1. Mass Spectrometry

2. Definition of Mass Spectrometry
Mass spectrometry (MS) :
An analytical technique by using mass spectrometry for the determination of the composition of a sample or molecule and elucidation of the chemical structures of molecules, such as peptides and other chemical compounds.
Mass spectrometry has been described as the smallest scale in the world, not because of the mass spectrometer’s size but because of the size of what it weighs -- molecules.

3.  Can work out molecular structure
Chemistry 101
Mass of each group is the combined mass of the atoms forming the group (often unique)
e.g. phenyl (C6H5) mass = 77, methyl (CH3) mass = 15, etc.
So:- If you break molecule up into constituent groups and measure the mass of the individual fragments (using MS) - Can determine what groups are present in the original molecule and how they are combined together
 Can work out molecular structure

4. What is Mass Spectrometry?
Mass spectrometry is a powerful technique for chemical analysis that is used to identify unknown compounds, to quantify known compounds, and to elucidate molecular structure
Principle of operation
A Mass spectrometer is a “Molecule Smasher”
Measures molecular and atomic masses of whole molecules, molecular fragments and atoms by generation and detection of the corresponding gas phase ions, separated according to their mass-to-charge ratio (m/z).
Measured masses correspond to molecular structure and atomic composition of parent molecule – allows determination and elucidation of molecular structure.

5. What is Mass Spectrometry?
May also be used for quantitation of molecular species.
Very sensitive technique - Works with minute quantities of samples (as low as 10-12g, moles) and is easily interfaced with chromatographic separation methods for identification of components in a mixture
Mass spectrometry provides valuable information to a wide range of professionals: chemists, biologists, physicians, astronomers, environmental health specialists, to name a few.
Limitation – is a “Destructive” technique – cannot reclaim sample

6. What is Mass Spectrometry Used For?
Chemical Analysis and Identification
Some Typical Applications
Enviromental Monitoring and Analysis (soil, water and air pollutants, water quality, etc.)
Geochemistry – age determination, Soil and rock Composition, Oil and Gas surveying
Chemical and Petrochemical industry – Quality control
Applications in Biotechnology
Identify structures of biomolecules, such as carbohydrates, nucleic acids
Sequence biopolymers such as proteins and oligosaccharides
Determination of drug metabolic pathways

7. How Does it Work?
Generate spectrum by separating gas phase ions of different mass to charge ratio (m/z)
m=molecular or atomic mass, z = electrostatic charge unit
In many cases (such as small molecules), z = 1
 measured m/z = mass of fragment

8. What is a Mass Spectrometer?
Many different types – each has different advantages, draw-backs and applications
All consist of 4 major sections linked together
Inlet – Ionization source – Analyser – Detector
All sections usually maintained under high vacuum
All functions of instrument control, sample acquisition and data processing under computer control
Data system and Computer Control is often overlooked – most significant advance in MS – allows 24/7 automation and development of modern powerful analytical techniques.

9. What is a Mass Spectrometer?
All Instruments Have:
Sample Inlet
Ion Source
Mass Analyzer
Detector
Data System

10. How does it work? +4000 V 0 V e- e- e- e- e- accelerate separate
ionise
+4000 V
0 V e-
Magnetic and/or electric field e- + +
heavy
vacuum
light
vapourise e- + A e-
sample + B + C A+ B+ C+
Mass spectrometry e-

11. Mass Spectrometer
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12. Analyser Types What is the analyser?
Analyser is the section of instrument that separates ions of different m/z
Many Different technologies
Magnetic Sector, Quadrupole, Ion Trap, ToF
All based on momentum separation

13. Analyser Types What is the analyser?
Analyser is the section of instrument that separates ions of different m/z
Many Different technologies
Magnetic Sector, Quadrupole, Ion Trap, ToF
All based on momentum separation

14. Analyser Types – Magnetic sector
Easiest Conceptually to understand
Separate electromagnetically
“Electromagnetic Prism”
Usually combined with ESA (energy focusing device) - enables high mass resolution (Double Focusing Instrument) – makes high accuracy mass measurements possible
Large (Heavy!!), Expensive to operate
Comparatively slow scan rates
High Skill level required to operate and maintain
Self-service use by users not possible

16. Mass Spectrometer Instrument Design
Different types of Ionization source
EI, CI, FAB, ESI, Maldi, (APCI, DESI, DART)
(Also sources for inorganic analysis – ICP, GD, etc.)
Different types of analyser
Magnetic Sector, Quadrupole, Ion Trap, ToF
Different sources and analysers have different properties, advantages and disadvantages
Selection of appropriate ionization method and analyzer are critical and defines MS applications.
Wide range of MS applications

17. Ionization Methods Electron bomb Ionization (EI
Chemical Ionization ( CI
Field ionization () FI
Matrix Assisted Laser Desorption Ionization ( MALDI
Fast atom bombardment ) FAB
Electro Spray Ionization () ESI

18. Electron Bomb Ionization ( EI )
Sample is heated and energized by a beam of electrons, usually gives a molecular ion (M+) and a lot of fragments。

19. Electron Bomb Ionization ( EI )

20. Properties of EI Hard ionization
Gas-phase molecules enter source through heated probe or
GC column
70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions
EI spectra can be matched to library stds CI (soft ionization)
Higher pressure of methane leaked into the source (mtorr)
Reagent ions transfer proton to analyte

21. Chemical Ionization (CI)
Electron ionization leads to fragmentation of the molecular ion, which sometimes prevents its detection.
Chemical ionization (CI):
A technique that produces ions with little excess energy.
Thus this technique presents the advantage of yielding a spectrum with less fragmentation in which the molecular species is easily recognized.
Consequently, chemical ionization is complementary to electron ionization.

22. Chemical Ionization (CI)

23. Properties of CI Disadvantages Advantages No Fragment Library
Need Volatile Sample
Need Thermal Stability
Quantitation Difficult
Low Mass Compounds
(<1000 amu)
Solids Probe Requires
Skilled Operator
Advantages
Parent Ion
Interface to GC
Insoluble Samples

24. Field ionization (FI)
Field ionization (FI) is a method that uses very strong electric fields to produce ions from gas-phase molecules. +
d<1mm

25. Field ionization (FI)

26. Matrix Assisted Laser Desorption Ionization (MALDI)
sample is co-crystallized with a matrix and then irradiated
with laser.
MALDI is achieved in two steps. In the first step, the compound to be analyzed is dissolved in a solvent containing in solution small organic molecules, called the matrix. The second step occurs under vacuum conditions inside the source of the mass spectrometer.

27. Properties of MALDI Good solubility
Vapour pressure must be sufficiently low to maintain vacuum conditions
Viscosity must allow diffusion of the analyte from the bulk to the surface
Polar : to solvate and separate preformed ion
Less Sensitive to Salts
Lower PRACTICAL detection limits
Easier to interpret spectra (less multiple charges)
Quick and easy
Higher mass detection
Higher Throughput (>1000 samples per hour)

28. Principle of MALDI
MALDI mass spectrometry has become a powerful analytical
tool for both synthetic polymers and biopolymers.

29. Fast atom bombardment ( FAB)
Softer than EI and CI. Ions are produced by bombardment with heavy atoms. Gives (M+H)+ ions and litle fragmentation. Good for more polar compounds.
Ar + e Ar acceleration (5-15 KeV)
Ar Ar Ar + Ar+
fast slow KeV
fast slow

30. Properties of FAB Disadvantages Advantages No Fragment Library
Solubility in Matrix (MNBA, Glycerol)
Quantitation Difficult
Needs Highly Skilled Operator
Relatively Low Sensitivity
Advantages
Parent Ion
High Mass Compounds (10,000 amu)
Thermally Labile Compounds (R.T.)

31. ElectroSpray Ionization (ESI)
Electrospray is abbreviated to ESI ，ample is sprayed out of a narrow nozzle in a high potential field. Generates positive (M+nH)n+ and negative (M - nH)n- ions and almost no fragmentation. Generates multiple charged ions.

32. 2. Principle

33. Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best)
Properties of ESI
Advantages
Electrospray Ionization can be
easily interfaced to LC.
Absolute signals from
Electrospray are more easily
reproduced, therefore, better
quantitation.
Mass Accuracy is considered
better.
Multiple charging is more
common then MALDI.
Disadvantages
No Fragmentation
Need Polar Sample
Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best)
Sensitive to Salts
Suppression

34. Types of Mass Analyzers
Magnetic sector analyzer Time of Flight analyzer (TOF) Quadrupole analyzers Fourier Transform Ion-Cyclotron

35. Magnetic Sector Analyzer
Magnetic sector analyzer – Uses electric and/or magnetic fields to separate ions

36. Principle of Magnetic Sector Analyzer
The ion source accelerates ions to a kinetic energy given by : (1/2)m2= zV Where m is the mass of the ion ,v is its velocity, z is the charge on the ion ,and V is the applied voltage of the ion optics.

37. Principle of Magnetic Sector Analyzer
Only ions of mass-to-charge ratio that have equal centripetal and centrifugal forces pass through the flight tube
It shows that the m/q ratio of the ions that reach the detector can be varied by changing either the magnetic field or the applied voltage of the ion optics.

38. In summary ,by varying the voltage or magnetic field of the magnetic-sector analyzer ,the individual ion beams are separated spatially and each has a unique radius of curvature according to its mass/charge ratio.

39. Advantages
Double focusing magnetic sector mass analyzers are the "classical" model against which other mass analyzers are compared.
Classical mass spectra
Very high reproducibility
Best quantitative performance of all MS analyzers
High resolution
High sensitivity
10,000 Mass Range
Linked scan MS/MS does not require another analyzer

40. Disadvantages Applications Requires Skilled Operator
Usually larger and higher cost than other mass analyzers
Difficult to interface to ESI
Low resolution MS/MS without multiple analyzers
Applications
All organic MS analysis methods
Accurate mass measurements
Quantitation
Isotope ratio measurements

41. Time of Flight Analyzer
TOF analyzer – ions are accelerated through a flight tube and the time of light to the detector is measured

42. Ions are accelerated and their time of flight to the detector is measured.

43. Principle of TOF Analyzer
Uses a pulse of ion mixtures, not steady stream
Ions accelerated into drift tube by a pulsed electric
field called the ion-extraction field
Drift Tube is usually 1-2 m long, under vacuum
Ions traverse the drift tube at different speeds
( L / t ) = v = ( 2zV / m )½

44. Advantages of TOF Analyzer
Good for kinetic studies of fast reactions and for
use with gas chromatography to analyze peaks
from chromatograph
High ion transmission
Can register molecular ions that decompose in the
flight tube
Extremely high mass range (>1MDa)
Fastest scanning

45. Disadvantages
Requires pulsed ionization method or ion beam switching (duty cycle is a factor)
Low resolution (4000)
Limited precursor-ion selectivity for most MS/MS experiments
Applications
Almost all MALDI systems
Very fast GC/MS systems

46. Quadrupole Analyzers
Quadrupole analyzers – ions are filtered or trapped in a
device consisting of several metal rods using specifically tailored electromagnetic fields

47. Quadrupole Analyzers Electric/magnetic fields trap, store, eject ions
Requires an in-line quadrupole to act as
mass pre-filter
Contains a single ring electrode and a top
and bottom cap electrode
Varying RF frequency will vary the m/z ratios
that are trapped
Additional fragmentation can be performed
on ions stored in the ion trap

48. Easy to use ,simple construction,fast
Advantages
Easy to use ,simple construction,fast
Good reproducibility
Relatively small and low-cost systems
Quadrupoles are now capable of routinely
analyzing up to a m/q ratio of 3000,which is
useful in electrospary ionization of biomolecules,
which commonly produces a charge distribution
below m/z 3000

49. Disadvantages
Low resolution(<4000)
Slow scanning
Low accuracy (>100ppm)
Applications
Majority of benchtop GC/MS and LC/MS systems
Separation of proteins and other biomolecules
with electrosprary
Sector / quadrupole hybrid MS/MS systems

50. Fourier Transform Ion Cyclotron Resonance (FT ICR) analyzers

51. Most FTICR mass spectrometers use superconducting magnets, which provide a relatively stable calibration over a long period of time.
Although some mass accuracy can be obtained without internal calibrant, mass accuracy and resolution are inversely proportional to m/z, and the best accurate mass measurements require an internal calibrant.
Unlike the quadrupole ion trap, the FTICR mass spectrometer is not operated as a scanning device.

52. Advantages The highest recorded mass resolution of all mass
spectrometers (>500,000)
Very good accuracy (<1ppm)
Well-suited for use with pulsed ionization
methods such as MALDI
Non-destructive ion detection; ion remeasurement
Stable mass calibration in superconducting
magnet FTICR systems

53. Disadvantages Expensive Requires superconducting magnet
Subject to space charge effects and ion molecule reactions
Artifacts such as harmonics and sidebands are present in the mass spectra
Many parameters (excitation, trapping, detection conditions) comprise the experiment sequence that defines the quality of the mass spectrum
Generally low-energy CID, spectrum depends on collision energy, collision gas, and other parameters.

54. Applications Ion chemistry High-resolution MALDI and electrospray
experiments for high-mass analytes
Laser desorption for materials and surface
characterizatio

55. The Mass Spectrum Presentation of data
The mass spectrum is presented in terms of ion abundance vs. m/e ratio (mass).
The most abundant ion formed in ionization gives rise to the tallest peak on the mass spectrum – this is the base peak.
base peak, m/e 43

56. Presentation of data
3. All other peak intensities are relative to the base peak as a percentage.
4. If a molecule loses only one electron in the ionization process, a molecular ion is observed that gives its molecular weight – this is designated as M+ on the spectrum.
M+, m/e 114

57. Presentation of data
5. In most cases, when a molecule loses a valence electron, bonds are broken, or the ion formed quickly fragment to lower energy ions
6. The masses of charged ions are recorded as fragment ions by the spectrometer – neutral fragments are not recorded !
fragment ions

58. B. Determination of Molecular Mass
1. When a M+ peak is observed it gives the molecular mass – assuming that every atom is in its most abundant isotopic form
2. Remember that carbon is a mixture of 98.9% 12C (mass 12), 1.1% 13C (mass 13) and <0.1% 14C (mass 14)
3. We look at a periodic table and see the atomic weight of carbon as – an average molecular weight
4. The mass spectrometer, by its very nature would see a peak at mass 12 for atomic carbon and a M + 1 peak at 13 that would be 1.1% as high
- We will discuss the effects of this later…

59. B. Determination of Molecular Mass
5. The Nitrogen Rule is another means of confirming the observance of a molecular ion peak
6. If a molecule contains an even number of nitrogen atoms (only ‘common’ organic atom with an odd valence) or no nitrogen atoms the molecular ion will have an even mass value
7. If a molecule contains an odd number of nitrogen atoms, the molecular ion will have an odd mass value
8. If the molecule contains chlorine or bromine, each with two common isotopes, the determination of M+ can be made much easier, or much more complex as we will see.