-9-2048.jpg)
DOC-20240320-WA0004.pptnuclear magnetic resonance spectroscopy presentation
- 1.
- 2. Mass spectrometry allowsus to determine the molecular mass and the molecular formula of a compound, as well as certain structural features of the compound. PRINCIPLE • In mass spectrometry, a small sample of a compound is introduced into an instrument called a mass spectrometer, where it is vaporized and then ionized as a result of an electron’s being removed from each molecule. Ionization can be accomplished in several ways. • The most common method bombards the vaporized molecules with a beam of high-energy electrons. The energy of the electron beam can be varied, but a beam of about 70 electron volts (eV) is commonly used. • When the electron beam hits a molecule, it knocks out an electron, producing a molecular ion, which is a radical cation—a species with an unpaired electron and a positive charge.
- 4. Molecular ion peak:Molecular weight of the compound Base Peak
- 6.
- 7.
- 8. MS of n-PentaneMS of Isopentane
- 9.
- 10. Rules For Fragmentationin MS MS of n-Pentane MS of Isopentane
- 11.
- 12.
- 13.
- 14.
- 16.
- 17.
- 18.
- 19. MS of NAPHTHALENEC10H8
- 20. MS of CYCLOHEXANEC6H12
- 21.
- 22. Fragmentation may involve Collisioninduced dissociation Electron capture dissociation Electron transfer dissociation Photo dissociation Surface induced dissociation
- 23. Inlet Systems: Inlet Systems: •LC & GC coupled to mass spectrometer LC & GC coupled to mass spectrometer • Permits separation and determination of components for Permits separation and determination of components for complex mixtures complex mixtures Requires specialized inlet systems Requires specialized inlet systems Major interface problem – carrier gas dilution Major interface problem – carrier gas dilution Jet separator (separates analyte from carrier gas) Jet separator (separates analyte from carrier gas)
- 24. Types of Atomicand Molecular MS Types of Atomic and Molecular MS • Thermal ionization & Spark source Thermal ionization & Spark source first MS first MS • Inductively coupled plasma (ICP) Inductively coupled plasma (ICP) current common approach current common approach - Differ by types ion sources and mass analyzer Differ by types ion sources and mass analyzer
- 25. Ion Sources: Ion Sources: •Formation of gaseous analyte ions Formation of gaseous analyte ions • Mass spectrometric methods are dictated by ionization techniques Mass spectrometric methods are dictated by ionization techniques • Appearance of spectrum highly dependant on ionization technique Appearance of spectrum highly dependant on ionization technique • Gas-phase Gas-phase Sample first vaporized then ionized Sample first vaporized then ionized Thermally stable compounds boiling points < 500 Thermally stable compounds boiling points < 500o o C C MW < 100 amu MW < 100 amu • Desorption Desorption Solid or liquid directly converted to gaseous ion Solid or liquid directly converted to gaseous ion MW as large as 10 MW as large as 105 5 daltons daltons Type Name and Acronym Ionizing Process Gas Phase Electron Impact (EI) Exposure to electron stream Chemical Ionization (CI) Reagent gaseous ions Field Ionization (FI) High potential electrode Desorption Field Desorption (FD) High potential electrode Electrospray Ionization (ESI) High electric field Matrix-assisted desorption ionization (MALDI) Laser beam Plasma Desorption (PD) Fission fragments from 252 Cf Fast Atom Bombardment (FAB) Energetic atomic beam Secondary Ion Mass Spectrometry (SIMS) Energetic beam of ions Thermospray Ionization (TS)
- 26. Ion Sources: Ion Sources: •Hard sources Hard sources Sufficient energy so analyte in highly excited energy state Sufficient energy so analyte in highly excited energy state Relaxation involves rupture of bonds Relaxation involves rupture of bonds - Produces fragment ions with Produces fragment ions with m m/ /z z < molecular ion < molecular ion - Kinds of functional groups Kinds of functional groups structural information structural information Hard Ionization
- 27. Ion Sources: Ion Sources: •Soft sources Soft sources Cause little fragmentation Cause little fragmentation Mass spectrum consists of molecular ion and only Mass spectrum consists of molecular ion and only few, if any, other peaks few, if any, other peaks Accurate mass Accurate mass Soft Ionization
- 28. IONIZATION TECHNIQUES EI: ElectronImpact CI: Chemical Ionization FAB: Fast Atom Bombardment ESI-TOF: Electron Spray Ionization MALDI-MALDI: Matrix Assisted Laser Desorption/Ionization
- 29. Ion Sources: Ion Sources: •Electron-Impact Source (EI) Electron-Impact Source (EI) Sample heated to produce molecular vapor Sample heated to produce molecular vapor Bombard with a beam of electrons Bombard with a beam of electrons - Electrons emitted from heated tungsten or rhenium filament Electrons emitted from heated tungsten or rhenium filament - Electrons accelerated by a potential of 70V Electrons accelerated by a potential of 70V - Path of electrons and molecular ion at right angles Path of electrons and molecular ion at right angles Form positive ions Form positive ions electron beam expels electron due to electron beam expels electron due to electrostatic repulsion electrostatic repulsion M + M + e e- - M M●+ ●+ + 2 + 2e e- - Positive ions attracted to first slit by small potential 5V Positive ions attracted to first slit by small potential 5V High potential applied at accelerator plates 10 High potential applied at accelerator plates 103 3 to 10 to 104 4 V V - Generates molecular ion velocity Generates molecular ion velocity
- 30. Ion Sources: Ion Sources: •Electron-Impact Source (EI) Electron-Impact Source (EI) Hard source 50V higher energy than chemical bond Hard source 50V higher energy than chemical bond Highly excited vibrational and rotational state Highly excited vibrational and rotational state - Electron beam does not increase translational Electron beam does not increase translational energy energy Relaxation results in extensive fragmentation Relaxation results in extensive fragmentation - Large number of positive ions of various masses Large number of positive ions of various masses - Typically less mass than molecular ion Typically less mass than molecular ion - Lower mass ions called Lower mass ions called daughter ions daughter ions - Sometimes molecular ion not present Sometimes molecular ion not present Base peak Base peak most intense peak most intense peak - Usually a daughter ion or fragment ion Usually a daughter ion or fragment ion
- 31. Advantages Advantages - Good sensitivity Goodsensitivity - Fragmentation Fragmentation unambiguous identification of unambiguous identification of analytes analytes Disadvantages Disadvantages - Need to volatize sample Need to volatize sample thermal thermal decomposition before ionization decomposition before ionization - Fragmentation Fragmentation disappearance of molecular ion disappearance of molecular ion peak peak - MW not determined MW not determined
- 32. Chemical Ionization (CI) ChemicalIonization is the second most common procedure Chemical Ionization is the second most common procedure for generating ions for generating ions Gaseous atoms from the sample are: Gaseous atoms from the sample are: -Heated from a probe Heated from a probe -Collide with ions produced reagent gas bombarded by Collide with ions produced reagent gas bombarded by electrons electrons -Usually positive ions are used Usually positive ions are used Soft Source Soft Source Methane is common reagent Methane is common reagent -Also use propane, isobutane and ammonia Also use propane, isobutane and ammonia -Reacts with high-energy electron beam to generate several Reacts with high-energy electron beam to generate several ions ions
- 33. Chemical Ionization (CI) Thevapourized sample is introduced into the mass spectrometer with an excess of a ‘reagent’ gas (commonly) at a pressure of about 1 torr. The excess carrier gas is ionized by electron impact to the primary ions CH4.+ and CH3 + . These react with the excess methane to give secondary ions.
- 34. • Fast AtomBombardment Sources (FAB) Fast Atom Bombardment Sources (FAB) Major role for MS studies of polar high molecular-weight species Major role for MS studies of polar high molecular-weight species Soft Ionization technique Soft Ionization technique Samples are in a condensed state Samples are in a condensed state - Glycerol solution matrix Glycerol solution matrix Ionized by bombardment with energetic (several keV) xenon or Ionized by bombardment with energetic (several keV) xenon or argon atoms argon atoms - Very rapid sample heating Very rapid sample heating - Reduces sample fragmentation Reduces sample fragmentation Positive & negative analyte ions are sputtered from the surface Positive & negative analyte ions are sputtered from the surface - Desorption process Desorption process
- 35. Beam offast energetic atoms are generated by: Beam of fast energetic atoms are generated by: - Passing accelerated argon or xenon ions from an ion source Passing accelerated argon or xenon ions from an ion source through a chamber through a chamber - Chamber contains argon or xenon atoms at 10 Chamber contains argon or xenon atoms at 10-5 -5 torr torr - High-velocity ions undergo a resonant electron-exchange reaction High-velocity ions undergo a resonant electron-exchange reaction without substantial loss of translational energy without substantial loss of translational energy Focusing Extraction plate Analyte ion beam (secondary ions) Probe tip Analyte metrix Atom beam
- 38. Matrix-Assisted Laser Desorption/Ionization(MALDI) Matrix-Assisted Laser Desorption/Ionization (MALDI) Accurate MW for polar biopolymers Accurate MW for polar biopolymers - DNA, RNA, Proteins DNA, RNA, Proteins - Few thousands to several hundred thousand Da Few thousands to several hundred thousand Da Sample is mixed with large excess of radiation-absorbing Sample is mixed with large excess of radiation-absorbing matrix material matrix material Solution is evaporated onto solid surface Solution is evaporated onto solid surface Sample exposed to pulsed laser beam Sample exposed to pulsed laser beam - Sublimation of analyte ions Sublimation of analyte ions - MS spectra recorded between laser beam pulses MS spectra recorded between laser beam pulses
- 40. Low background noise Lowbackground noise Multiple charged ions (+2, +3) Multiple charged ions (+2, +3) Observe dimers trimers Observe dimers trimers Mechanism is not completely understood Mechanism is not completely understood -Matrix compound must absorb the laser radiation Matrix compound must absorb the laser radiation -Soluble enough in sample solvent to be present in large excess Soluble enough in sample solvent to be present in large excess -Analyte should not absorb laser radiation Analyte should not absorb laser radiation Fragmentation will occur Fragmentation will occur
- 42. Mass analyzers: Mass analyzers:Time of Flight (TOF) Mass Analyzers Time of Flight (TOF) Mass Analyzers • Ions generated by bombardment of the sample with a brief pulse of: Ions generated by bombardment of the sample with a brief pulse of: Electrons, secondary ions, laser-generated photons Electrons, secondary ions, laser-generated photons Ions accelerated by electric field pulse 10 Ions accelerated by electric field pulse 103 3 to 10 to 104 4 V V Same frequency of ionization pulse, but lags behind Same frequency of ionization pulse, but lags behind • Accelerated particle enter Accelerated particle enter field-free field-free drift tube drift tube Ions enter tube with Ions enter tube with same same kinetic energy kinetic energy Ion velocity vary inversely with mass Ion velocity vary inversely with mass - Lighter particles arrive at detector before heavier particles Lighter particles arrive at detector before heavier particles - Flight times are 1 to 30 Flight times are 1 to 30 s, Limits resolution compared to s, Limits resolution compared to quadrupole, Less widely used than quadrupole quadrupole, Less widely used than quadrupole - Advantages: unlimited mass range, rapid data acquisition, Advantages: unlimited mass range, rapid data acquisition, simplicity, ruggedness, ease of access to ion source simplicity, ruggedness, ease of access to ion source
- 43. Time-of-Flight (TOF) MSis a pulsed MS. It has a simple construction, consisting of an accelerator, a field-free region, a reflectron and detector inside a high vacuum chamber called a flight tube TOF MS separates and detects ions of different m/z by measuring the time taken for the ions to travel through a field-free region. First, ions generated in an ionization unit are accumulated and introduced in pulses to a flight tube. These ions are accelerated by applying a high acceleration voltage between the electrodes. The corresponding kinetic energy is obtained. Given a constant acceleration voltage as well as kinetic energy, each ion flies at its unique velocity inside the flight tube to reach the ion detector, which is higher for ions with smaller masses and lower for ions with larger masses.
- 44. Time of flight(T) is proportional to the square root of m/z, i.e. for a fixed flight distance (L), ions with smaller m/z reach the detector sooner than those with larger m/z. Therefore, by keeping all other parameters constant, the time of flight (T) can be converted directly to m/z, which is how a mass spectrum is generated in a TOF MS. Since there is no limit to the time of flight in TOF MS, it can theoretically measure an unlimited mass range. This analytical technique has been extremely useful for proteomics using MALDI-TOF MS systems, where proteins are identified by comparing measurements of fragmented peptides with a database
- 45. Mass analyzers: Mass analyzers: •Quadrupole mass analyzer Quadrupole mass analyzer More compact, less expensive, rugged More compact, less expensive, rugged High scan rate High scan rate spectrum in < 100ms spectrum in < 100ms • Four parallel cylindrical rods serve as electrodes Four parallel cylindrical rods serve as electrodes Opposite rods are connected electrically Opposite rods are connected electrically - One pair attached to positive side of variable dc source One pair attached to positive side of variable dc source - One pair attached to negative side of variable dc source One pair attached to negative side of variable dc source Variable radio-frequency ac potential applied to each pair Variable radio-frequency ac potential applied to each pair of rods of rods • Ions accelerated through space between rods Ions accelerated through space between rods Potential of 5 to 10 V, ac and dc voltages increased Potential of 5 to 10 V, ac and dc voltages increased simultaneously with ratio being constant. All ions without simultaneously with ratio being constant. All ions without specific specific m/z m/z strike rods and become neutral strike rods and become neutral - only ions having a limited range of only ions having a limited range of m m/ /z z reach transducer (detector) reach transducer (detector)
- 46. Striking a roddepends on: Striking a rod depends on: - rate of movement through rod rate of movement through rod - Mass to charge ratio Mass to charge ratio - More difficult to deflect heavy ions than lighter ions More difficult to deflect heavy ions than lighter ions - Prevents heavier atoms from striking rods Prevents heavier atoms from striking rods When a combination of the direct current voltage and high-frequency alternating current voltage is applied to each pole, an electric field with a rapidly varying phase is generated within the quadrupole. Consequently, ions passing through this electric field oscillate in the x- and ydirections. When a given set of parameters are applied to the poles, certain ions of a specific m/z range maintain a stable oscillation and pass through the quadrupole to reach the detector. In the contrary, the oscillations of ions with other m/z values become unstable, causing them
- 47. Mass Spectrometry Mass Spectrometry AdvantagesOver Atomic Optical Advantages Over Atomic Optical Spectrometric Spectrometric • Detection limits three orders of Detection limits three orders of magnitude better magnitude better • Remarkably simple spectra that Remarkably simple spectra that are unique and easily are unique and easily interpreted interpreted • Ability to measure isotopic ratios Ability to measure isotopic ratios Disadvantages Disadvantages • Instrument costs are two to Instrument costs are two to three times higher three times higher • Interference effects Interference effects
- 48.