Mass Spectrometry (MS) • Technique for studying the masses of atoms or molecules or fragments of molecules • Provides information about … o o o o o

Elemental composition Molecular structure Composition of complex mixtures Structure & composition of solid surfaces Isotopic ratios of atoms • Discriminate between 79Br & 81Br

Mass Spectrometry Applications

Skoog, Table 20-5

Mass Spectrometry Beginnings • Aston (Cambridge) o

1919 – Discovered two isotopes of neon (20Ne & 22Ne)

• 1922 – Nobel Prize in Chemistry o

“for his discovery, by means of his mass spectrograph, of isotopes, in a large number of non-radioactive elements, and for his enunciation of the whole-number rule"

• Discovered 212 of 281 naturally occurring isotopes www.nobel.se

Francis William Aston

Obtaining a Mass Spectrum • Gaseous molecules desorbed from condensed phases are ionized • Ions are accelerated by an electric field • Ion separation by mass-to-charge ratio (m/z)

ethyl benzene

Skoog, Fig. 20-1

The Instrument • Sample ionization o o

Gas-phase Desorption

• Mass analyzer ~ monochromator o o o o

Quadrupole (Q) Time-of-flight (TOF) Double-focusing (B, E) Ion trap

• Ion transducer o o

Skoog, Fig. 11-1

Electron multiplier Faraday cup

Ion Transducers • Electron multiplier o

o o

Analogous to a photomultiplier tube (PMT) Rugged & reliable Current gain ~ 107

• Faraday cup o o

o

Inexpensive Simple mechanically & electrically Less sensitive than electron multiplier Skoog, Fig. 11-2(b), 11-3

Ion Sources Basic Type

Name and Acronym

Ionizing Agent

Gas phase

Electron impact (EI)

Energetic electrons

Chemical ionization (CI)

Reagent gaseous ions

Field ionization (FI)

High-potential electrode

Field desorption (FD)

High-potential electrode

Electrospray ionization (ESI)

High electrical field

Matrix-assisted desorption/ionization (MALDI)

Laser beam

Plasma desorption (PD)

Fission fragments from 252Cf

Fast atom bombardment (FAB)

Energetic atomic beam

Secondary ion mass spectrometry (SIMS)

Energetic beam of ions

Thermospray ionization (TS)

High temperature

Desorption

Skoog, Table 20-1

Electron Ionization (EI) • Electrons accelerated through potential of 70 V and interact with incoming molecules

M

+

e– 70 eV

→

M+• Molecular ion

• Interaction with 70-eV electron will likely remove electron with lowest ionization energy o

+

e– ~55 eV

Formaldehyde

n < < 

Harris, 6th ed., Fig. 22-3

+

e– 0.1 eV

Electron Ionization (EI)

• Path of electrons & molecules are at right angles o

Collide to produce mostly singly-charged positive ions

• Inefficient process Skoog, Fig. 20-3

Chemical Ionization (CI) • Ionization source is filled with a reagent gas o

CH4, C4H10, NH3, H2, CH3OH, NO

• Energetic electrons (100 – 200 eV) convert CH4 to a variety of reactive products: CH 4 e   CH 42 e     CH   CH  CH 4 4 5  CH 3    CH   CH  H 4 3  CH   CH  C H 3 4 2 5 H 2

 CH   M  CH  MH 5 4  5

C 2 H M  C 2 H 4 MH



EI vs. CI Mass Spectra BASE PEAK

• Hard source o

• Soft source

More fragmentation

o

• Structural information o

Functional groups

Less fragmentation

• Molecular weight information

Harris, Fig. 21-14

EI vs. CI Mass Spectra • 1-Decanol mass spectra • Hard source o o

More fragments Structural info

• Soft source o o

Skoog, Fig. 20-2

Less fragments MW info

Electrospray Ionization (ESI) • Sample typically in form of solution (organic or aqueous) • Excess solvent must be removed before entering MS o

Large increase in pressure from solvent vaporization

• Differential solvent removal o o o

o

Solution passed through stainless steel capillary tube Apply high electric potential (3 – 5 kV) Solvent evaporates rapidly from droplet surface and droplets get smaller and smaller Solvent molecules diffuse away

Electrospray Ionization (ESI)

Harris, 6th ed., Fig. 22-16 (b); (Skoog, Fig. 20-8)

Laser Desorption Ionization (LDI) • Molecular system exposed to laser beam has its internal energy greatly increased o o o o

Melting Vaporization Ionization Decomposition

• Process of beaming laser light onto small area of sample specimen to desorb ions Herbert, C. G.; Johnstone, R. A. W.; Mass Spectrometry Basics; 2003, p. 8

Matrix-Assisted Laser Desorption Ionization (MALDI) • Aqueous/alcohol solution of sample is mixed with radiation-absorbing matrix material o

Matrices (Skoog, Table 20-4)

• Solution evaporated on metallic probe surface • Solid mixture is exposed to pulsed laser beam o

Analyte is sublimed as ions

• Useful for obtaining accurate molecular weights of biopolymers Source: http://www.srsmaldi.com

to TOF-MS

ESI & LDI Pioneers • Fenn (Virginia Commonwealth) & Tanaka (Shimadzu) • 2002 – Nobel Prize in Chemistry o

o

"for the development of methods for identification and structure analyses of biological macromolecules" "for their development of soft desorption ionization methods for mass spectrometric analyses of biological macromolecules"

Source: http://www.nobel.se

John B. Fenn

Koichi Tanaka

Fast Atom Bombardment (FAB) • Focus a high primary current beam of neutral atoms or molecules on sample • Sample dissolved in non-volatile liquid matrix • Inert gas atoms are ionized to give positive ions • As ions collide with other inert gas atoms (He, Ar, Xe), charge exchange occurs o

Fast-moving ions become fastmoving atoms

Source: http://www-methods.ch.cam.ac.uk/meth/ms/theory/fab.gif

Secondary Ion Mass Spectrometry (SIMS) • Focus a high primary current beam of ions on sample • Sample dissolved in nonvolatile liquid matrix • Dynamic SIMS o o

Current beam high enough to damage surface Elemental and isotopic information obtained

• Static SIMS o o

Dedicated to analysis of top monolayer of surface Fresh layer of new ions continuously brought to surface

Source: http://www.chemistry.wustl.edu/~walker/sims_exp.gif; http://www.ulb.ac.be/sciences/cpmct/images/logosims.gif

Magnetic Sector (B) • Ions deflected according to their mass • Spectrum obtained by changing the field strength 2 2

m Br  z 2V

m/z = mass-to-charge ratio B = magnetic field strength r = radii of curvature (trajectory) V = accelerating voltage Harris, 6th ed., Fig. 22-2; (Skoog, Fig. 20-12)

Electrostatic Sector (E) • Ions deflected according to their kinetic energy o

KE = ½mv2

Harris, 6th ed., Fig. 22-12

Double-Focusing • Combination of magnetic & electrostatic sectors o

Improved resolving power • Resolution of 105

o o

Compatible with chromatographic columns Compact

• Configurations o

Mattauch-Herzog • Skoog, Fig. 11-9

o

Nier-Johnson • Skoog, Fig. 20-13

Mattauch-Herzog

Source: http://www.oup.com/images/booksites/higson/higson_fig9.8.jpg

Double-Focusing Ion Optics Forward Geometry EB Configuration (Nier-Johnson)

Reverse Geometry BE Configuration Herbert, C. G.; Johnstone, R. A. W.; Mass Spectrometry Basics; 2003, p. 178-179

Time-of-Flight (TOF)

Harris, 6th ed., Fig. 22-14; (Skoog, Fig. 11-8)

Quadrupole (Q)

Harris, Fig. 21-13; (Skoog, Fig. 11-4)

Ion-Trap

Harris, 6th ed., Fig. 22-15; (Skoog, Fig. 20-15)

Hyphenated MS Methods • Mass spectrometer = detector for other analytical techniques o

Mass spectra collected as compounds exit

• Chromatography/MS o o

Gas Chromatography/MS (GC-MS) Liquid Chromatography/MS (LC-MS)

• Capillary Electrophoresis/MS (CE-MS)

Chromatography/Mass Spectrometry • MS requires high vacuum o

Avoid molecular collisions during ion separation

• Chromatography is high-pressure technique o

Must remove huge excess matter between the chromatograph and the spectrometer

• For GC, narrow capillary column connected directly to inlet of the mass spectrometer • For LC, liquid from column creates huge volume of gas when vaporized o o

Pneumatically assisted electrospray Atmospheric pressure chemical ionization (APCI)

APCI • Uses heat and coaxial flow of N2 to convert eluate into a fine aerosol mist • Creates new ions from gas-phase reactions between ions & molecules • High voltage is applied to metal needle in the path of the aerosol

Harris, 6th ed., Fig. 22-18

Liquid Chromatography/MS (LC-MS)

Harris, 6th ed., Fig. 22-16 (a)

Gas Chromatography/MS (GC-MS) • Must remove most of the carrier gas from the analyte • Quadrupole or ion trap mass analyzers used

Skoog, Fig. 27-13, 27-14

Capillary Electrophoresis/MS (CE-MS) • Capillary effluent is passed into an electrospray ionization device • Products enter quadrupole mass analyzer • Detection limits: tens of femtomoles (10–14 M)

Skoog, Fig. 30-7

Chromatography/MS Spectra

GC CE

Herbert, C. G.; Johnstone, R. A. W.; Mass Spectrometry Basics; 2003, p. 264

Tandem Mass Spectrometry (MS/MS) First MS • Isolates molecular ions • Soft ionization source o

Molecular ions or protonated molecular ions • “Parent” ions

• Analogous to chromatographic column o

Provides pure ionic species for second spectrometer

Second MS • Fragments ions o

Collisions between ions & He atoms cause further fragmentation • “Daughter” ions

• Provides series of mass spectra for each molecular ion produced

QQQ Tandem Instrument

• Q1 & Q3 are regular quadrupole filters • Q2 is a collision focusing chamber o o

Helium pumped into chamber & collides with parent ions Operates in rf-mode only • Focuses scattered ions but does not act as a mass filter Skoog, Fig. 20-24

The Mass Spectrum • Molecular ion (M+•) = unknown molecular mass • M+• breaks apart efficiently with EI o

Fragments provides clues about structure

• CI mass spectrum has strong MH+ peak o

Molecular mass information

• Nitrogen Rule o

Odd nominal mass for M+• • Odd # N atoms

o

Even nominal mass for M+• • Even # N atoms Harris, Fig. 21-14

Molecular Ion & Isotope Patterns • M+●peak is base peak for aromatic compounds o

EI spectra

• Next higher mass peak provides elemental composition info o

M + 1 peak

• Carbon o o

12C

98.92 % 1.08 % 13C

Intensity of M + 1 relative to M+● for CnHm: Intensity = n × 1.08 % + m × 0.012 %

• Hydrogen o

0.012 % 2H

13C

2H

Benzene: Intensity = 6 × 1.08 % + 6 × 0.012 % = 6.55 % Biphenyl: Intensity = 12 × 1.08 % + 10 × 0.012 % = 13.1 % Harris, Fig. 21-18

Molecular Ion & Isotope Patterns

Harris, Table 21-1

Rings and Double Bonds • Rings + double bonds (R + DB) formula o

Used if composition of a molecular ion is known

h n R DB c   1 2 2 c = # of Group 14 atoms (e.g., C, Si) [make 4 bonds] h = # of (H + halogen) atoms [make 1 bond] n = # of Group 15 atoms (e.g., N, P) [make 3 bonds]

 22 11  11 R DB  14 1   1 5 rings & double bonds 2

2

Harris, 6th ed., p. 526 figure

Identifying the Molecular Ion (M+●) Peak • Highest m/z value of any “significant” peaks o

~ 5 – 20% of base peak intensity

• Isotopic peak intensity (M+1, M+2, etc.) must be consistent with proposed chemical composition • Heaviest fragment ion must correspond to a probable mass loss o o

Loss in 3 – 14 or 21 – 25 Da range rare Common mass losses 15 Da (●CH3)

29 Da (●C2H5)

17 Da (●OH or NH3)

31 Da (●OCH3)

18 Da (H2O)

43 Da (●C3H7 or CH3CO●)

Fragmentation Patterns

Harris, Fig. 21-26, 21-17

Interpreting Fragmentation Patterns

• Highest peak of “significant” intensity = m/z 100 • Next highest peak at m/z 85 (loss of ●CH3) • M+●has an even mass o

Nitrogen rule  cannot be an odd number of N atoms in molecule Harris, 6th ed., Fig. 22-10

Interpreting Fragmentation Patterns

observed (M 1)/M intensity 6% Number of C atoms   6 contribution per carbon atom 1.08 %

Intensity = 6 × 1.08 % + 12 × 0.0012 % + 1 × 0.038 % = 6.7 % of M+● 13C

2H

17O

R + DB = c – h/2 + n/2 + 1 = 6 – 12/2 + 0 + 1 = 1 ring or double bond Harris, 6th ed., Fig. 22-10

Fragmentation of 2-Hexanone

Harris, 6th ed., Fig. 22-11