Analytical Chemistry Section E.14 Mass Spectrometer
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Contents 1. Basic of Mass Spectrometer 2. Advanced Mass
Spectrometer 3. Conclusion
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1. Basic of Mass Spectrometer Terms of Mass Spectrometer
Principles Mass Spectrometry Ionization Techniques Fragmentation
Mass Analyzer Resolution Isotope Peaks
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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.
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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) Terms of Mass
Spectrometer (2)
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-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) Principles Base
Peak : the most abundant ion Fig. 1. Mass Spectrum of Methanol
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Mass Spectrometry Fig. 2. Block diagram of a Mass Spectrometer
- EI, CI - ESI - MALDI - Single Focusing - Double Focusing -
Quadrupole & Quadrupole Ion Trap - TOF - FT-ICR - Tandem
(MS/MS) Fragment(in MS n ) - CID - ETD
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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. Ionization Techniques (1) Fig. 3. Diagram of EI (70eV)
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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) Ionization Techniques (2)
Fig. 4. Mechanism of CI
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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. Ionization
Techniques (3) Fig. 4-1. Diagram of ESI Fig. 4-2. Two processes of
the conversion of ions from droplets into the gas phase (a) Charge
Residue Model (b) Ion Desorption Model
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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. Ionization Techniques (4)
Fig. 5. Diagram of MALDI
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- 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 (MS n ) Fragmentation (1) Fig. 6. Diagram of
Fragmentation of peptides
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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. Fragmentation (2)
Fig. 7. Diagram of comparison between CID & ETD 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.
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Mass Analyzer (1) Fig. 8-1. Diagram of a Principle of Single
Focusing Magnetic Mass Analyzer Fig. 8-2. Diagram of a principle
Double Focusing Magnetic Mass Analyzer
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- The same principle both Quadrupole and Quadrupole Ion Trap -
Advantages of Quadrupole Ion Trap High Sensitivity, Trapping the
specific ions, Specialized for Qualitative Analysis. Mass Analyzer
(2) Fig. 9-1. Diagram of a Principle Quadrupole Mass Analyzer Fig.
9-2. Diagram of a Principle Quadrupole Ion Trap Mass Analyzer
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- The analytical technique has been extremely useful for
proteomics using MALDI-TOF/MS systems. Mass Analyzer (3) Fig. 10.
Diagram of comparison of Linear and Reflector - Reflector-TOF Low
Sensitivity High Resolution - Linear-TOF High Sensitivity Low
Resolution
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- 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 Mass Analyzer (4) Fig. 11-1. Diagram of FT-ICR Fig. 11-2.
Diagram of a Principle of Ion Cyclotron Resonance
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- Hyphenated techniques (MS-MS) - Tandem MS modes Precursor Ion
Scan, Product Ion Scan, Neutral Loss Scan, Selected Reaction
Monitoring - High Resolution, Selectivity Mass Analyzer (5) Fig.
12. Diagram of a Principle of Tandem MS
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Resolution m2m2 m1m1
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- 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. Isotope
Peaks (1) Table. 1. Empirical formulae and isotope peak ratios for
a nominal RMM value of 70 (M=100%)
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- The intensities in mass spectra of Isotope Peaks of C 24 H 22
O 7 (Using the Table. 2.) Isotope Peaks (2) Table. 2. Natural
isotopic abundances of some common elements as a percentage of the
most abundant isotope (M+1) +, (M+2) + (M) +
- 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 EI/MS
Fig. 13. Agilent 7000 Series Triple Quadrupole GC/MS
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Characteristics - GC/MS/MS - Gold Quadrupole - Hexapole
Collision Cell - Triple-Axis HED-EM Detector EI/MS -
Characteristics Fig. 14. Diagram of a Principle of Agilent 7000
Series Triple Quadrupole GC/MS Video
- Using a Helium buffer gas - Reduction of Chemical Noise -
High Sensitivity, Resolution EI/MS Agilent Techniques (2) Fig.
16-1. Diagram of Agilent 7000 Series Triple Quadrupole GC/MS Fig.
16-2. Diagram of a Principle of Helium Quenching
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Ultra low neutrals noise Long life and high linearity Superior
sensitivity EI/MS Agilent Techniques (3) Fig. 17-1. Diagram of
Agilent 7000 Series Triple Quadrupole GC/MS Fig. 17-2. Diagram of a
Principle of Triple-Axis Detector
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- 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 ESI/ETD/MS Fig.
18. Thermo Orbitrap Elite
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Characteristics - Ion Optics - Ion Trap with Neutral Blocker -
Trap-HCD - Orbitrap ESI/ETD/MS - Characteristics Fig. 19. Diagram
of a Principle of Thermo Orbitrap Elite Video
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- Variable Spaced Stacked Lenses. Increasing spacing =
increasing field penetration to focus ion beam - Robustness, High
Sensitivity ESI/ETD/MS Thermo Techniques (1) Fig. 20-2. Ion Optics
Fig. 20-1. Diagram of Thermo Orbitrap Elite
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- Rotated 45 o Quadrupole - Blocking the Neutral Beams. -
Separation of Neutrals and Ions - More Robustness ESI/ETD/MS Thermo
Techniques (2) Fig. 21-2. Ion Trap (Square Quadrupole with Neutral
Blocker) Fig. 21-1. Diagram of Thermo Orbitrap Elite
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- Trap-HCD fragmentation (HCD,CID, PQD,ETD) - Dual Pressure
Trap - No low mass cut off - High Resolution ESI/ETD/MS Thermo
Techniques (3) Fig. 22-2. Diagram of Trap-HCD Fig. 22-1. Diagram of
Thermo Orbitrap Elite
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- New type of Ion Trap - Faster Scanning - High Resolution
ESI/ETD/MS Thermo Techniques (4) Fig. 23-2. Diagram of Orbitrap
Fig. 23-1. Diagram of Thermo Orbitrap Elite
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- 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 MALDI/MS Fig. 24. Waters MALDI SYNAPT G2-S HDMS
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Characteristics - Variable Ionization Techniques - T-Wave Ion
Guide - TRIWAVE - QUANTOF - HDMS instruments MALDI/MS -
Characteristics Fig. 25. Diagram of a Principle of Waters MALDI
SYNAPT G2-S HDMS Video
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-Very simple to exchange Ion Source -Extend Compound Coverage
-High Flexibility MALDI/MS Waters Techniques (1) Fig. 26. Diagram
of Ion Source of Waters MALDI SYNAPT G2-S HDMS
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- Positive and Negative RF fields are applied to each ring
electrode pair. - New type of Ion Optics - Outstanding linearity,
Sensitivity MALDI/MS Waters Techniques (2) Fig. 27-1. Diagram of
Waters MALDI SYNAPT G2-S HDMS Fig. 27-2. Diagram of T-Wave Ion
Guide
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- Ion Mobility Separation Being separated by Size, Shape and
Charge - Fragmentation(CID, ETD) - Increasing Peak Capacity and
Detection limit MALDI/MS Waters Techniques (3) Fig. 28-1. Diagram
of Waters MALDI SYNAPT G2-S HDMS Fig. 28-2. Diagram of TRIWAVE
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-Dual Stage Reflectron -Hybrid Ion Detection System -Compatible
with HDMS analysis -High Resolution : Over 40,000 FWHM MALDI/MS
Waters Techniques (3) Fig. 29. Diagram of Waters MALDI SYNAPT G2-S
HDMS
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3. Conclusion Summary Reference
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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.