1. <Instant Notes, D. Kealey & P.J. Haines>
Analytical Chemistry
Section E.14
Mass Spectrometer
<Instant Notes, D. Kealey & P.J. Haines>

2. Contents 1. Basic of Mass Spectrometer 2. Advanced Mass Spectrometer
3. Conclusion

3. 1. Basic of Mass Spectrometer
Terms of Mass Spectrometer
Principles
Mass Spectrometry
Ionization Techniques
Fragmentation
Mass Analyzer
Resolution
Isotope Peaks

4. Terms of Mass Spectrometer (1)
Principles
Mass Spectrometry (MS) is a technique whereby materials are ionized and dissociated into fragments characteristic of the molecule(s) or element(s) present in the sample. The numbers of ions of each mass provide information for qualitative and quantitative analysis.
Mass Spectrometric
A Mass Spectrometer, which is operated under high vacuum, incorporates a sample inlet and ion source, a mass analyzer, an ion detector and a data processing system.
Ionization Techniques
Alternative ionization techniques are available differing in energy and applicability. Some produce a high degree of dissociation of molecules, while others are used primarily to establish an accurate relative molecular mass of a compound or to facilitate elemental analysis.

5. Terms of Mass Spectrometer (2)
Fragmentation
After ionization, molecules may dissociate into fragments of smaller mass, some carrying a charge. The presence and relative abundances of the various charged fragments provide structural information and enable unknown compounds to be identified.
Isotope peaks
These are peaks in a mas spectrum arising from fragments containing naturally occurring heavier isotopes of one or more elements.
Mass Spectra
Spectral data is either tabulated or shown graphically as a plot of the numbers of ions of each mass detected. For ease of interpretation, these are presented as line diagrams.
Related Topics : Inductively coupled plasma spectrometry (E5)
Combined techniques (Section F)

6. Principles
MS is an analytical technique in which gaseous ions formed from the molecules or atoms of a sample are separated in space or time and detected according to their mass-to-charge ratio, m/z.
Example of Mass Spectrum (m/z 31 for Methanol)
Base Peak
: the most abundant ion
Fig. 1. Mass Spectrum of Methanol

7. Mass Spectrometry - Single Focusing - Double Focusing
- Quadrupole & Quadrupole Ion Trap
- TOF
- FT-ICR
- Tandem (MS/MS)
Fragment(in MSn)
- CID
- ETD
- EI, CI
- ESI
- MALDI
Fig. 2. Block diagram of a Mass Spectrometer

8. Ionization Techniques (1)
1. EI (Electron Ionization)
- EI Process (in gas phase)
M + e- → M+• + 2e-
(M is the analyte molecule being
ionized, e- is the electron and M+•
is the resulting ion.)
- Only possible in gas phase.
(70eV)
Fig. 3. Diagram of EI

9. Ionization Techniques (2)
2. CI (Chemical Ionization)
- Collision of the analyte with
ions of a reagent gas.
- Reagent gas : Methane,
Ammonia, Isobutane
- Advantage to analyze mixture
compounds
- Variations : NCI(Negative),
APCI(Atmospheric Pressure)
Fig. 4. Mechanism of CI

10. Ionization Techniques (3)
Fig Two processes of the conversion of ions from droplets into the gas phase
(a) Charge Residue Model (b) Ion Desorption Model
Fig Diagram of ESI
3. ESI (Electrospray Ionization)
- Using the solvents. (water + Organic solvents + Acid)
- Possible to analyze biological molecules and Polymers.
- Using the nebulizer gas (inert). → rapid evaporation of solvents.
- Being produced multiply charged ions.

11. Ionization Techniques (4)
4. MALDI (Matrix-Assisted
Laser Desorption Ionization)
- Sample is mixed with a
compound capable of absorbing
energy from the laser.
⇒Analyte/Matrix Mixture
- Possible to analyze solid phase
samples.
- Soft ionization technique.
- Being produced proton ions.
- Possible to use Genomics,
Proteomics.
Fig. 5. Diagram of MALDI

12. Fragmentation (1)
Fig. 6. Diagram of Fragmentation of peptides
- The backbone of a peptide can fragment at three bonds CH-CO, CO- NH and NH-CH with each dissociation producing two fragments named according to the location of the charge and the amino acid position
(n-terminus = a-, b-, c-; c-terminus = x-, y-, z-)
- Using in a Tandem MS (MSn)

13. Fragmentation (2) CID (Collision-Induced Dissociation)
Fig. 7. Diagram of comparison between CID & ETD
CID (Collision-Induced Dissociation)
- Breaking the weakest bonds and producing a characteristic series of fragments.
- Many PTMs are fragile and are lost in the CID process.
ETD (Electron-Transfer Dissociation)
- ETD cleaves selectively on the peptide backbone, leaving PTMs intact.
- ETD produces a different set of fragments that are complementary to CID, so sequence coverage is more complete.

14. Mass Analyzer (1) - Related Equations 𝒎 𝒛 = 𝐁 𝟐 𝐫 𝟐 𝟐𝐕
Fig Diagram of a Principle of Single Focusing Magnetic Mass Analyzer
Fig Diagram of a principle Double Focusing Magnetic Mass Analyzer
- Related Equations
𝒎 𝒛 = 𝐁 𝟐 𝐫 𝟐 𝟐𝐕
(r : radii of curvature, B : magnetic field strength, V : accelerating voltage)

15. Mass Analyzer (2)
Fig Diagram of a Principle Quadrupole Mass Analyzer
Fig Diagram of a Principle Quadrupole Ion Trap Mass Analyzer
- The same principle both Quadrupole and Quadrupole Ion Trap
- Advantages of Quadrupole Ion Trap
→ High Sensitivity, Trapping the specific ions, Specialized for
Qualitative Analysis.

16. Mass Analyzer (3) - Linear-TOF • High Sensitivity • Low Resolution
- Reflector-TOF
• Low Sensitivity
• High Resolution
- The analytical technique has been
extremely useful for proteomics
using MALDI-TOF/MS systems.
Fig. 10. Diagram of comparison of Linear and Reflector

17. Mass Analyzer (4)
Fig Diagram of a Principle of Ion Cyclotron Resonance
Fig Diagram of FT-ICR
- The excited ions pass a set of metal detector plates with each orbit.
- Very Strong Magnetic Field : 5~12 Tesla
- The image current is recorded and Fourier Transformed to produce
the mass spectrum.
- Extremely High Price, Vacuum needs, Resolution and Mass Accuracy

18. Mass Analyzer (5) - Hyphenated techniques (MS-MS) - Tandem MS modes
Fig. 12. Diagram of a Principle of Tandem MS
- Hyphenated techniques (MS-MS)
- Tandem MS modes
→ Precursor Ion Scan, Product Ion Scan, Neutral Loss Scan,
Selected Reaction Monitoring
- High Resolution, Selectivity

19. Resolution - MS are designed to give a specified Resolving Power.
→ The minimum acceptable Resolution = One mass unit.
- Two masses are considered to be resolved when the valley
between their peaks is less than 10% of the smaller peak height.
Resolving Power= m 2 m 2 − m 1
- Requirement for unit mass resolution increase with the magnitudes of m1 and m2. m1 m2

20. Isotope Peaks (1)
- Most elements occur naturally as a mixture of isotopes, all of
which contribute to peaks in a mass spectrum.
- Isotope Peaks are of importance in the interpretation of mass
spectra.
Table. 1. Empirical formulae and isotope peak ratios for a nominal RMM value of 70 (M=100%)

21. Isotope Peaks (2)
- The intensities in mass spectra of Isotope Peaks of C24H22O7
(Using the Table. 2.)
(M)+
(M+1)+, (M+2)+
Table. 2. Natural isotopic abundances of some common elements as a percentage of the most abundant isotope

22. 2. Advanced Mass Spectrometer
EI/MS
Agilent 7000 Series Triple Quadrupole GC/MS
ESI/ETD/MS
Thermo Orbitrap Elite
MALDI/MS
Waters MALDI SYNAPT G2-S HDMS

23. EI/MS - Manufacturer Agilent - Model 7000 Series Triple Quadrupole
GC/MS
- Ionization Type
EI, PCI, NCI
- Resolution
0.7 to 2.5 Da
- Scanning Speed
Up to 6250 u/s
- Mass Range
1.2 to 1050 m/z
Fig. 13. Agilent 7000 Series Triple Quadrupole GC/MS

24. EI/MS - Characteristics
Fig. 14. Diagram of a Principle of Agilent 7000 Series Triple Quadrupole GC/MS
• Characteristics
- GC/MS/MS
- Gold Quadrupole
- Hexapole Collision Cell
- Triple-Axis HED-EM Detector
Video

25. EI/MS – Agilent Techniques (1)
Fig Gold Plated Hyperbolic Quartz Quadrupole
- Heated gold plated hyperbolic
quartz quadrupoles
- Reliability, Stability
Fig Diagram of Agilent 7000 Series Triple Quadrupole GC/MS

26. EI/MS – Agilent Techniques (2)
Fig Diagram of a Principle of Helium Quenching
- Using a Helium buffer gas
- Reduction of Chemical Noise
- High Sensitivity, Resolution
Fig Diagram of Agilent 7000 Series Triple Quadrupole GC/MS

27. EI/MS – Agilent Techniques (3)
Fig Diagram of a Principle of Triple-Axis Detector
– Ultra low neutrals noise
– Long life and high linearity
– Superior sensitivity
Fig Diagram of Agilent 7000 Series Triple Quadrupole GC/MS

28. ESI/ETD/MS - Manufacturer Thermo Scientific - Model Orbitrap Elite
- Ionization Type
ESI
- Fragmentation Type
CID, ETD, HCD
- Resolution
>240,000 at m/z 400
- Mass Accuracy
< 3 ppm with external calibration
< 1 ppm with internal calibration
Fig. 18. Thermo Orbitrap Elite

29. ESI/ETD/MS - Characteristics
Fig. 19. Diagram of a Principle of Thermo Orbitrap Elite
• Characteristics
- Ion Optics
- Ion Trap with Neutral Blocker
- Trap-HCD
- Orbitrap
Video

30. ESI/ETD/MS – Thermo Techniques (1)
Fig Diagram of Thermo Orbitrap Elite
- Variable Spaced Stacked Lenses.
→ Increasing spacing = increasing
field penetration to focus ion beam
- Robustness, High Sensitivity
Fig Ion Optics

31. ESI/ETD/MS – Thermo Techniques (2)
Fig Diagram of Thermo Orbitrap Elite
- Rotated 45o Quadrupole
- Blocking the Neutral Beams.
- Separation of Neutrals and Ions
- More Robustness
Fig Ion Trap (Square Quadrupole with Neutral Blocker)

32. ESI/ETD/MS – Thermo Techniques (3)
Fig Diagram of Thermo Orbitrap Elite
- Trap-HCD fragmentation (HCD,CID,
PQD,ETD)
- Dual Pressure Trap
- No low mass cut off
- High Resolution
Fig Diagram of Trap-HCD

33. ESI/ETD/MS – Thermo Techniques (4)
Fig Diagram of Thermo Orbitrap Elite
- New type of Ion Trap
- Faster Scanning
- High Resolution
Fig Diagram of Orbitrap

34. MALDI/MS - Manufacturer Waters - Model MALDI SYNAPT G2-S HDMS
- Ionization Type
MALDI, ESI, APPI, APCI, ESCi®
- Fragmentation Type
CID, ETD
- Resolution
> 40,000 FWHM
- Mass Range
Max. 100,000 m/z
Fig. 24. Waters MALDI SYNAPT G2-S HDMS

35. MALDI/MS - Characteristics
Fig. 25. Diagram of a Principle of Waters MALDI SYNAPT G2-S HDMS
• Characteristics
- Variable Ionization Techniques
- T-Wave Ion Guide
- TRIWAVE
- QUANTOF
- HDMS™ instruments
Video

36. MALDI/MS – Waters Techniques (1)
Fig. 26. Diagram of Ion Source of Waters MALDI SYNAPT G2-S HDMS
-Very simple to exchange Ion Source
-Extend Compound Coverage
-High Flexibility

37. MALDI/MS – Waters Techniques (2)
Fig Diagram of Waters MALDI SYNAPT G2-S HDMS
- Positive and Negative RF fields are applied to each ring electrode pair.
- New type of Ion Optics
- Outstanding linearity, Sensitivity
Fig Diagram of T-Wave Ion Guide

38. MALDI/MS – Waters Techniques (3)
Fig Diagram of Waters MALDI SYNAPT G2-S HDMS
- Ion Mobility Separation
→ Being separated by Size, Shape and Charge
- Fragmentation(CID, ETD)
- Increasing Peak Capacity and Detection limit
Fig Diagram of TRIWAVE

39. MALDI/MS – Waters Techniques (3)
Fig. 29. Diagram of Waters MALDI SYNAPT G2-S HDMS
-Dual Stage Reflectron
-Hybrid Ion Detection System
-Compatible with HDMS™ analysis
-High Resolution : Over 40,000 FWHM

40. 3. Conclusion
Summary
Reference

41. Summary - Basic principle of Mass Spectrometer
- Ionization, Fragmentation
- Several Types of Mass Analyzer
- Identification of Mass Spectra
- Application
⇒ Future Works : Advanced Hybrid Mass Spectrometer
Contributing to analyze and interpret
Biological Molecules in Proteomics quickly and
accurately.

42. Reference - Agilent Technologies - Thermo Scientific - Waters
ms/systems/7000triplequadrupolegcms/pages/default.aspx
- Thermo Scientific
2_L10710_87170_ _-1
- Waters
- Instant Notes, Analytical Chemistry, Kealey & Haines