11

Chemical Analysis by Chemical Analysis by Mass SpectrometryMass Spectrometry

22

• 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.

=>

99

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.

1010

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

1111

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

1212

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

1313

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)

1414

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

1515

What is a Mass Spectrometer?

All Instruments Have:

1. Sample Inlet

2. Ion Source

3. Mass Analyzer

4. Detector

5. Data System

1616

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?

1717

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

1818

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

1919

2020

Peptide Mass FingerprintPeptide Mass Fingerprint

Trypsin Digest

2121

Peptide Mass FingerprintPeptide Mass Fingerprint

MS

2222

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

2424

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