Mass Spectrometry (MS)

• Technique for studying the masses of atomsor molecules or fragments of molecules

• Provides information about …o Elemental compositiono Molecular structureo Composition of complex mixtureso Structure & composition of solid surfaceso 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 hismass spectrograph, of isotopes, ina large number of non-radioactiveelements, and for his enunciation ofthe whole-number rule"

• Discovered 212 of 281 naturallyoccurring isotopes

www.nobel.se

Francis William Aston

Obtaining a Mass Spectrum

• Gaseous molecules desorbed from condensedphases are ionized

• Ions are accelerated by an electric field• Ion separation by mass-to-charge ratio (m/z)

Skoog, Fig. 20-1

ethyl benzene

The Instrument• Sample ionization

o Gas-phaseo Desorption

• Mass analyzer ~monochromatoro Quadrupole (Q)o Time-of-flight (TOF)o Double-focusing (B, E)o Ion trap

• Ion transducero Electron multipliero Faraday cup

Skoog, Fig. 11-1

Ion Transducers

• Electron multipliero Analogous to a

photomultiplier tube (PMT)o Rugged & reliableo Current gain ~ 107

• Faraday cupo Inexpensiveo Simple mechanically

& electricallyo Less sensitive than

electron multiplier

Skoog, Fig. 11-2(b), 11-3

Ion Sources

Skoog, Table 20-1

High temperatureThermospray ionization (TS)

Energetic beam of ionsSecondary ion mass spectrometry (SIMS)

Energetic atomic beamFast atom bombardment (FAB)

Fission fragments from 252CfPlasma desorption (PD)

Laser beamMatrix-assisted desorption/ionization (MALDI)

High electrical fieldElectrospray ionization (ESI)

High-potential electrodeField desorption (FD)Desorption

High-potential electrodeField ionization (FI)

Reagent gaseous ionsChemical ionization (CI)

Energetic electronsElectron impact (EI)Gas phase

Ionizing AgentName and AcronymBasic Type

Electron Ionization (EI)

• Electrons accelerated through potential of 70 Vand interact with incoming molecules

• Interaction with 70-eVelectron will likely removeelectron with lowestionization energyo n < <

M + e– → M+• + e– + e–

70 eV Molecular ~55 eV 0.1 eVion

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

Formaldehyde

Electron Ionization (EI)

• Path of electrons & molecules are at right angleso Collide to produce mostly singly-charged positive ions

• Inefficient process

Skoog, Fig. 20-3

Chemical Ionization (CI)

• Ionization source is filled with a reagent gaso CH4, C4H10, NH3, H2, CH3OH, NO

• Energetic electrons (100 – 200 eV) convert CH4 toa variety of reactive products:

MHHCMHC

MHCHMCH

4252

45

25243

34

3544

44

HHCCHCH

HCHCH

CHCHCHCH

e2CHeCH

EI vs. CI Mass Spectra

• Hard sourceo More fragmentation

• Structural informationo Functional groups

Harris, Fig. 21-14

BASE PEAK

• Soft sourceo Less fragmentation

• Molecular weightinformation

EI vs. CI Mass Spectra

• 1-Decanol massspectra

• Hard sourceo More fragmentso Structural info

• Soft sourceo Less fragmentso MW info

Skoog, Fig. 20-2

Electrospray Ionization (ESI)

• Sample typically in form of solution (organic oraqueous)

• Excess solvent must be removed before enteringMSo Large increase in pressure from solvent vaporization

• Differential solvent removalo Solution passed through stainless steel capillary tubeo Apply high electric potential (3 – 5 kV)o Solvent evaporates rapidly from droplet surface and

droplets get smaller and smallero Solvent molecules diffuse away

Electrospray Ionization (ESI)

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

Laser Desorption Ionization (LDI)

• Molecular systemexposed to laser beamhas its internal energygreatly increasedo Meltingo Vaporizationo Ionizationo Decomposition

• Process of beaminglaser light onto smallarea of samplespecimen to desorb ions

Herbert, C. G.; Johnstone, R. A. W.; Mass Spectrometry Basics; 2003, p. 8

Matrix-Assisted Laser DesorptionIonization (MALDI)

• Aqueous/alcohol solution ofsample is mixed withradiation-absorbing matrixmaterial

o Matrices (Skoog, Table 20-4)

• Solution evaporated onmetallic probe surface

• Solid mixture is exposed topulsed laser beam

o Analyte is sublimed as ions

• Useful for obtaining accuratemolecular weights ofbiopolymers

Source: http://www.srsmaldi.com

to TOF-MS

ESI & LDI Pioneers

• Fenn (Virginia Commonwealth) &Tanaka (Shimadzu)

• 2002 – Nobel Prize in Chemistryo "for the development of methods for

identification and structure analysesof biological macromolecules"

o "for their development of softdesorption ionization methods formass spectrometric analyses ofbiological macromolecules"

Source: http://www.nobel.se

Koichi Tanaka

John B. Fenn

Fast Atom Bombardment (FAB)• Focus a high primary current

beam of neutral atoms ormolecules on sample

• Sample dissolved in non-volatileliquid matrix

• Inert gas atoms are ionized togive positive ions

• As ions collide with other inertgas atoms (He, Ar, Xe), chargeexchange occurs

o Fast-moving ions become fast-moving 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 non-

volatile liquid matrix• Dynamic SIMS

o Current beam high enough todamage surface

o Elemental and isotopic informationobtained

• Static SIMSo Dedicated to analysis of top

monolayer of surfaceo 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 deflectedaccording to theirmass

• Spectrum obtainedby changing thefield strength

Harris, 6th ed., Fig. 22-2; (Skoog, Fig. 20-12)

2VrB

zm 22

m/z = mass-to-charge ratioB = magnetic field strengthr = radii of curvature (trajectory)V = accelerating voltage

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

sectorso Improved resolving power

• Resolution of 105

o Compatible with chromatographic columnso Compact

• Configurationso Mattauch-Herzog

• Skoog, Fig. 11-9

o Nier-Johnson• Skoog, Fig. 20-13

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

Mattauch-Herzog

Double-Focusing Ion Optics

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

ForwardGeometry

EBConfiguration

(Nier-Johnson)

ReverseGeometry

BEConfiguration

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 otheranalytical techniqueso Mass spectra collected as compounds exit

• Chromatography/MSo Gas Chromatography/MS (GC-MS)o Liquid Chromatography/MS (LC-MS)

• Capillary Electrophoresis/MS (CE-MS)

Chromatography/Mass Spectrometry

• MS requires high vacuumo Avoid molecular collisions during ion separation

• Chromatography is high-pressure techniqueo Must remove huge excess matter between the

chromatograph and the spectrometer

• For GC, narrow capillary column connected directlyto inlet of the mass spectrometer

• For LC, liquid from column creates huge volume ofgas when vaporizedo Pneumatically assisted electrosprayo 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 ofthe carrier gas from theanalyte

• Quadrupole or ion trapmass analyzers used

Skoog, Fig. 27-13, 27-14

Capillary Electrophoresis/MS (CE-MS)

• Capillary effluent is passed into an electrosprayionization device

• Products enter quadrupole mass analyzer• Detection limits: tens of femtomoles (10–14 M)

Skoog, Fig. 30-7

Chromatography/MS Spectra

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

GC

CE

Tandem Mass Spectrometry (MS/MS)

First MS• Isolates molecular ions• Soft ionization source

o Molecular ions orprotonated molecular ions

• “Parent” ions

• Analogous tochromatographic columno Provides pure ionic

species for secondspectrometer

Second MS• Fragments ions

o Collisions between ions& He atoms causefurther fragmentation

• “Daughter” ions

• Provides series of massspectra for eachmolecular ion produced

QQQ Tandem Instrument

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

o Helium pumped into chamber & collides with parent ionso Operates in rf-mode only

• Focuses scattered ions but does not act as a mass filterSkoog, Fig. 20-24

The Mass Spectrum• Molecular ion (M+•) =

unknown molecular mass• M+• breaks apart efficiently

with EIo Fragments provides clues

about structure

• CI mass spectrum hasstrong MH+ peako Molecular mass information

• Nitrogen Ruleo 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 compoundso EI spectra

• Next higher mass peakprovides elementalcomposition infoo M + 1 peak

• Carbono 98.92 % 12Co 1.08 % 13C

• Hydrogeno 0.012 % 2H

Intensity of M + 1 relative to M+●for CnHm:

Intensity = n × 1.08 % + m × 0.012 %

13C 2H

Harris, Fig. 21-18

Benzene: Intensity = 6 × 1.08 % + 6 × 0.012 % = 6.55 %

Biphenyl: Intensity = 12 × 1.08 % + 10 × 0.012 % = 13.1 %

Molecular Ion & Isotope Patterns

Harris, Table 21-1

Rings and Double Bonds

• Rings + double bonds (R + DB) formulao Used if composition of a molecular ion is known

12n

2h

cDBR

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]

bondsdouble&rings51

211

21122

114DBR

Harris, 6th ed., p. 526 figure

Identifying the Molecular Ion (M+●) Peak

• Highest m/z value of any “significant” peakso ~ 5 – 20% of base peak intensity

• Isotopic peak intensity (M+1, M+2, etc.) must beconsistent with proposed chemical composition

• Heaviest fragment ion must correspond to aprobable mass losso Loss in 3 – 14 or 21 – 25 Da range rareo Common mass losses

43 Da (●C3H7 or CH3CO●)18 Da (H2O)

31 Da (●OCH3)17 Da (●OH or NH3)

29 Da (●C2H5)15 Da (●CH3)

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 inmolecule

Harris, 6th ed., Fig. 22-10

Interpreting Fragmentation Patterns

Harris, 6th ed., Fig. 22-10

6%1.08

%6atomcarbonperoncontributi

intensity1)/M(MobservedatomsCofNumber

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

Fragmentation of 2-Hexanone

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