1 Chemical Analysis by Mass Spectrometry

2 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.

3 Mass of each group is the combined mass of the atoms forming the group (often unique) e.g. phenyl (C 6 H 5 ) mass = 77, methyl (CH 3 ) 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? 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 Ionization A high-energy electron can dislodge an electron from a bond, creating a radical cation (a positive ion with an unpaired e - ). =>

Separation of Ions Only the cations are deflected by the magnetic field. Only the cations are deflected by the magnetic field. Amount of deflection depends on m/z. Amount of deflection depends on m/z. The detector signal is proportional to the number of ions hitting it. The detector signal is proportional to the number of ions hitting it. By varying the magnetic field, ions of all masses are collected and counted. => By varying the magnetic field, ions of all masses are collected and counted. =>

Mass Spectrometer =>

The Mass Spectrum Masses are graphed or tabulated according to their relative abundance. =>

9 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.

10 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 g, 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

11 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

12 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

13 Mass Measurement Mass Spectrometers measure isotopic mass. They DO NOT measure average molecular mass!! (MW) e.g For a molecule with empirical formula C 60 H 122 N 20 O 16 S2 Average MW = (weighted average for each isotope) Exact mass = (exact mass of most abundant isotope)

14 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

15 What is a Mass Spectrometer? All Instruments Have: 1.Sample Inlet 2.Ion Source 3.Mass Analyzer 4. Detector 5. Data System

16 e-e- + e-e- e-e V0 V + e-e- e-e- heavy light Magnetic and/or electric field sample vapourise ionise accelerateseparate + A + B + C A+A+ B+B+ C+C+ vacuumvacuum Mass spectrometry How does it work?

17 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

18 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 1 19

20 Peptide Mass Fingerprint Trypsin Digest

21 Peptide Mass Fingerprint MS

22 Peptide Mass Fingerprint

The Mass Spectrum Plot mass of ions (m/z) (x-axis) versus the intensity of the signal (roughly corresponding to the number of ions) (y-axis) Plot mass of ions (m/z) (x-axis) versus the intensity of the signal (roughly corresponding to the number of ions) (y-axis) Tallest peak is base peak (100%) Tallest peak is base peak (100%) Other peaks listed as the % of that peakOther peaks listed as the % of that peak

Example 2 24

EI Fragmentation of CH 3 OH CH 3 OHCH 3 OH + CH 3 OHCH 2 O=H + + H CH 3 OH + CH 3 + OH CHO=H + + HCH 2 O=H +

Molecular ion Electron Impact MS of CH 3 OH