Analytical Chemistry Section E.14 Mass Spectrometer.

Analytical Chemistry Section E.14 Mass Spectrometer

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

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

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.

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)

-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

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

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)

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

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

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

- 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

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.

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

- 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

- 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

- 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

- 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

Resolution m2m2 m1m1

- 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%)

- 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) +

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

- 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

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

- Heated gold plated hyperbolic quartz quadrupoles - Reliability, Stability EI/MS Agilent Techniques (1) Fig. 15-1. Diagram of Agilent 7000 Series Triple Quadrupole GC/MS Fig. 15-2. Gold Plated Hyperbolic Quartz Quadrupole

- 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

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

- 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

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

- 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

- 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

- 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

- 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

- 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

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

-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

- 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

- 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

-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

3. Conclusion Summary Reference

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.

Reference - Agilent Technologies http://www.chem.agilent.com/en-US/Products/Instruments/ms/gc- ms/systems/7000triplequadrupolegcms/pages/default.aspx - Thermo Scientific http://www.thermoscientific.com/ecomm/servlet/productsdetail_11152_L10 710_87170_13901130_-1 - Waters http://www.waters.com/waters/nav.htm?cid=134614100 - Instant Notes, Analytical Chemistry, Kealey & Haines

Analytical Chemistry Section E.14 Mass Spectrometer.

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