<Instant Notes, D. Kealey & P.J. Haines> Analytical Chemistry Section E.14 Mass Spectrometer <Instant Notes, D. Kealey & P.J. Haines>

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.

Terms of Mass Spectrometer (2) 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)

Principles 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) Base Peak : the most abundant ion Fig. 1. Mass Spectrum of Methanol

Mass Spectrometry - Single Focusing - Double Focusing - Quadrupole & Quadrupole Ion Trap - TOF - FT-ICR - Tandem (MS/MS) Fragment(in MSn) - CID - ETD - EI, CI - ESI - MALDI Fig. 2. Block diagram of a Mass Spectrometer

Ionization Techniques (1) 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. (70eV) Fig. 3. Diagram of EI

Ionization Techniques (2) 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) Fig. 4. Mechanism of CI

Ionization Techniques (3) Fig. 4-2. Two processes of the conversion of ions from droplets into the gas phase (a) Charge Residue Model (b) Ion Desorption Model Fig. 4-1. Diagram of ESI 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 (4) 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. Fig. 5. Diagram of MALDI

Fragmentation (1) Fig. 6. Diagram of Fragmentation of peptides - 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 (MSn)

Fragmentation (2) CID (Collision-Induced Dissociation) Fig. 7. Diagram of comparison between CID & ETD 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. 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) - Related Equations 𝒎 𝒛 = 𝐁 𝟐 𝐫 𝟐 𝟐𝐕 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 - Related Equations 𝒎 𝒛 = 𝐁 𝟐 𝐫 𝟐 𝟐𝐕 (r : radii of curvature, B : magnetic field strength, V : accelerating voltage)

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 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 (3) - Linear-TOF • High Sensitivity • Low Resolution - Reflector-TOF • Low Sensitivity • High Resolution - The analytical technique has been extremely useful for proteomics using MALDI-TOF/MS systems. Fig. 10. Diagram of comparison of Linear and Reflector

Mass Analyzer (4) Fig. 11-2. Diagram of a Principle of Ion Cyclotron Resonance Fig. 11-1. Diagram of FT-ICR - 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 (5) - Hyphenated techniques (MS-MS) - Tandem MS modes Fig. 12. Diagram of a Principle of Tandem MS - Hyphenated techniques (MS-MS) - Tandem MS modes → Precursor Ion Scan, Product Ion Scan, Neutral Loss Scan, Selected Reaction Monitoring - High Resolution, Selectivity

Resolution - MS are designed to give a specified Resolving Power. → The minimum acceptable Resolution = One mass unit. - Two masses are considered to be resolved when the valley between their peaks is less than 10% of the smaller peak height. Resolving Power= m 2 m 2 − m 1 - Requirement for unit mass resolution increase with the magnitudes of m1 and m2. m1 m2

Isotope Peaks (1) - 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. Table. 1. Empirical formulae and isotope peak ratios for a nominal RMM value of 70 (M=100%)

Isotope Peaks (2) - The intensities in mass spectra of Isotope Peaks of C24H22O7 (Using the Table. 2.) (M)+ (M+1)+, (M+2)+ Table. 2. Natural isotopic abundances of some common elements as a percentage of the most abundant isotope

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

EI/MS - 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 Fig. 13. Agilent 7000 Series Triple Quadrupole GC/MS

EI/MS - Characteristics Fig. 14. Diagram of a Principle of Agilent 7000 Series Triple Quadrupole GC/MS • Characteristics - GC/MS/MS - Gold Quadrupole - Hexapole Collision Cell - Triple-Axis HED-EM Detector Video

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

EI/MS – Agilent Techniques (2) Fig. 16-2. Diagram of a Principle of Helium Quenching - Using a Helium buffer gas - Reduction of Chemical Noise - High Sensitivity, Resolution Fig. 16-1. Diagram of Agilent 7000 Series Triple Quadrupole GC/MS

EI/MS – Agilent Techniques (3) Fig. 17-2. Diagram of a Principle of Triple-Axis Detector – Ultra low neutrals noise – Long life and high linearity – Superior sensitivity Fig. 17-1. Diagram of Agilent 7000 Series Triple Quadrupole GC/MS

ESI/ETD/MS - 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 Fig. 18. Thermo Orbitrap Elite

ESI/ETD/MS - Characteristics Fig. 19. Diagram of a Principle of Thermo Orbitrap Elite • Characteristics - Ion Optics - Ion Trap with Neutral Blocker - Trap-HCD - Orbitrap Video

ESI/ETD/MS – Thermo Techniques (1) Fig. 20-1. Diagram of Thermo Orbitrap Elite - Variable Spaced Stacked Lenses. → Increasing spacing = increasing field penetration to focus ion beam - Robustness, High Sensitivity Fig. 20-2. Ion Optics

ESI/ETD/MS – Thermo Techniques (2) Fig. 21-1. Diagram of Thermo Orbitrap Elite - Rotated 45o Quadrupole - Blocking the Neutral Beams. - Separation of Neutrals and Ions - More Robustness Fig. 21-2. Ion Trap (Square Quadrupole with Neutral Blocker)

ESI/ETD/MS – Thermo Techniques (3) Fig. 22-1. Diagram of Thermo Orbitrap Elite - Trap-HCD fragmentation (HCD,CID, PQD,ETD) - Dual Pressure Trap - No low mass cut off - High Resolution Fig. 22-2. Diagram of Trap-HCD

ESI/ETD/MS – Thermo Techniques (4) Fig. 23-1. Diagram of Thermo Orbitrap Elite - New type of Ion Trap - Faster Scanning - High Resolution Fig. 23-2. Diagram of Orbitrap

MALDI/MS - 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 Fig. 24. Waters MALDI SYNAPT G2-S HDMS

MALDI/MS - Characteristics Fig. 25. Diagram of a Principle of Waters MALDI SYNAPT G2-S HDMS • Characteristics - Variable Ionization Techniques - T-Wave Ion Guide - TRIWAVE - QUANTOF - HDMS™ instruments Video

MALDI/MS – Waters Techniques (1) Fig. 26. Diagram of Ion Source of Waters MALDI SYNAPT G2-S HDMS -Very simple to exchange Ion Source -Extend Compound Coverage -High Flexibility

MALDI/MS – Waters Techniques (2) Fig. 27-1. Diagram 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 Fig. 27-2. Diagram of T-Wave Ion Guide

MALDI/MS – Waters Techniques (3) Fig. 28-1. Diagram of Waters MALDI SYNAPT G2-S HDMS - Ion Mobility Separation → Being separated by Size, Shape and Charge - Fragmentation(CID, ETD) - Increasing Peak Capacity and Detection limit Fig. 28-2. Diagram of TRIWAVE

MALDI/MS – Waters Techniques (3) Fig. 29. Diagram of Waters MALDI SYNAPT G2-S HDMS -Dual Stage Reflectron -Hybrid Ion Detection System -Compatible with HDMS™ analysis -High Resolution : Over 40,000 FWHM

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 - Thermo Scientific - Waters 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_1115 2_L10710_87170_13901130_-1 - Waters http://www.waters.com/waters/nav.htm?cid=134614100 - Instant Notes, Analytical Chemistry, Kealey & Haines