1. Mass Spectrometry Instrumentation and Applications
Monzir S. Abdel-Latif
Chemistry Department
IUG

2. Analytical method to measure the molecular or atomic weight of samples
Mass Spectrometry
Analytical method to measure the molecular or atomic weight of samples

3. Different compounds can be uniquely identified by their masses
Butorphanol L-dopa Ethanol N OH HO
-CH2-
-CH2CH-NH2
COOH HO
CH3CH2OH
MW = MW = MW = 46.1

4. MS History
MS concept first put into practice by Francis Aston, a physicist working in Cambridge, England in 1919
Designed to measure masses of elements
Aston was awarded Nobel Prize in 1922
1920s - Electron impact ionization and magnetic sector mass analyzer were introduced

5. MS History 1948-52 - Time of Flight (TOF) mass analyzers introduced
Quadrupole ion filters introduced by Wolfgang Paul, also invented the ion trap in 1983 (wins 1989 Nobel Prize)
Tandem mass spectrometers were presented
Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY

6. MS Principles Find a way to “charge” an atom or molecule (ionization)
Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature (as current) relative to its mass-to-charge ratio (mass analyzer)
Detect ions using microchannel plate or electron multiplier tube

7. Mass Spec Principles
Sample + _
Ionizer
Mass Analyzer
Detector

8. Typical sample: isolated
A Typical Mass Spectrum O C H 3
Mass
Spectrometer H C C N 3 N C C H C C O N N H
Typical sample: isolated
compound (~1 nanogram)
Mass Spectrum
194 67
109
Abundance 55 82 42 94
136
165 40 60 80
100
120
140
160
180
200
Mass (amu)

9. Resolution Width of peak indicates the resolution of the MS instrument
The better the resolution or resolving power, the better the instrument and the better the mass accuracy
Resolving power is defined as: M/DM
M is the mass number of the observed average of two adjacent masses and DM is the difference between the two masses

10. Resolution
If we have 5000 resolution for a mass spectrometer, we can separate m/z from m/z , or separate m/z from m/z , or separate m/z from m/z or m/z 9999 from m/z (all down to a 10% valley between the two peaks).

11. monoisotopic mass 1296.68518 R = 1000 R = 5000 average mass 1297.50248
Mass (m/z)
average mass I
R = 1000
R = 5000
monoisotopic mass . I
Mass (m/z)
12C62H90N17O14
12C6113C1H90N17O14
12C6013C2 H90N17O14
12C5913C3H90N17 O14

14. Mass Spectrometer Schematic
Inlet
Ion
Source
Mass
Analyzer
Detector
Data
System
High Vacuum System
Rough pumps
Rotary pumps
Turbo pumps
Diffusion pumps
Vapor
HPLC GC
Solids probe
MALDI
ESI
FAB
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
PC’s
UNIX
Mac

15. Different Ionization Methods
Electron Impact (EI - Hard method)
small molecules, Daltons
Chemical Ionization (CI) and Fast Atom Bombardment, polar high molecular weight species (FAB – Softer)
peptides, sugars, up to Daltons
Electrospray Ionization (ESI - Soft)
peptides, proteins, up to 200,000 Daltons
Matrix Assisted Laser Desorption (MALDI-Soft)
peptides, proteins, DNA, up to 500 kD

16. Fragmentation  Break Of Covalent Bond
Fragment ion 
An electrically charged dissociation product of an ionic fragmentation. Such an ion may fragmentate further to produce other electrically charged molecular or atomic moieties of successively lower formula weight.
Fragmentation  Break Of Covalent Bond
„Hard“
Ionization
„Soft“ EI
CI,FAB
MALDI
ESI

17. Electron Impact Ionization Source
Electron Collector (Trap)
Positive
Ions +
Repeller
Neutral
Molecules
Inlet _ _ + to
Analyzer + + + + + + e- e- e- _
Electrons
Filament
Extraction
Plate

18. Electron Impact Ionization
Sample introduced into instrument by heating it until it evaporates
Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV)
Molecules are “shattered” into fragments (70 eV >> bond energy)
Fragments travel to mass analyzer
The primary weakness is the loss of the molecular ion peak

19. Why it isn’t Wise to Use EI For Analyzing large molecules like proteins
EI shatters chemical bonds
Any given protein contains many different amino acids
EI would shatter the protein into peptides of 2,3,4… amino acids and amino acid sub-fragments
Result is a huge number of different signals from a single protein -- too complex to analyze

20. Chemical Ionization (CI)
Electron ionization leads to fragmentation of the molecular ion, which sometimes prevents its detection.
Chemical ionization (CI):
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.

21. Chemical Ionization (CI)
Typical reagent gases (ex. CH4, isobutane, or NH3) are present in a millionfold excess with respect to the analyte.
Analyte is ionized by ion-molecule chemical reactions:
Primary Ion Formation:
CH4 + e-  CH4+ + 2e-
Secondary Reagent Ions:
CH4 + CH4+  CH5+ + CH3
CH4 + CH3+  C2H5+ + H2
Product Ion Formation:
M + CH5+  CH4 + [M + H] + (protonation)
AH + CH3+  CH4 + A+ (H− abstraction)
M + CH5+  [M+ CH5] + (adduct formation)
A + CH4+  CH4 + A+ (charge exchange)

22. Fast Atom Bombardment (FAB)
Material to be analyzed is mixed with a non-volatile chemical protection environment called a matrix
This is bombarded under vacuum with a high energy (4 – 10 keV) beam of atoms.
Atoms are typically an inert gas (Ar or Xe)

24. Soft Ionization
Soft ionization techniques keep the molecule of interest fully intact
Electro-spray ionization first conceived in 1960’s by Malcolm Dole but put into practice in 1980’s by John Fenn (Yale)
MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt)
Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF

25. Soft Ionization Methods
337 nm UV laser
Fluid (no salt) + _
cyano-hydroxy
cinnamic acid
Gold tip needle
MALDI
ESI

26. Electrospray Ionization
Sample dissolved in polar, volatile buffer and pumped through a stainless steel capillary ( mm) at a rate of mL/min!!!
Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the droplets to pick charges and nebulize (aerosol)
Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)

27. Electrospray Ionization
Can be modified to “nanospray” system with flow < 1 mL/min
Very sensitive technique, requires less than a picomole of material
Strongly affected by salts & detergents

28. Electrospray (ESI)

29. Matrix-Assisted Laser Desorption Ionization
337 nm UV laser
cyano-hydroxy
cinnamic acid

30. MALDI Sample is ionized by bombarding sample with laser light
Sample is mixed with a UV absorbant matrix (like 4-hydroxycinnaminic acid)
Light wavelength matching the absorbance maximum of absorbant matrix will be absorbed and the matrix transfers some of its energy to the analyte (leads to ion sputtering)

31. MALDI
Generally tolerates salts and nonvolatile components, an advantage over ESI
Easier to use and maintain
Requires as low as 10 mL of 1 pmol/mL sample

32. Mass Spectrometer Schematic
Rough pumps
Rotary pumps
Turbo pumps
Diffusion pumps
High Vacuum System
Inlet
Ion
Source
Mass
Filter
Detector
Data
System
Vapor
HPLC GC
Solids probe
MALDI
ESI
FAB
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
PC’s
UNIX
Mac

33. Different Mass Analyzers
Magnetic Sector Analyzer + Double Focusing
High resolution, exact mass, original MA
Quadrupole Analyzer
Low (1 amu) resolution, fast, cheap
Time-of-Flight Analyzer
No upper m/z limit, high throughput
Tandem Mass Spectrometers (MS-MS)
V. good resolution
Ion Trap Mass Analyzer
Good resolution, simple
Ion Cyclotron Resonance (FT-ICR)
Highest resolution, exact mass, costly

34. Magnetic Sector Mass Analyzer

35. Double focusing mass analyzer

36. Quadrupole Mass Analyzer
A quadrupole mass filter consists of four parallel metal rods with different charges
The applied voltages affect the trajectory of ions traveling down the flight path
For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path

37. Quadrupole Ion Filter resonant ion non-resonant ion _ Detector + + _
Source
DC and AC
Voltages

39. TOF

40. Principle Of Reflector-TOF
acceleration
region
Field free drift region
sample target 1 2
reflector
m = m
E < E 12
m/z
detector

41. Ion Trap Mass Analyzer Invented by Wolfgang Paul (Nobel Prize1989)
Offer good mass resolving power
The two end-cap electrodes are grounded while the doughnut shaped ring electrode is connected to a variable RF voltage source

42. FT-ICR Uses powerful magnet (5-10 Tesla) to create miniature cyclotron
Originally developed in Canada (UBC) by A.G. Marshal in 1974
FT approach allows many ion masses to be determined simultaneously (efficient)
Has higher mass resolution than any other MS analyzer available

43. FT-ICR-MS instrument general scheme

44. A circulating ion in a magnetic field is capable of absorbing energy from an ac electric field provided the frequency of the field matches the cyclotron frequency
the cyclotron frequencyA circulating ion perpendicular to field has a frequency called

45. FTICR: New Dimensions of High Performance Mass Spectrometry
High mass resolution >
Accuracy of mass determination < 0.1 ppm
Sensitivity (ESI, Octapeptide) ca. 50 attomol
Structure-specific fragmentation MS/MS , MSn

46. Mass Spectrometer Schematic
Rough pumps
Rotary pumps
Turbo pumps
Diffusion pumps
High Vacuum System
Inlet
Ion
Source
Mass
Filter
Detector
Data
System
Vapor
HPLC GC
Solids probe
MALDI
ESI
FAB
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Microch plate
Electron Mult.
PC’s
UNIX
Mac

47. Mass Detectors
Electron Multiplier

48. Electron multipliers with discrete dynodes
In this device, positive ions strike a conversion cathode liberating electrons which are then accelerated and multiplied’ via a series of up to twenty dynodes. This type of detector is extremely sensitive, having a gain of up to 108. Aluminium-based dynodes have improved performances of the traditional materials (Cu/Be alloys) which age rather badly in the residual atmosphere of the spectrometers, or during non working periods (returning to atmospheric pressure).

50. Continuous dynode multipliers with a channeltron®
The ions are directed towards a collector whose entrance, in the form of a horn, is made of a lead doped glass with which acts as the conversion cathode. The ejected electrons are attracted towards a positive electrode and their collisions against the internal walls give rise to multiplication, as with the separated dynodes. The assembly is usually mounted off-axis to avoid the impact of neutral species as well as photons emitted by the filament, equally susceptible to the removal of the electrons.

52. Microchannel plate detectors(MCP)
They consist of the union of a large number microchanneltrons arranged like honeycombs. This resembles an electronic version of a photographic plate. Each individual detector is formed from a portion of microtube (25mm diameter) whose interior is coated by a semiconductor material acting as a continuous dynode. This system preserves the spatial resolution of the input charged ions.

54. Thank You for your Attention