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Mass Spectrometry (Mass Spec.). Prof. Yonghai Chai. School of Chemistry & Materials Science. For Bilingual Chemistry Education. OUTLINE. Introduction to Mass Spectrometry Ionization Methods Mass Analyzer Fragmentation and MS Interpretation Hyphenated MS Techniques. By James Crawford.

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mass spectrometry mass spec

Mass Spectrometry(Mass Spec.)

Prof. Yonghai Chai

School of Chemistry & Materials Science

For Bilingual Chemistry Education


Introduction to Mass Spectrometry

Ionization Methods

Mass Analyzer

Fragmentation and MS Interpretation

Hyphenated MS Techniques


By James Crawford

How do two people with different languages communicate with each other?

Then, how can I catch up, Ms.?

chemical identification
Chemical Identification
  • Comparison of
  • Physical Properties
    • Boiling Point
    • Melting Point
    • Density
    • Optical rotation
    • Appearance
    • Odor
  • Elemental Analysis
      • Burn the compound and measure the amounts of CO2, H2O and other components that are produced to determine the empirical formula
spectroscopic methods for structure determination
Spectroscopic Methods for Structure Determination

Ultraviolet-Visible (UV/Vis) spectroscopy:

determination of solutions of transition metal ions and highly conjugated organic compounds

Infrared (IR) spectroscopy:

Functional groups

Mass spectrometry (MS):

Molecular mass and formula and structure information

Nuclear magnetic resonance (NMR) spectroscopy:

Map of carbon-hydrogen framework

definition of mass spectrometry
Definition of Mass Spectrometry

Mass spectrometry (MS) :

An analytical technique by using mass spectrometry for the determination of the composition of a sample or molecule and elucidation of the chemical structures of molecules, such as peptides and other chemical compounds.

Mass spectrometry has been described as the smallest scale in the world, not because of the mass spectrometer’s size but because of the size of what it weighs -- molecules.

timeline for ms development
Timeline for MS Development
  • 1897 Early Mass Spectrometry
  • 1919 The observation of isotopes using mass spectrometry
  • 1934 Double Focusing Analyzer
  • 1939 Accelerator Mass Spectrometry
  • 1946 Time-of-Flight Mass Spectrometry
  • 1947 Preparative Mass Spectrometry
  • 1949 Ion Cyclotron Resonance (ICR)
  • Reverse Geometry Double
  • focusing MS
  • 1953 Quadrupole Analyzers

Joseph John Thomson

"In recognition of the great merits of his theoretical and experimental investigations on the conduction of electricity by gases.“

1906 Nobel Prize

"At first there were very few who believed in the existence of these bodies smaller than atoms. I was even told long afterwards by a distinguished physicist who had been present at my [1897] lecture at the Royal Institution that he thought I had been 'pulling their legs."

Cited from:

Replica of J.J. Thomson's third mass spectrometer.

continuation of timeline
Continuation of Timeline
  • 1956 Gas Chromatography Mass Spectrometry (GC/MS)
  • Identifying Organic Compounds with Mass
  • Spectrometry
  • 1962 Mass Spectrometry Imaging
  • 1966 Chemical Ionization
  • 1966 Peptide Sequencing
  • 1966 Tandem Mass Spectrometry
  • 1966 Metabolomics
  • 1968 Electrospray Ionization
  • Collision Induced Dissociation
  • 1969 Field Desorption-MS of Organic Molecule

Francis William Aston

"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."

Mass spectrometry of isotopes

1922 Nobel Prize

Cited from:

continuation of timeline9
Continuation of Timeline

1974 Fourier Transform Ion Cyclotron Resonance

1974 Extra-Terrestrial Mass Spectrometry

1975 Atmospheric Pressure Chemical Ionization (APCI)

1976 Californium-252 Plasma Desorption MS

1978 GC-C-IRMS

1978 Triple Quadrupole Mass Analyzer

1980 Inductively Coupled Plasma MS

1981 Matrix-Assisted Desorption Ionization

1984 Quadrupole/Time-Of-Flight Mass Analyzer

1985 Matrix-Assisted Laser Desorption Ionization (MALDI)

Wolfgang Paul

Hans Georg Dehmelt

“For the development

of the ion trap technique.”

1989Nobel prize

Cited from:

continuation of timeline10
Continuation of Timeline


1987 Soft Laser Desorption of Proteins

1989 ESI on Biomolecules

1989 Monitoring Enzyme Reactions with ESI-MS

1990 Protein Conformational Changes with ESI-MS

1990 Clinical Mass Spectrometry

1991 MALDI Post-Source Decay

1991 Non-covalent Interactions with ESI

1992 Low Level Peptide Analysis

1993 Oligonucleotide Ladder Sequencing

1993 Protein Mass Mapping

1996 Intact Virus Analyses

John B. Fenn


Koichi Tanaka

"For the development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules."

Cited from:

continuation of timeline11
Continuation of Timeline
  • 1998 Electron Capture Dissociation (ECD)
  • 1999 Nanostructure Desorption/Ionization
  • Quantitative Proteomics and Metabolomics with
  • Isotope Labels
  • 2000 Orbitrap
  • 2004 Desorption Electrospray Ionization (DESI)
  • 2004 Electron Transfer Dissociation (ETD)
  • 2005 Direct Analysis in Real Time (DART)

Fred W. McLafferty

Alfred O.C. Nier

Alan G. Marshall

Klaus Biemann

R. Graham Cooks

Donald F. Hunt

Catherine Fenselau

Franz Hillenkamp

Carol V. Robinson

Michael Karas

Malcolm Dole

Brian T. Chait

Cited from:

what information can be determined
What information can be determined?
  • Molecular weight
  • Molecular formula (HRMS)
  • Structure (from fragmentation fingerprint)
  • Isotopic incorporation / distribution
  • Protein sequence (MS-MS)
schematic mass spectrometer
Schematic Mass Spectrometer






what s in a mass spectrum
What’s in a Mass Spectrum

Mass-to-charge ratios of a molecule or its fragment are graphed or tabulated according to their relative abundance

Fragment Ions

Fragment Ions: derived from molecular ion or higher weight fragments


Biomolecule characterization

Pharmaceutical analysis

  • Proteins and peptides
  • Oligonucleotides

Paleoclimatology and Archeology

  • Forensic analysis/clinical
  • Environmental analysis
    • Pesticides on foods
    • Soil and groundwater contamination


O16 and O18


relative abundance of isotopes
Relative Abundance of Isotopes

Atomic weight of an element is a weighted average of the naturally occurring isotopes.

isotopic ratio from the spectra
Isotopic Ratio from the Spectra

Mass spec. can be used to measure the isotopic ratios

continuation of isotopes
Continuation of Isotopes
  • Chlorine (35Cl to 37Cl is 3:1, give M + 2)
ionization methods

Ionization Methods

Electron bomb Ionization (电子轰击离子化) EI

Chemical Ionization (化学电离) CI

Field ionization (场电离) FI

Matrix Assisted Laser Desorption Ionization (基质协助的激光解吸) MALDI

Fast atom bombardment (快原子轰击) FAB

Electro Spray Ionization(电喷雾) ESI

electron bomb ionization ei


Mass Spectrum




Electron Bomb Ionization ( EI )

Sample is heated and energized by a beam of electrons, usually gives a molecular ion (M+) and a lot of fragments。


Properties of EI

  • Hard ionization
    • Gas-phase molecules enter source through heated probe or
    • GC column
    • 70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions
    • EI spectra can be matched to library stds CI (soft ionization)
    • Higher pressure of methane leaked into the source (mtorr)
    • Reagent ions transfer proton to analyte
chemical ionization ci
ChemicalIonization (CI)

Electron ionization leads to fragmentation of the molecular ion, which sometimes prevents its detection.

Chemical ionization (CI):

A technique that produces ions with little excess energy.

Thus this technique presents the advantage of yielding a spectrum with less fragmentation in which the molecular species is easily recognized.

Consequently, chemical ionization is complementary to electron ionization.


Parent Ion

Interface to GC

Insoluble Samples


No Fragment Library

Need Volatile Sample

Need Thermal Stability

Quantitation Difficult

Low Mass Compounds

(<1000 amu)

Solids Probe Requires

Skilled Operator

Properties of CI

field ionization fi

















Field ionization (FI)

Field ionization (FI) is a method that uses very strong electric fields to produce ions from gas-phase molecules.

matrix assisted laser desorption ionization maldi
Matrix Assisted Laser Desorption Ionization (MALDI)

sample is co-crystallized with a matrix and then irradiated

with laser.

MALDI is achieved in two steps. In the first step, the compound to be analyzed is dissolved in a solvent containing in solution small organic molecules, called the matrix. The second step occurs under vacuum conditions inside the source of the mass spectrometer.

properties of maldi
Properties of MALDI

Good solubility

Vapour pressure must be sufficiently low to maintain vacuum conditions

Viscosity must allow diffusion of the analyte from the bulk to the surface

Polar : to solvate and separate preformed ion

Less Sensitive to Salts

Lower PRACTICAL detection limits

Easier to interpret spectra (less multiple charges)

Quick and easy

Higher mass detection

Higher Throughput (>1000 samples per hour)

MALDI mass spectrometry has become a powerful analytical

tool for both synthetic polymers and biopolymers.

Principle of MALDI

fast atom bombardment fab
Fast atom bombardment (FAB)

Softer than EI and CI. Ions are produced by bombardment with heavy atoms. Gives (M+H)+ ions and litle fragmentation. Good for more polar compounds.

Ar + e Ar+ acceleration (5-15 KeV)

Ar+ + Ar Ar + Ar+

fast slow + 8 KeV

fast slow

properties of fab

Parent Ion

High Mass Compounds (10,000 amu)

Thermally Labile Compounds (R.T.)


No Fragment Library

Solubility in Matrix (MNBA, Glycerol)

Quantitation Difficult

Needs Highly Skilled Operator

Relatively Low Sensitivity

Properties of FAB
electrospray ionization esi
ElectroSpray Ionization (ESI)

Electrospray is abbreviated to ESI ,ample is sprayed out of

a narrow nozzle in a high potential field. Generates positive

(M+nH)n+ and negative (M - nH)n- ions and almost no

fragmentation. Generates multiple charged ions.

properties of esi

Electrospray Ionization can be

easily interfaced to LC.

Absolute signals from

Electrospray are more easily

reproduced, therefore, better


Mass Accuracy is considered


Multiple charging is more

common then MALDI.


No Fragmentation

Need Polar Sample

Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best)

Sensitive to Salts


Properties of ESI
types of mass analyzers
Types of Mass Analyzers

Magnetic sector analyzer (磁分析器)

Time of Flight analyzer (TOF) (飞行时间分析器)

Quadrupole analyzers (四极滤质器)

Fourier Transform Ion-Cyclotron (傅立叶离子回旋共


magnetic sector analyzer
Magnetic SectorAnalyzer

Magnetic sector analyzer – Uses electric and/or magnetic fields to separate ions

principle of magnetic sector analyzer
Principle of Magnetic SectorAnalyzer

The ion source accelerates ions to a kinetic

energy given by : (1/2)m2= zV

Where m is the mass of the ion ,v is its velocity, z

is the charge on the ion ,and V is the applied

voltage of the ion optics.

principle of magnetic sector analyzer40
Principle of Magnetic SectorAnalyzer

Only ions of mass-to-charge ratio that have equal centripetal and centrifugal forces pass through the flight tube: m v 2 / r = Bzv

By rearranging the equation, m/z = B2r2/2V

It shows that the m/q ratio of the ions that reach the detector can be varied by changing either the magnetic field or the applied voltage of the ion optics.

In summary ,by varying the voltage or magnetic field of the magnetic-sector analyzer ,the individual ion beams are separated spatially and each has a unique radius of curvature according to its mass/charge ratio.

Double focusing magnetic sector mass analyzers are the "classical" model against which other mass analyzers are compared.

  • Classical mass spectra
  • Very high reproducibility
  • Best quantitative performance of all MS analyzers
  • High resolution
  • High sensitivity
  • 10,000 Mass Range
  • Linked scan MS/MS does not require another analyzer
  • Requires Skilled Operator
  • Usually larger and higher cost than other mass analyzers
  • Difficult to interface to ESI
  • Low resolution MS/MS without multiple analyzers
  • Applications
  • All organic MS analysis methods
  • Accurate mass measurements
  • Quantitation
  • Isotope ratio measurements
time of flight analyzer
Time of Flight Analyzer

TOF analyzer – ions are accelerated through a flight tube and the time of light to the detector is measured

principle of tof analyzer
Principle of TOF Analyzer
  • Uses a pulse of ion mixtures, not steady stream
  • Ions accelerated into drift tube by a pulsed electric
  • field called the ion-extraction field
  • Drift Tube is usually 1-2 m long, under vacuum
  • Ions traverse the drift tube at different speeds
  • ( L / t ) = v = ( 2zV / m )½
advantages of tof analyzer
Advantages of TOF Analyzer
  • Good for kinetic studies of fast reactions and for

use with gas chromatography to analyze peaks

from chromatograph

  • High ion transmission
  • Can register molecular ions that decompose in the

flight tube

  • Extremely high mass range (>1MDa)
  • Fastest scanning
  • Requires pulsed ionization method or ion beam switching (duty cycle is a factor)
  • Low resolution (4000)
  • Limited precursor-ion selectivity for most MS/MS experiments
  • Applications
  • Almost all MALDI systems
  • Very fast GC/MS systems
quadrupole analyzers
Quadrupole Analyzers

Quadrupole analyzers – ions are filtered or trapped in a

device consisting of several metal rods using specifically tailored electromagnetic fields

quadrupole analyzers50
Quadrupole Analyzers
  • Electric/magnetic fields trap, store, eject ions
  • Requires an in-line quadrupole to act as
  • mass pre-filter
  • Contains a single ring electrode and a top
  • and bottom cap electrode
  • Varying RF frequency will vary the m/z ratios
  • that are trapped
  • Additional fragmentation can be performed
  • on ions stored in the ion trap
  • Easy to use ,simple construction,fast
  • Good reproducibility
  • Relatively small and low-cost systems
  • Quadrupoles are now capable of routinely

analyzing up to a m/q ratio of 3000,which is

useful in electrospary ionization of biomolecules,

which commonly produces a charge distribution

below m/z 3000

  • Low resolution(<4000)
  • Slow scanning
  • Low accuracy (>100ppm)
  • Applications
  • Majority of benchtop GC/MS and LC/MS systems
  • Separation of proteins and other biomolecules

with electrosprary

  • Sector / quadrupole hybrid MS/MS systems
Most FTICR mass spectrometers use superconducting magnets, which provide a relatively stable calibration over a long period of time.

Although some mass accuracy can be obtained without internal calibrant, mass accuracy and resolution are inversely proportional to m/z, and the best accurate mass measurements require an internal calibrant.

Unlike the quadrupole ion trap, the FTICR mass spectrometer is not operated as a scanning device.

  • The highest recorded mass resolution of all mass

spectrometers (>500,000)

  • Very good accuracy (<1ppm)
  • Well-suited for use with pulsed ionization

methods such as MALDI

  • Non-destructive ion detection; ion remeasurement
  • Stable mass calibration in superconducting

magnet FTICR systems

  • Expensive
  • Requires superconducting magnet
  • Subject to space charge effects and ion molecule reactions
  • Artifacts such as harmonics and sidebands are present in the mass spectra
  • Many parameters (excitation, trapping, detection conditions) comprise the experiment sequence that defines the quality of the mass spectrum
  • Generally low-energy CID, spectrum depends on collision energy, collision gas, and other parameters.
  • Ion chemistry
  • High-resolution MALDI and electrospray

experiments for high-mass analytes

  • Laser desorption for materials and surface



The Mass Spectrum

    • Presentation of data
      • The mass spectrum is presented in terms of ion abundance vs. m/e ratio (mass).
      • The most abundant ion formed in ionization gives rise to the tallest peak on the mass spectrum – this is thebase peak.

base peak, m/e 43


Presentation of data

    • 3.All other peak intensities are relative to the base peak as a percentage.
    • 4. If a molecule loses only one electron in the ionization process, a molecular ion is observed that gives its molecular weight – this is designated as M+ on the spectrum.

M+, m/e 114


Presentation of data

    • 5. In most cases, when a molecule loses a valence electron, bonds are broken, or the ion formed quickly fragment to lower energy ions
    • 6. The masses of charged ions are recorded asfragment ionsby the spectrometer –neutral fragments are not recorded !

fragment ions


B. Determination of Molecular Mass

    • 1. When a M+ peak is observed it gives the molecular mass –assuming that every atom is in its most abundant isotopic form
    • 2. Remember that carbon is a mixture of 98.9% 12C (mass 12), 1.1% 13C (mass 13) and <0.1% 14C (mass 14)
    • 3. We look at a periodic table and see the atomic weight of carbon as 12.011 – an average molecular weight
    • 4. The mass spectrometer, by its very nature would see a peak at mass 12 for atomic carbon and a M + 1 peak at 13 that would be 1.1% as high
    • - We will discuss the effects of this later…

B. Determination of Molecular Mass

    • 5. The Nitrogen Rule is another means of confirming the observance of a molecular ion peak
    • 6. If a molecule contains an even number of nitrogen atoms (only ‘common’ organic atom with an odd valence) or no nitrogen atoms the molecular ion will have an even mass value
    • 7. If a molecule contains an odd number of nitrogen atoms, the molecular ion will have an odd mass value
    • 8. If the molecule contains chlorine or bromine, each with two common isotopes, the determination of M+ can be made much easier, or much more complex as we will see.

The Rule of Thirteen – Molecular Formulas from Molecular Mass – Lecture 1

When a molecular mass, M+, is known, a base formula can be generated from the following equation:

M / 13 = ( n + r ) / 13

The base formula being:CnHn + r

For this formula, the HDI can be calculated from the following formula:

HDI = ( n – r + 2 ) / 2

Remember and Review


The Rule of Thirteen

The following table gives the carbon-hydrogen equivalents and change in HDI for elements also commonly found in organic compounds:

Remember and Review


C. High Resolution Mass Spectrometry

    • 1. If sufficient resolution (R > 5000) exists, mass numbers can be recorded to precise values (6 to 8 significant figures)
    • 2. From tables of combinations of formula masses with the natural isotopic weights of each element, it is often possible to find anexactmolecular formula from HRMS
    • Example: HRMS gives you a molecular ion of 98.0372; from mass 98 data:
      • C3H6N4 98.0594
      • C4H4NO2 98.0242
      • C4H6N2O 98.0480
      • C4H8N3 98.0719
      • C5H6O2 98.0368  gives us the exact formula
      • C5H8NO 98.0606
      • C5H10N2 98.0845
      • C7H14 98.1096

D. Exact Mass Determination

1. Need Mass Spectrometer with a high mass accuracy – 5 ppm (sector or TOF)

2. C9H15NO4, FM 201.1001 (mono-isotopic)

3. Mass accuracy = {(Mass Error)/FM}*106

4. Mass Error = (5 ppm)(201.1001)/106 =  0.0010 amu

E. Mass accuracy

  • 1. Mass Error = (5 ppm)(201.1001)/106 =0.0010 amu
  • 2. 201.0991 to 201.1011 (only 1 possibility)
  • 3. Sector instruments, TOF mass analyzers
  • 4. How many possibilities with MA = 50 ppm? with 100 ppm?

F. Important fragmentation patterns in EI

Fragmentation leads to smaller ions by the cleaving of parts of molecule

  • Unreasonable losses from molecular ion:

M – [3~ 14] and M – [21~26] are unraesonable losses!

  • Reasonable losses from molecular ion:

Neutral fragments expelled by simple cleavage

OE+· → EE+ + OE·

Neutral fragments expelled by multi-centered fragments

OE+· → EE + OE +·


1. Simple cleavage

  • Radical Remote Fragmentation (a-cleavage)

i. Compounds containing saturated heteroatoms

ii. Compounds containing unsaturated heteroatoms

iii. Compounds containing unsaturated carbon-carbon bonds


Charge Remote Fragmentation ( i-cleavage)

a-Cleavage and i-cleavage are competitive reactions.

  • The sequence of a-cleavage tendency:

N > S, O, p bond, R > Cl > Br > I

  • The sequence of i-cleavage tendency:

halogen > O, S >> N, C


Compounds without heteroatoms ( s-cleavage)





2. Rearrangement

  • McLafferty rearrangement

Pattern I


Pattern II


Second McLafferty rearrangement


G. Patterns of different organic compounds’ fragmentation

  • Saturated hydrocarbons(饱和烃)

1. Alkanes (直链烷烃)


Figure 1 Mass spectrum of dodecane.


2. Branched Alkanes (支链烷烃)

Figure 2 Mass spectrum of 5-methylpentadecane.



Figure 3 Mass spectrum of 4-methylundecane.

Figure 4 Mass spectrum of 2,2,4,6,6-pentamethylheptane.


3. Cycloalkanes (环烷烃)

Figure 5 Mass spectrum of cyclohexane.



Figure 6 Mass spectrum of 1-methyl-3-pentylcyclohexane.


Aromatic hydrocarbons (芳烃)

Figure 7 Mass spectrum of 1-phenylhexane


Alcohols, Phenol and Ether

1. Alcohol

Figure 8 Mass spectrum of 1-dodecanol


3. Ether

Figure 9 Mass spectrum of hexyl ether.


Ketone and Aldehyde (酮和醛)

Figure 10 Mass spectrum of 2-dodecanone.



Figure 12 Mass spectrum of hexyl benzoate.


Other compounds (其它化合物)

Figure 13 Mass spectrum of 1-chlorododecane.


Exercise 1:

HRMS shows exact mass of compound A is 136.0886 and the formula of this

compound is C9H12O, please confirm the structure of compound A.


DEB: Ω = (2*9+2-12)/2 = 4

m/z: 118 M-18 M-H2O


39, 51, 77

107 M-29 M-C2H5



Exercise 2:

Please confirm the structure of compound A.

I158 = 13%

I157 = 3.7%

I156 = 41%



1. I156/I158 = 3/1

Containing one Cl atom

Containing eight C atoms

2. Nc = 3.7/41÷1.1%≈8

3. m/z: 39, 51, 77

; 94



4. 156-35-77-16-14 = 14


Exercise 3:

Based on the EI mass spectrum of compound A, please write the

process of fragmentation


Hyphenated Mass Techniques

Chromatography: Separation

Mass: Detection

Chromatography-Mass Spectroscopy :

Separation + Detection














Mass Spectrometer

Gas Chromatograph (GC)

























Gas chromatography-mass spectrometry (GC-MS) is a method that combines

the features of gas-liquid chromatography and mass spectrometry to identify

different substances within a test sample.







  • The sample is injected into the GC inlet where it is heated and swept onto a chromatographic column by a carrier gas.
  • The pure compounds in a mixture are separated by interacting with the coating or packing of the column (stationary phase) and the carrier gas (mobile phase).
  • This separation is often improved by programming changes in column temperature and pressure.






Liquid chromatography-mass spectrometry (LC-MS) is an analytical

chemistry technique that combines the physical separation capabilities of

liquid chromatography with the mass analysis capabilities of mass


Different compounds exit

at different time

Identification of each molecule




Peak A: mass1

Peak B: mass2

Peak C: mass3


Hyphenated LC-MS

Liquid chromatography-mass spectrometry (Ion trap LCMS system )


Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS, involves multiple

steps ofmass spectrometry selection, with some form of fragmentation

occurring in between the stages.