1 chemical analysis by mass spectrometry. 2 all chemical substances are combinations of atoms. atoms...
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Chemical Analysis by Chemical Analysis by Mass SpectrometryMass Spectrometry
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• All chemical substances are combinations of atoms.
• Atoms of different elements have different masses (H = 1, C = 12, O = 16, S = 32, etc.)
• An element is a substance that cannot be broken down into a simpler species by chemical means - has a unique atomic number corresponding to the number of protons in the nucleus
• Different atoms combine in different ways to form molecular sub-units called functional groups.
33
• 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
HOW DO WE ACHIEVE THIS?HOW DO WE ACHIEVE THIS?
• PERSUADE THE MOLECULE TO ENTER THE VAPOR PHASE (CAN BE DIFFICULT)
• PRODUCE IONS FROM THE MOLECULES THAT ENTER THE GAS PHASE
• SEPARATE THE IONS ACCORDING TO THEIR MASS-TO-CHARGE RATIOS (m/z))
• MEASURE AND RECORD THESE IONS
Electron Impact IonizationElectron Impact Ionization
A high-energy electron can dislodge an A high-energy electron can dislodge an electron from a bond, creating a radical electron from a bond, creating a radical cation (a positive ion with an unpaired cation (a positive ion with an unpaired ee--).).
e- + H C
H
H
C
H
H
H
H C
H
H
C
H
H
H
H C
H
H
C
H
H
+ H
H C
H
H
C
H
H
H
+ =>
Separation of IonsSeparation of Ions
Only the cations are deflected by the Only the cations are deflected by the magnetic field.magnetic field.
Amount of deflection depends on Amount of deflection depends on m/zm/z.. The detector signal is proportional to The detector signal is proportional to
the number of ions hitting it.the number of ions hitting it. By varying the magnetic field, ions of By varying the magnetic field, ions of
all masses are collected and counted. all masses are collected and counted. =>=>
Mass SpectrometerMass Spectrometer
=>
The Mass SpectrumThe Mass Spectrum
Masses are graphed or tabulated according to Masses are graphed or tabulated according to their relative abundance.their relative abundance.
=>
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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.
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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, 10-15 moles)
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
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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
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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
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Mass Measurement
Mass Spectrometers measure isotopic mass.
They DO NOT measure average molecular mass!! (MW)
e.g For a molecule with empirical formula C60H122N20O16S2
Average MW = 1443.8857(weighted average for each isotope)
Exact mass = 1442.8788(exact mass of most abundant isotope)
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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
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What is a Mass Spectrometer?
All Instruments Have:
1. Sample Inlet
2. Ion Source
3. Mass Analyzer
4. Detector
5. Data System
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e-
+
e-
e-
+4000 V 0 V
+
e-
e-
heavy
light
Magnetic and/or electric field
sample
vapourise
ioniseaccelerate separate
+A
+B
+C
A+ B+ C+
vacuum
Mass spectrometryMass spectrometry
How does it work?
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Mass Spectrometer Instrument Design
Different types of Ionization source
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
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Biotechnology applications
Advances in Proteomics and other areas in biotechnology made possible by development of soft ionisation techniques
Protein and peptide analysis for MW determination
Protein Identification and profiling using digests and data base searching – major development in Proteomics
Protein post-translational modification Protein structure characterisation
Oligo-nucleotide analysis –
Carbohydrate analysis
Example 1Example 1
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2020
Peptide Mass FingerprintPeptide Mass Fingerprint
Trypsin Digest
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Peptide Mass FingerprintPeptide Mass Fingerprint
MS
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Peptide Mass FingerprintPeptide Mass Fingerprint
The Mass SpectrumThe Mass Spectrum
Plot mass of ions (m/z) (x-axis) versus Plot mass of ions (m/z) (x-axis) versus the intensity of the signal (roughly the intensity of the signal (roughly corresponding to the number of ions) corresponding to the number of ions) (y-axis)(y-axis)
Tallest peak is base peak (100%)Tallest peak is base peak (100%)• Other peaks listed as the % of that Other peaks listed as the % of that
peakpeak
Example 2Example 2
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EI Fragmentation of CHEI Fragmentation of CH33OHOH
CH3OH CH3OH+
CH3OH CH2O=H+ + H
CH3OH + CH3 + OH
CHO=H+ + HCH2O=H+
Molecular ion
Electron Impact MS of CHElectron Impact MS of CH33OHOH
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