MASS SPECTROMETRY LECTURE

NOTES 2O18
Instrumentation, Ionization methods, Mass analyzers,
qualitative and quantitative analysis, Fourier
transform Mass spectrometry, tandem mass spectroscopy,
secondary ion mass spectroscopy.
Coupled
techniques such as GC-MS, CE-MS.
Basic theory of mass spectrometry

The mass spectrometer(MS) is an instrument that serves for establishment of

the molecular weight and structure of both inorganic and organic compounds,
and the identification and determination of analytes in complex mixtures.
The MS is an instrument capable of producing a beam of ions from sample
under investigation, separating these ions according to their mass-to charge
(m/z) ratios and recording the relative abundances of the separated ion species
as a mass spectrum. The ion-currents corresponding to the different species are
amplified and either displayed on an oscilloscope or a chart-recorder, or are
stored in a computer. The peak intensities are plotted as ordinates, in arbitrary
units or normalized with respect to the most important peak, which is assigned a
value of 100.
 100

128
80

Relative abundance (%)

a

60

40

20 102
64 77 c
c c
51 b
b b b
0
50 100 150
Mass to charge ratio m/z

Mass spectrum of CO2. Note that the molecular
ion appears at m/z = 44 (C = 12, O = 16). Frag-
ment ions appear at m/z values of 28, 16, and
12. These correspond to CO+, O+, and C+, resp- ,
ectively.
The mass spectrum of naphthalene with
electron impact ionization by 70 eV electrons.
a, molecular ion and base peak(C10H+8, 100%);
b, 13C isotope peak;
c, fragment ion peaks.
Introduction to mass spectrometry
Mass spectrometers use the difference in mass-to-charge ratio (m/e) of ionized
atoms or molecules to separate them from each other. Mass spectrometry is
therefore useful for quantitation of atoms or molecules and also for determining
chemical and structural information about molecules. Molecules have distinctive
fragmentation patterns that provide structural information to identify structural
components.
All commonly used mass analyzers use electric and magnetic fields to apply a
force on charged particles (ions). The relationship between force, mass, and the
applied fields can be summarized in Newton's second law and the Lorentz force
law:
F = ma (Newton's second law)
F= e(E+ v × B) (Lorentz force law)
where F is the force applied to the ion, m is the mass of the ion, a is the
acceleration, e is the ionic charge, E is the electric field , v B is the vector cross
product of the ion velocity and the applied magnetic field.
From Newton's second law, it is apparent that the force causes an acceleration that
is mass-dependent, and the Lorentz force law tells us that the applied force is also
dependent on the ionic charge. Therefore, it should be understood that mass
spectrometers separate ions according to their mass-to-charge ratio (m/z) rather
than by their mass alone.
The general operation of a mass spectrometer is:
• create gas-phase ions
• separate the ions in space or time based on their mass-to-charge ratio
• measure the quantity of ions of each mass-to-charge ratio
The ion separation power of a mass spectrometer is described by the resolution R,
which is usually defined as: 

where m is the ion mass and delta m is the difference in mass between two
resolvable peaks in a mass spectrum.
 Mass Spectrometry
Theory
In mass spectrometry, a small sample of a chemical compound is vaporized,
bombarded with high energy electrons to ionize the sample, and the ions
produced are detected based on the charge to mass ratio of the ions.

Ionization process in mass spectrometry.

Several different types of ions are produced in this process. If the compound loses
only one electron, then a molecular ion, having the same mass as the original
compound, is produced. This molecular ion is called the M+ ion (and it gives us the
exact molecular weight of the compound).
The stream of high energy electrons is sufficiently powerful to break chemical
bonds in the molecule, producing a series of molecular fragments. These positively
charged fragments are also detected by the instrument, producing the mass
spectrum. Organic chemical compounds will fragment in very specific ways
depending on what functional groups are present in the molecule. Analysis of the
fragmentation pattern can lead to the determination of the structure of the molecule.

Fragments produced by benzamide.

• the analyte (A-B-C) undergoing ionization and fragmentation
• the charged fragments (A+ B+ C+) being separated by mass
• the fragments which are focused on the mass filter's exit slit passing into
the detector
• and the charged ions being detected.
 Instrumentation
Components of a mass spectrometry
1) Inlet system : introduce a very small amount of sample ( micromole or less) into the
mass spectrometer, where its components are converted to gaseous ions.
2) Ion source : converts the components of a sample into ions by bombardment with
electrons, ions, molecules, or photon.
3) Mass analyzer : separates the analyte ions according to their m/z ratios.
4) Detector : converts the beam of ions into an electrical signal(currents) ; the detector
output can be displayed or stored, to yield the mass spectrum.
5) Electronics of power supply and control of the systems.
6) Vacuum systems : maintain low pressures ( 10–5 to 10–8 torr); rotary vacuum oil
pump, diffusion pump, turbomolecular pump.
 Mass Spectrometer Components

Sample Data
Interfacing Ion Source Analyzer Detector
introduction System

 EI  Quadrupole
 GC  Library
 CI  Sector
 LC  FAB  TOF
 SFC  MALDI  FT-ICR
 IC  ESI  Ion Trap
 CE Vacuum System:
 APCI
 DLI DP or TMP
 Thermospray
 ICP  Particle Beam 10–5 ~ 10–8 torr
 Solid probe
 MS/MS – Q/TOF, TOF/TOF
 HRMS
 LRMS
Schematic diagram of mass spectrometer.
Vacuum system

- Oil rotary pump : 50 ~ 200 ml/min

- Turbomolecular pump : no cooling water, 10 min.
- Oil diffusion pump : cooling water, 40~50 min. 600 ~ 1000 l/sec

Vacuum in MS 10-6 to 10-5 Torr

- Increase sensitivity
- avoid ion-molecule reaction
- collision free ion trajectories
- avoid background interference

Vacuum System:
- Pumps must be maintained ; Ultra-clean ; Vacuum regulation and
maintenance
MS Configuration for Ionization

EI
High vacuum

Interface Ion Mass Data

Detector
to vacuum Source Analyzer system

API, APCI, ESI

High vacuum
Ion Interface Mass Data
Detector
Source to vacuum Analyzer system
Sample introduction
For EI and CI there are four main options
Heated reservoir septum inlet for pure gases or volatile liquids. Basically this is a heated
reservoir (~200oC) with a small restriction ‘bleed’ into the ion source. Sample is injected
into the reservoir through a septum.
Direct insertion probe for volatile solids. Sample is loaded into a quartz tube at the end of a
probe and inserted directly into the ion source. The end of the probe can then be heated, if
required, up to temperatures in excess 400oC.
Gas chromatograph (GC) for volatile, non-polar mixtures. The mixture is injected into the
top of the GC column the end of which is passed, via a heated interface, directly into the ion
source. The components of the mixture separate out during their passage through the
column and enter the ion source sequentially.
Particle Beam interface for semi-volatile compounds that are amenable to EI and CI. This is
a useful LC/MS interface that can also be used for rapid introduction of samples by flow
injection. Samples are dissolved in a suitable solvent and the solution is introduced into the
mass spectrometer using a suitable (e.g. HPLC) pump. The liquid is nebulised with helium
gas to form an aerosol of solvent droplets. The stream of liquid droplets passes through a
desolvation chamber and then a series of nozzles and skimmers that remove the solvent and
helium, allowing a stream of solid particles (the sample) to enter the EI/CI ion source.
Chromatographic inlet systems

GC-MS : Thermal vaporization of

A> Interfacing GC with MS
the analyte, separation, transfer of
1. Molecular jet separator (all-glass) : Ryhage, 1966 analyte to the MS, ionization and
detection
2. Membrane separator : Llewellyn, 1966
GC Transfer line MS
3. Effusion separator : Watson-Bieman, 1964
Injection Transfer Ionization
4. Open split coupling Ion filtering
Separation
Detection
5. Capillary direct interface

B> LC-MS interface LC-MS : Separation, nebulization of

the analyte, transfer of ions to the MS,
1. Moving belt wire interface and detection
2. Thermospray LC Interface MS
3. Atmospheric pressure inlet (API)
Injection Transfer Ion filtering
4. Electrospray Separation Ionization Detection
5. Particle beam
6. Dynamic FAB
Ionizer
In the GC-MS discussed in this introduction, the charged particles (ions) required
for mass analysis are formed by Electron Impact (EI) Ionization.  The gas
molecules exiting the GC are bombarded by a high-energy electron beam (70
eV).  An electron which strikes a molecule may impart enough energy to remove
another electron from that molecule.  Methanol, for example, would undergo the
following reaction in the ionizing region:
        CH3OH + 1 e  CH3OH+. + 2 e
        note:  the symbols +. indicate that a radical cation (radical ion) was
formed
EI Ionization usually produces singly charged ions containing one unpaired
electron.  A charged molecule which remains intact is called the molecular ion. 
Energy imparted by the electron impact and, more importantly, instability in a
molecular ion can cause that ion to break into smaller pieces (fragments).  The
methanol ion may fragment in various ways, with one fragment carrying the
charge and one fragment remaining uncharged.  For example:
         CH3OH+. (molecular ion)  CH2OH+(fragment ion) + H.
(or)   CH3OH+. (molecular ion)  CH3+(fragment ion) + .OH
Ionization techniques

- EI (electron impact)
- CI (chemical ionization)
- FAB (fast atom bombardment)
- ESI (electrospray ionization)
- MALDI (matrix assisted laser desorption ionization)
- APCI (atmospheric pressure chemical ionization)
 Sample introduction / ionization method:

Ionization Typical Sample Mass Method

method Analytes Introduction Range Highlights
Relatively  GC or  to  Hard method 
versatile 
Electron  Impact (EI) small  liquid/solid  1,000  provides 
volatile probe Daltons structure info
Relatively  GC or  to  Soft method 
molecular ion 
Chemical Ionization (CI) small  liquid/solid  1,000 
volatile probe Daltons peak [M+H]+
Peptides  to  Soft method 
Liquid 
200,000  ions often 
Electrospray (ESI) Proteins  Chromatography 
multiply 
nonvolatile or syringe Daltons charged
Carbohydrates  Sample mixed  Soft method 
to 
Fast Atom Bombardment Organometallics  but harder 
Peptides 
in viscous  6,000 
than ESI or 
(FAB) matrix Daltons
nonvolatile MALDI
Matrix Assisted Laser Sample mixed  to 
Peptides  Soft method 
500,000 
Desorption  Proteins  in solid  very high 
(MALDI) Nucleotides matrix mass
Daltons
Electron Ionization (EI) 
Theory
Electrons are produced by thermionic emission from a tungsten or rhenium filament. A
typical filament current is 1x10-4Amps. These electrons leave the filament surface and are
accelerated towards the ion source chamber which is held at a positive potential (equal to
the accelerating voltage). The electrons acquire an energy equal to the voltage between the
filament and the source chamber - typically 70 electron volts (70 eV). The electron trap is
held at a fixed positive potential with respect to the source chamber. A proportion of the
electron beam will strike the electron trap producing the trap current. This is used as a
feedback circuit to stabilize the electron beam.
A permanent magnet is positioned across the ion chamber to produce a magnetic flux in
parallel to the electron beam (see figure). This causes the electron beam to spiral from the
filament to the trap, increasing the chance and efficiency of analyte ionization. Gaseous
analyte molecules are introduced into the path of the electron beam where they may be
ionized by electronic interactions with this electron beam. Ionization can also be brought
about by direct impaction of an electron with an analyte molecule. The positive ion
repeller voltage and the negative excitation voltage work together to produce an electric
field in the source chamber such that ions will leave the source through the ion exit slit.
The ions are directed through the various focussing and centering lenses and are focussed
onto the source exit slit. The ions can then be mass analysed.
http://www-methods.ch.cam.ac.uk/meth/ms/theory/eims.html
Schematic of a Kratos Analytical Electron Ionization source (as used on the
MS890).
http://www-methods.ch.cam.ac.uk/meth/ms/theory/eims.html
http://masspec.scripps.edu/information/intro/chapter3.html#3.2.1.
Mechanisms of ion formation
Consider the ionization of the analyte species AB:
1. AB + e-*  A+ + B- + e-
2. AB + e-*  A+ + B° + 2e-
3. AB + e-*  [AB+°*] + 2e- followed by [AB+°*]  AB+°
4. AB + e-*  [AB2+*]" + 3e- followed by [AB2+*]"  A+ + B+
- very low abundance
5. AH + e-*  AH* + e- followed by AH* + AH  [AH+H]+ + A-
- 'self chemical ionization'
Notes:
Species with a * superscript are in high energy sates. Species with a ° superscript are radicals.
Species with a " superscript are short lived intermediates which are not seen in the spectra.
1 and 2 are the highest abundance and are termed instantaneous fragmentation. This is the reason why EI is considered a hard
ionization process.
3 is fairly high abundance and is the process responsible for the molecular ion formation. Unfortunately the highly energetic radical
intermediate [AB+°*] tends to undergo fragmentation (or rearrangement) as a stabilizing process, this is responsible for the
lower mass fragment ions present in the spectra. 4 is a very low abundance process, but theoretically it can occur.
5 is a process which can occur at higher pressures (self Chemical Ionization), this is especially problematic in the ionization of
alcohols and amines where you may find that the dominant ionization process is proton exchange between two analyte
molecules, leading to the formation of the [M+H]+ pseudo-molecular ion.
EI

M + e–  M+· + e – + e –
70 eV Molecular ion ~ 55 eV 0.1 eV

Ionization energies of valence electrons in

formaldehyde.
Here are other first ionization energies:
CH3CH2CH2CH3 10.6 eV (sigma)
CH2=CHCH2CH3 9.6 eV (pi)
(CH3CH2)2O 9.6 eV (nonbonding)
C5H10NH 8.6 eV (nonbonding)
C6H6 9.2 eV (pi)
Electron ionization (70 eV) mass spectra of two isomeric ketones with
the composition C6H12O.
The Molecular Ion Peak
In the example above, involving benzamide (C7H7NO), the molecular ion (M+) peak
had a mass to charge ratio (m/e) of 121. This value is calculated using the most
abundant isotopes of the elements present in the molecule:

7 × 12C = 84 7 × 1H = 7
1 × 14N = 14
1 × 16O = 16
Total 121
This type of calculation is called the unit mass molecular ion. Please refer to the
spectrum for benzamide.

Mass spectrum for benzamide.

The observed abundance of the suspected molecular ion peak must correspond to
expectations based on the assumed molecular structure. For example, highly
branched substances undergo fragmentation readily; therefore, these types of
substances will not have a very intense molecular ion peak. The intensity of
molecular ion peaks will usually correspond to the following sequence:
aromatic compounds > conjugated alkenes > alicyclic compounds > sulfides >
unbranched hydrocarbons > mercaptans > ketones > amines > esters > ethers >
carboxylic acids > branched hydrocarbons > alcohols
For instance, if an unknown compound is suspected to be an alcohol, then the
molecular ion peak will be quite small or completely absent.
Interpreting spectra
A simple spectrum, that of methanol, is shown here.   CH3OH+. (the molecular ion) and
fragment ions appear in this spectrum.  Major peaks are shown in the table next to the
spectrum.   The x-axis of this bar graph is the increasing m/z ratio.  The y-axis is the
relative abundance of each ion, which is related to the number of times an ion of that m/z
ratio strikes the detector.  Assignment of relative abundance begins by assigning the
most abundant ion a relative abundance of 100% (CH2OH+ in this spectrum).  All other
ions are shown as a percentage of that most abundant ion.  For example, there is
approximately 64% of the ion CHO+ compared with the ion CH2OH+ in this spectrum. 
The y-axis may also be shown as abundance (not relative).  Relative abundance is a way
to directly compare spectra produced at different times or using different instruments.
EI ionization introduces a great deal of energy into molecules.  It is known as a "hard"
ionization method.  This is very good for producing fragments which generate
information about the structure of the compound, but quite often the molecular ion does
not appear or is a smaller peak in the spectrum.
Of course, real analyses are performed on compounds far more complicated than
methanol.  Spectra interpretation can become complicated as initial fragments undergo
further fragmentation, and as rearrangements occur.  However, a wealth of information is
contained in a mass spectrum and much can be determined using basic organic chemistry
"common sense".
Following is some general information which will aid EI mass spectra
interpretation:
Molecular ion (M .+):If the molecular ion appears, it will be the highest mass in an EI
spectrum (except for isotope peaks discussed below).  This peak will represent the
molecular weight of the compound.  Its appearance depends on the stability of the
compound.  Double bonds, cyclic structures and aromatic rings stabilize the molecular ion
and increase the probability of its appearance.
Reference Spectra: Mass spectral patterns are reproducible.  The mass spectra of many
compounds have been published and may be used to identify unknowns.  Instrument
computers generally contain spectral libraries which can be searched for matches.
Fragmentation:General rules of fragmentation exist and are helpful to predict or interpret
the fragmentation pattern produced by a compound.  Functional groups and overall
structure determine how some portions of molecules will resist fragmenting, while other
portions will fragment easily.  A detailed discussion of those rules is beyond the scope of
this introduction, and further information may be found in your organic textbook or in mass
spectrometry reference books.  A few brief examples by functional group are described
(see examples).
Isotopes:Isotopes occur in compounds analyzed by mass spectrometry in the same
abundances that they occur in nature.  A few of the isotopes commonly encountered in the
analyses of organic compounds are below along with an example of how they can aid in
peak identification.
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htm
Four possible fragmentation pathways for the molecular ion of 2-hexanone.
 Mass Spectrometry
The Nitrogen Rule
If a compound contains an even number of nitrogen atoms (or no nitrogen atoms), its
molecular ion will appear at an even mass number. If, however, a compound contains
an odd number of nitrogen atoms, then its molecular ion will appear at an odd mass
value. This rule is very useful for determining the nitrogen content of an unknown
compound.
In the case of benzamide, the molecular ion appears at m/e = 121, indicating an odd
number of nitrogen atoms in the structure.
The masses of molecular and fragment ions also reflect the electron count, depending
on the number of nitrogen atoms in the species.

Ions with no nitrogen odd-electron ions even-electron ions

or an even # N atoms even-number mass odd-number mass

Ions having an odd-electron ions even-electron ions

odd # N atoms odd-number mass even-number mass
4-methyl-3-pentene-2-one has no nitrogen so the mass of the molecular ion (m/z = 98) is an even
number

N,N-diethylmethylamine has one nitrogen and its molecular mass (m/z = 87) is an odd number.
A majority of the fragment ions have even-numbered masses (ions at m/z = 30, 42, 56 & 58 are
not labeled), and are even-electron nitrogen cations.
Electron ionization (70 eV) mass spectra of molecular ion region of benzene
(C6H6) and biphenyl (C12H10).
Intensity of M+1 relative to molecular ion for C nHm :
Intensity = n × 1.08% + m × 0.012%
Contribution from 13C Contribution from 2H
Methyl Bromide:  An example of how isotopes can aid in
peak identification.
The ratio of peaks containing 79Br and its isotope 81Br (100/98) confirms
the presence of bromine in the compound.
 Relative Isotope Abundance of Common Elements:

  Relative Relative Relative

Element Isotope Abundance Isotope Abundance Isotope Abundance

Carbon 12
C 100 13
C 1.11
Hydrogen H
1
100 2
H .016
Nitrogen 14
N 100 15
N .38
Oxygen 16
O 100 17
O .04 18
O .20
Sulfur 32
S 100 33
S .78 34
S 4.40
Chlorine 35
Cl 100  37
Cl 32.5
Bromine 79
Br 100 81
Br 98.0
Electron ionization mass spectrum Mass spectrum showing natural isotopes
(70 eV) of 1-bromobutane. of Pb observed as an impurity of brass.
Molecular Mass and Nominal Mass
The unit of atomic mass is the dalton, Da, defined as 1/12 of the mass of 12C.
Mass specrometrists perfer the symbol “u” for “unified atomic mass unit”.
“Da” and “u” are synonymous.
Atomic mass is the weighted average of the masses of the isotopes of an
element.
Ex. Bromine 79
Br 78.91834 Da 50.69%
81
Br 80.91629 Da 49.31%
Br = 78.91834 × 0.5069 + 80.91629 × 0.4931
= 79.904 Da
The molecular Mass of a molecule or ion is the sum of atomic masses listed in
a periodic table.
Ex. C2H5Br = 12.0107 × 2 + 1.00794 × 5 + 79.904 × 1 = 108.965
The nominal mass of a molecule or ion is the the integer mass of the species
with the most abundant isotope of each of the constituent atoms.
Ex. C2H5Br = 12 × 2 + 1 × 5 + 79 × 1 = 108
Chemical ionization (CI)
CI uses a reagent ion to react with the analyte molecules to form ions by either
a proton or hydride transfer:

MH + C2H5+  MH2+ + C2H4

MH + C2H5+  M+ + C2H6

The reagent ions are produced by introducing a large excess of methane

(relative to the analyte) into an electron impact (EI) ion source. Electron
collisions produce CH4+ and CH3+ which further react with methane to form
CH5+ and C2H5+:

CH4+ + CH4  CH5+ + CH3

CH3+ + CH4  C2H5+ + H2

Chemical ionization uses ion-molecule reactions to produce ions from the
analyte. The chemical ionization process begins when a reagent gas such as
methane, isobutane, or ammonia is ionized by electron impact. A high reagent
gas pressure (or long reaction time) results in ion-molecule reactions between the
reagent gas ions and reagent gas neutrals. Some of the products of these ion-
molecule reactions can react with the analyte molecules to produce analyte ions.
Example
(R = reagent, S = sample, e = electron, . = radical electron , H = hydrogen):
R + e  R+. + 2e
R+. + RH  RH+ + R.
RH+ + S  SH+ + R
(of course, other reactions can occur)
 CI Reagent Gases
Methane:
• good for most organic compounds
• usually produces [M+H]+, [M+CH3]+ and [M+C3H5]+ adducts
• adducts are not always abundant
• extensive fragmentation
Isobutane:
• usually produces [M+H]+, [M+C4H9]+ adducts and some fragmentation
• A dducts are relatively more abundant than for methane CI
• not as universal as methane
Ammonia:
• fragmentation virtually absent
• polar compounds produce [M+NH4]+ adducts
• basic compounds produce [M+H]+ adducts
• non-polar and non-basic compounds are not ionized
a positive-ion CI reagent-gas mass spectrum of ammonia
Mass spectra of the sedative pentobarbital, using EI (left) or CI (right).
The molecular ion ( m/z 226) is not evident with EI. The dominant ion
from CI is MH+(protonated molecule). The peak at m/z 255 in the CI
spectrum is from M(C2H5)+.
Fast Atom Bombardment (FAB)/Liquid Secondary Ionisation
(LSIMS)
This technique, developed in the early 1980s, revolutionised the range of
compounds analysable by mass spectrometry and opened up the field to most
areas of biomedical research. Although now considered insensitive by
comparison with more recently introduced ionisation modes, FAB still has a role
as a rapid, reliable and robust technique for samples where quantity and purity
are not a problem.
The sample is first dissolved in a liquid matrix. This is typically a viscous, low
vapour pressure liquid such as glycerol or 3-nitrobenzyl alcohol. A few micro-
litres of this liquid are placed on a small metal target at the end of a probe which
is inserted into the mass spectrometer.
The liquid surface is then bombarded with a beam of high kinetic energy atoms
(xenon or argon) or ions (caesium). Molecules are sputtered from the surface,
enter the gas phase and ionise, either by protonation or deprotonation.
The resulting ions tend to be stable and exhibit little fragmentation.
Theory
The techniques of FAB and LSIMS involve the bombardment of a solid analyte +
matrix mixture by a fast particle beam (see figure). The matrix is a small organic
species (glycerol or 3-nitrobenzyl alcohol, 3-NBA) which is used to keep a 'fresh'
homogeneous surface for bombardment, thus extending the spectral lifetime and
enhancing sensitivity. In FAB, the particle beam is a neutral inert gas, typically Ar
or Xe, at bombardment energies of 4-10 KeV, whereas in LSIMS, the particle beam
is an ion, typically Cs+, at bombardment energies of 2-30KeV.
The particle beam is incident at the analyte surface, where it transfers much of its
energy to the surroundings, setting up momentary collisions and disruptions. Some
species are ejected off the surface as positive and negative ions by this process, and
these 'sputtered' or secondary ions are then extracted from the source and analysed
by the mass spectrometer. The polarity of the source extraction can be switched
depending on what species are to be analysed. In the figure, the extraction is
negative and thus only the positive ions are being analysed, this is termed +FAB.
For LSIMS the atom beam is replaced by an ion beam.
Both FAB and LSIMS are comparatively soft ionization techniques, and are thus
well suited to the analysis of low volatility species, typically producing large peaks
for the pseudo-molecular ion species [M+H]+ and [M–H]-, along with structurally
significant fragment ions and some higher mass cluster ions and dimers.
Schematic of a Fast Atom Bombardment source. The figure shows a
schematic representation of a fast atom bombardment ion source, operating in
the positive ion mode. The fast atom beam is incident at the analyte+matrix
surface where it causes the 'sputtering' off of secondary ions. The positive ions
are then extracted from the source into the mass spectrometer.
Fast Atom Bombardment (FAB).
The Cesium ions bombard the
matrix.
Matrix-assisted Laser Desorption/Ionization (MALDI)
Unlike FAB/LSIMS, this process uses a crystalline, rather than liquid, matrix,
and a beam of photons, rather than atoms or ions. The net result is a dramatic
increase, compared with FAB, in both sensitivity and mass range of analysable
compounds. The sample is dissolved in a matrix and is allowed to crystallise
on a stainless steel target. Suitable matrices include 2, 5 -dihydroxybenzoic
acid and sinapinic acid. The target is then inserted into the mass spectrometer
and the surface bombarded with a pulsed laser beam (typically generated by
inexpensive nitrogen lasers with a beam wavelength of 337nm). Molecules are
desorbed from the surface and ionise, usually by protonation or deprotonation.
Any fragment or multiply charged ions are generally of low abundance in this
ionisation mode.
(i) The Formation of a 'Solid Solution'. The analyte molecules are distributed throughout the matrix
so that they are completely isolated from one other. This is necessary if the matrix is to form a
homogenous 'solid solution' (any liquid solvent(s) used in preparation of the solution are removed
when the mixture is dried before analysis).
(ii) Matrix Excitation. Some of the laser energy incident on the solid solution is absorbed by the
matrix, causing rapid vibrational excitation, bringing about localized disintegration of the solid
solution, forming clusters made up of a single analyte molecule surrounded by neutral and excited
matrix molecules. The matrix molecules evaporate away from these clusters to leave the excited
analyte molecule.
(iii) Analyte Ionization. The analyte molecules can become ionized by simple protonation by the
photo-excited matrix, leading to the formation of the typical [M+X] + type species (where X= H,
Li, Na, K, etc.). Some multiply charged species, dimers and trimers can also be formed. Negative
ions are formed from reactions involving deprotonation of the analyte by the matrix to form [M-
H]- and from interactions with photoelectrons to form the [M] -° radical molecular ions.
These ionization reactions occur in the first tens of nanoseconds after irradiance, and within the initial
desorbing matrix/analyte cloud. It is in this way that the characteristic MALDI spectra are
created, typically giving large signals for species of the type [NM+X] n+ (N * n).
MALDI. Formation of ions by laser desorption
http://masspec.scripps.edu/information/intro/chapter3.html#3.2.1.
Sequence of events in MALDI.
(a) Dried mixture of analyte and matrix on sample probe inserted into
backplate of ion source.
(b1) Enlarged view of laser pulse striking sample.
(b2) Matrix is ionized and vaporized by laser and transfers some charge to
analyte.
(b3) Vapor expands in a supersonic plume
Peptides and glycopeptides Peptides and Peptides, small proteins
proteins and oligonucleotides
(<10 bases)

The key aspect of MALDI MS is to dilute and isolate macromolecules in a

suitable matrix of highly laser light absorbing small organic molecules,
such as a-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA) and
2,5-dihydroxybenzoic acid (DHB), and then allowing it to dry on a
MALDI-target into a crystalline deposit throughout which the molecules of
the analyte are dispersed.
MALDI-MS Sample preparation
Materials
For peptide analysis:
20 mg/ ml a-cyano-4-hydroxy-trans-cinnamic acid in 0.1% TFA/ 50%
acetonitrile or in acetone-water (99:1, v/v).
20 mg/ ml 2,5-dihydroxybenzoic acid (DHB) in 0.1% TFA/ 30%* acetonitrile.
*The percentage of acetonitrile may be varied, depending on the
hydrophobicity of the peptides analyzed. The matrix additive 2-methoxy-5-
benzoic acid may be added (1 mg/ml) to the DHB matrix solution for better
detection of larger (>3000 Da) peptides.
 For protein analysis:
sinapinic acid (20 mg/ ml) in 0.1% TFA/ 50% acetonitrile or in acetone-water
(99:1, v/v).
 
http://www.bio.vu.nl/mnb/proteomics/8.maldisampleprep.html
Electrospray Ionization (ESI)
Theory
The production of ions by evaporation of charged droplets obtained through spraying or
bubbling, has been known about for centuries, but it was only fairly recently discovered that
these ions may hold more than one charge4. A model for ion formation in ESI, containing the
commonly accepted themes, is described below5:
Large charged droplets are produced by 'pneumatic nebulization'; i.e. the forcing of the
analyte solution through a needle (see figure), at the end of which is applied a potential - the
potential used is sufficiently high to disperse the emerging solution into a very fine spray of
charged droplets all at the same polarity. The solvent evaporates away, shrinking the droplet
size and increasing the charge concentration at the droplet's surface. Eventually, at the
Rayleigh limit, Coulombic repulsion overcomes the droplet's surface tension and the droplet
explodes. This 'Coulombic explosion' forms a series of smaller, lower charged droplets. The
process of shrinking followed by explosion is repeated until individually charged 'naked'
analyte ions are formed. The charges are statistically distributed amongst the analyte's
available charge sites, leading to the possible formation of multiply charged ions under the
correct conditions. Increasing the rate of solvent evaporation, by introducing a drying gas
flow counter current to the sprayed ions (see figure), increases the extent of multiple-
charging. Decreasing the capillary diameter and lowering the analyte solution flow rate i.e. in
nanospray ionization, will create ions with higher m/z ratios (i.e. it is a softer ionization
technique) than those produced by 'conventional' ESI and are of much more use in the field
of bioanalysis.
Pneumatically assisted electrospray interface for mass spectrometry.
 Gas-phase ion formation. Electrospray interface for
capillary electrophoresis /
mass spectrometry.

Electrospray from a silica capillary.

Atmospheric Pressure Chemical Ionization (APCI)
Similar to electrospray ionization, liquid effluent is introduced directly into
the Atmospheric Pressure Chemical Ionization (APCI) source, however the
similarity with electrospray stops there. The APCI source contains a heated
vaporizer which facilitates rapid desolvation/vaporization of the droplets.
Vaporized sample molecules are carried through an ion-molecule reaction
region at atmospheric pressure. The ionization occurs through a corona
discharge, creating reagent ions from the solvent vapor. Chemical
ionization of sample molecules is very efficient at atmospheric pressure due
to the high collision frequency. Proton transfer (protonation MH+
reactions) occurs in the positive mode, and either electron transfer or
proton transfer (proton loss, [M-H]-) in the negative mode. The moderating
influence of the solvent clusters on the reagent ions, and of the high gas
pressure, reduces fragmentation during ionization and results in primarily
molecular ions. APCI is widely used in the pharmaceutical industry to
analyze relatively nonpolar, semi volatile samples of less than 1200
Daltons and it is an especially good ionization source for liquid
chromatography.
Atmospheric pressure chemical ionization interface between a liquid
chromatography column and a mass spectrometer.
A fine aerosol is produced by the nebulizing gas flow and the heater. The
electric discharge from the corona needle creates gaseous ions from the
analyte.
Mass analyzer: A device that separates a mixture of ions by their mass-
to- charge ratios.

mass-to-charge ratio m/z: 

The abbreviation m/ z is used to denote the dimensionless quantity formed by
dividing the mass number of an ion by its charge number. It has long been
called the mass- to- charge ratio although m is not the ionic mass nor is z a
multiple or the elementary (electronic) charge e. The abbreviation m/z
therefore, is not recommended. [IUPAC Compendium] 

mass analysis:
A process by which a mixture of ionic or neutral species is identified according
to the mass- to- charge (m/ z) ratios (ions) or their aggregate atomic masses
(neutrals). The analysis may be qualitative and/ or quantitative. [IUPAC
Compendium]
Common mass analyzer designs:
Quadrupole Mass Spectrometer

Introduction
A quadrupole mass filter consists of four parallel metal rods arranged as in the
figure below. Two opposite rods have an applied potential of (U+Vcos(wt))
and the other two rods have a potential of -(U+Vcos(wt)), where U is a dc
voltage and Vcos(wt) is an ac voltage. The applied voltages affect the
trajectory of ions traveling down the flight path centered between the four
rods. For given dc and ac voltages, only ions of a certain mass-to-charge ratio
pass through the quadrupole filter and all other ions are thrown out of their
original path. A mass spectrum is obtained by monitoring the ions passing
through the quadrupole filter as the voltages on the rods are varied. There are
two methods: varying w and holding U and V constant, or varying U and V
(U/V) fixed for a constant w.
Instrumentation
Quadrupole mass spectrometers consist of an ion source, ion optics to
accelerate and focus the ions through an aperture into the quadrupole filter,
the quadrupole filter itself with control voltage supplies, an exit aperture, an
ion detector and electronics, and a high-vacuum system.

Quadrupole mass spectrometer.

Magnetic-sector mass spectrometer
The ion optics in the ion-source chamber of a mass spectrometer extract and accelerate
ions to a kinetic energy given by:
K.E. = 0.5 mv2 = eV

where m is the mass of the ion, v is it's velocity, e is the charge of the ion and V is the
applied voltage of the ion optics.

The ions enter the flight tube between the poles of a magnet and are deflected by the
magnetic field, H. Only ions of mass-to-charge ratio that have equal centrifugal and
centripetal forces pass through the flight tube:

mv2 / r = Hev

centrifugal = centripetal forces.

Where r is the radius of curvature of the ion path:

r= mv / eH

This equation shows that the m/e of the ions that reach the detector can be varied by
changing either H or V.
Magnetic sector mass spectrometer.
Instrumentation

Single Focusing analyzers:

A circular beam path of 180, 90, or 60 degrees can be used. The
various forces influencing the particle separate ions with different
mass-to-charge ratios.

Double Focusing analyzers:

An electrostatic analyzer is added in this type of instrument to separate
particles with difference in kinetic energies.
Electrostatic sector of a double-focusing mass spectrometer. Positive ions
are attracted toward the negative cylindrical plate. Trajectories of high-
energy ions are changed less than trajectories of low-energy ions. Ions
reaching the exit slit have a narrow range of kinetic energies.
Ion-Trap Mass Spectrometry

Introduction
The ion-trap mass spectrometer uses three electrodes to trap ions in a small
volume. The mass analyzer consists of a ring electrode separating two
hemispherical electrodes. A mass spectrum is obtained by changing the
electrode voltages to eject the ions from the trap. The advantages of the ion-
trap mass spectrometer include compact size, and the ability to trap and
accumulate ions to increase the signal-to-noise ratio of a measurement.
Ion Trap Analysis 
Theory
The quadrupole ion trap mass analyser (see figure) consists of three hyperbolic
electrodes: the ring electrode, the entrance endcap electrode and the exit endcap
electrode. These electrodes form a cavity in which it is possible to trap and analyse
ions. Both endcap electrodes have a small hole in their centres through which the
ions can travel. The ring electrode is located halfway between the two endcap
electrodes.
Ions produced from the source enter the trap through the inlet focussing system
and the entrance endcap electrode. Various voltages are applied to the electrodes to
trap and eject ions according to their mass-to-charge ratios. The ring electrode RF
potential, an a.c. potential of constant frequency and variable amplitude, is applied
to the ring electrode to produce a 3D quadrupolar potential field within the
trapping cavity. This will trap ions in a stable oscillating trajectory confined within
the trapping cell. The nature of the trajectory is dependent on the trapping potential
and the mass-to-charge ratio of the ions. During detection, the electrode system
potentials are altered to produce instabilities in the ion trajectories and thus eject
the ions in the axial direction. The ions are ejected in order of increasing mass-to-
charge ratio, focussed by the exit lens and detected by the ion detector system.
Ion-trap mass spectrometer.
(a) Mass analyzer consists of two end caps (left and right) and central ring
electrode. (b) Schematic diagram.
Time-of-Flight Mass Spectrometry (TOF-MS)

Introduction

A time-of-flight mass spectrometer uses the differences in transit time through a

drift region to separate ions of different masses. It operates in a pulsed mode so
ions must be produced or extracted in pulses. An electric field accelerates all
ions into a field-free drift region with a kinetic energy of qV, where q is the ion
charge and V is the applied voltage. Since the ion kinetic energy is 0.5mv2,
lighter ions have a higher velocity than heavier ions and reach the detector at
the end of the drift region sooner.

Theory

K.E. = qV
1/2 mv2 = qV
v = (2qV/m)1/2

The transit time (t) through the drift tube is L/V where L is the length of the
drift tube.
t = L / (2V/m/q)1/2
Instrumentation
This schematic shows ablation of ions from a solid sample with a pulsed
laser. The reflectron is a series of rings or grids that act as an ion mirror.
This mirror compensates for the spread in kinetic energies of the ions as
they enter the drift region and improves the resolution of the instrument.
The output of an ion detector is displayed on an oscilloscope as a function
of time to produce the mass spectrum.

Schematic of a reflectron TOF-MS

Fourier Transform Ion Cyclotron Resonance (FT-ICR)
Theory
Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry is probably the most
complex method of mass analysis. The technique has gone through a very complex development
since it's conception in the mid. 1950s, this is reviewed in the history section. It is the most
sensitive of the techniques in common use today, and has almost unlimited mass resolution, >10 6
is observable with most instruments and resolutions in the 10 4 to 105 range are routinely
available.
The FT-ICR mass spectrometer consists of three main sections. The first is the sample source,
which can be practically any of the available techniques, although ESI and MALDI are the most
common. The second section is the ion transfer region, where the ions produce in the source are
focussed, bunched and transferred into the high vacuum region before entering the analyser cell.
The final region is the cell itself. It is possible in some cases (especially with MALDI) to produce
ions directly in the cell, this does away with the need for the complex ion focussing and pumping
regions.
The standard arrangement for the analyser region, of the FT-ICR instrument, is an ion-trap
located within a spatially uniform static magnetic field of strength, B 0. The effect of the magnetic
field is to constrain the incident ions in a circular orbit (see figure), the frequency of which is
determined by the mass, mi, charge, zi, and velocity, v, of the ion, by action of the Lorentz force,
defined in equation 1. The analyte ion is bent into a circular path in a plane perpendicular to the
magnetic field. The ion's angular frequency, wc, is defined by equation 2. The opposite sense of
rotation is experienced by ions of opposite charge.
Equation 1:

Equation 2:

Equation 3:
 
Where:
• F = Lorentz force (observed by the incident ion)
               
 
            
• B0 = ICR magnetic field

• mi = mass of the ion

• zi = charge on the ion

• v = incident velocity of the ion
• wc = induced rotational (cyclotron) frequency
The presence of ions between a pair of detector electrodes (in the trapping cell)
will not actually produce any measurable signal. It is necessary to excite the
ions of a given m/z as a coherent package to a larger orbital radius, by applying
an RF sweep of a few milliseconds across the cell (see figure). One frequency
will excite one particular m/z (Fourier transformation allows for all frequencies
to measure simultaneously). Measurement of the angular frequency (equation
2) leads to values for m/z (equation 3) and thus to the mass spectrum. Because
frequency can be measured more accurately than any other physical property,
the technique has a very high mass resolution. After excitation, the ions are
allowed to relax back to their natural ICR motion for later remeasurement, if
required.
A schematic diagram of the trapping, excitation and detection of
a ions, to produce a mass spectrum, in FT-ICR mass
spectrometry:

http://www-methods.ch.cam.ac.uk/meth/ms/theory/fticr.html
Ion Detection
Once the ion passes through the mass analyzer it is then detected by the ion
detector, the final element of the mass spectrometer. The detector allows a
mass spectrometer to generate a signal current from incident ions by
generating secondary electrons, which are further amplified. Alternatively,
some detectors operate by inducing a current generated by a moving charge.
Among the detectors described, the electron multiplier and scintillation
counter are the most commonly used and convert the kinetic energy of
incident ions into a cascade of secondary electrons

Figure. After an ion

passes through the Mass
Analyzer a signal is
produced by the detector.
Ion Detectors

- Faraday cup
- Electron Multiplier
- High-energy dynodes with electron multiplier
- Array
- FT-MS
Tandem Mass Spectrometry
In contrast to electron ionization (EI) which produces many fragment ions, the
new ionization techniques are relatively gentle and do not produce a significant
amount of fragment ions. To obtain more information on the molecular ions
generated in the electrospray ionization and MALDI ionization sources, it has
been necessary to apply techniques such as tandem mass spectrometry (MS/MS)
to induce fragmentation. Tandem mass spectrometry (abbreviated MSn - where n
refers to the number of generations of fragment ions being analyzed) allows one
to induce fragmentation and mass analyze the fragment ions. This is
accomplished by collisionally generating fragments from a selected ion and then
mass analyzing the fragment ions. Fragmentation can be achieved by inducing
ion/molecule collisions by a process known as collision-induced dissociation
(CID) (also known as collision-activated dissociation (CAD)). Collision-induced
dissociation is accomplished by selecting an ion of interest with a mass analyzer
and introducing that ion into a collision cell. The selected ion then collides with a
collision gas (typically argon or helium) resulting in fragmentation. The
fragments are then analyzed to obtain a fragment ion spectrum. The abbreviation
MSn is applied to processes which analyze beyond the initial ions (MS) to the
fragment ions (MS2) and subsequent generations of fragment ions (MS3, MS4 and
…). Tandem mass analysis is primarily used to obtain structural information.
Multiple Mass Spectrometry ; MS/MS/MS:
Provides even greater certainty of identification and additional characterization
information than electrospray ionization/ tandem mass spectrometry. Fully
automated. [CHI Proteomics report] 
When more than two stages are involved, the technique is called multi-
dimensional MS (MS n where n indicates the number of stages).

Principle of selected reaction monitoring, also called tandem mass spectrometry,

mass spectrometry / mass spectrometry, or MS/MS
Fragments are analyzed to
obtain a fragment ion
spectrum. The
abbreviation MSn is
applied to processes
which analyze beyond the
initial ions (MS) to the
fragment ions (MS2) and
subsequent generations of
fragment ions (MS3, MS4
and …).
http://masspec.scripps.edu/information/intro
/chapter3.html#3.2.1.
 THE END

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