HEJ Research Institute of Chemistry,

International Center for Chemical and

Biological Sciences, University of Karachi
Karachi

CHEM - 701 Marks Distribution

Mass / CD/ORD

NMR

IR

35 Marks

40 Marks

8 Marks

X Ray

10 Marks

UV

7 Marks

Total

100 Marks

Mass Spectrometry

Mass spectrometry for Chemists and Biochemists

M.E. Rose
Mass Spectrometry
William Kemp
Mass Spectrometry Principles and Application
Edmond de Hoffmann and Vincent Stroobant

Mass Spectrometry: Techniques and Applications of Tandem Mass

Spectrometry
Kenneth L. Busch, Gary L. Glish and Scott A. McLuckey

Biochemical Applications of Mass spectrometry

George R. Waller, Otis C. Dermer

Why Mass Spectrometry?

Versatility:

Sensitivity:
Selectivity:

High Speed:

Analyze most types of materials

Small and large molecules
Polar and non polar and nuteral
materials
Proteins, sugars and natural products
Detect<10-12 g of material
Detect specific components of mixtures
Drug metabolities in blood
Environmental pollutants
Crude plant extracts
Peptides in protein digests
Easily automated
Run several thousand samples per day
No operator required

-e

M+.

-N.

C+

-Nc

M+.
A+

A+. + N
-Na

B+

-Nb

etc.

Various pathways together compose fragmentation pattern

One pathway

Multiple pathways

Simple fragmentation

Complex Fragmentation

Simple spectrum

Complex spectrum

1.

Amount of internal energy originally imparted to the

molecular ion (M+)
2.
Structure
3.
Time allowed between ion formation and detection
Mass spectrum Appearance of the fragmentation pattern at specified
energies and times.

Abundances of ions
x M+.
y M+.
sufficient internal energies
insufficient
internal
energies
fragment further to
A+, B+, C+ etc

do not fragment further

y M+.

(initial energy imparted to M+. and individual rates of

formation of A+, B+, C+ etc. lead to varying amounts of
these ions measured as relative abundances.

Table 1.1. Normalized ion abundances in the mass spectrum of 1.3-dimethylbenzene ( % RA)
m/z
Abundance
m/z
Abundance
m/z
Abundance
38
1.2
65
5.5
102
1.1
39
7.1
74
1.1
103
6.1
51
2.8
77
11.3
104
2.6
52
9.1
78
5.1
105
28.7
53
5.0
79
6.5
106
61.7
62
1.6
91
100.0
107
5.4
63
4.2
92
7.8
a
ion abundances less than one per cent of the base peak have been omitted.
Table 1.2 ion abundances in the mass spectrum of 1,3-dimethylbenzene as %
TIC
m/z
Abundance
m/z
Abundance
m/z
Abundance
39
2.5
65
1.9
92
2.7
50
1.0
77
3.9
103
2.1
51
3.2
78
1.8
105
10.0
52
1.7
79
2.2
106
21.4
63
1.5
91
34.8
107
1.9
a
ion abundances of less than one per cent are omitted

A+ + B+

M2+
C

2+

+N

Isotopes:

1.08 percent natural abundance in

carbon
CH4
M+.. m/z 16
(12CH4)
M++1 m/z 17
(13CH4)
Ratio : 99:1
C10H18
M+.
100%
M+ +1
10.8%
M+ +2
0.45%
M+ +3
0.01%
Some elements like S, Cl, Br have particularly abundant isotopes
separated by 2 mass units and hence readily recognized.
13

The isotope pattern helps to determine the number of Cl and Br

atoms in the original molecule.
Isotopic Pattern:
(a+b)n
n = no. of atoms of
isotopes
a & b = rel.abudance

Table 1.4. The more important natural isotope abundance for

elements commonly occurring in mass spectrometry4
Element

Isotope (percentage of natural abudance)b

Hydrogen
Boron
Carbon
Nitrogen
Oxygen
Fluorine
Silicon

Phosphorus
Sulphur
Chlorine
Bromine
Iodine

31

H (99.99)
10
B (19.8)
12
C (98.9)
14
N (99.6)
16
O (99.8)
19
F (100.0)
28
Si (92.2)
P (100.0)
32
S (95.0)
35
CI (75.5)
79
Br (50.5)
127
I (100.0)

B (80.2)
13
C (1.1)
15
N (0.4)
18
O (0.2)
11

Si (4.7)

30

Si (3.1)

S (0.7)
37
CI (24.5)
81
Br (49.5)

34

S (4.2)

29

33

Calculation of Isotope Peaks

The isotope pattern of the molecular ion of Cl2 is given by the
binomial expansion of: (a + b) n where a and b are the relative
abundances of the two isotopes of chlorine, and n is the number
of atoms of chlorine. For Cl2, this expression gives:
(0.75 + 0.24)2
(0.75)2:2(0.24x0.75): (0.24)2
0.56:0.36:0.06
1.00:0.64:0.11
Calculation of isotope peak intensities for real molecules
involving multiple elements is far more complicated.
Calculations assume a Natural Abundance of stable isotopes.

 Chlorine is an excellent example of how isotope

distributions are useful for interpretation. The
molecular weight of chlorine is 35.45 u. The natural
abundance of these two isotopes is observed in the
mass spectrum as two peaks are separated by m/z 2
with a relative intensity of 3:1. The mass spectrum of
CH3Cl clearly shows two peaks with the isotope
distribution pattern for an ion with a single chlorine
atom. CH3 35Cl (m/z 50) and CH3 37Cl (m/z 52) are
separated by m/z 2 and have the 3:1 abundance ratio
characteristic of an ion with a single chlorine atom.

Metastable ions
The ions, which at the detector, have less than the full
kinetic energy orginally imparted at the ion source.
Ions spend 10-6 s in the ion source and take 10-5 s after
leaving the source to reach the detector.
Ions,
i)
with large excess of int. E have high rates of
decompn. (> 106/sec) and fragment in the ion
source.
ii)
small excess of int E have low rates of
decompn(< 105/sec) they are stable within the
life time of the analysis.
iii)
intermediate int E have rate consts b/w 105106/sec give rise to metastable ions.

Metastable ions are used in:

1.Defocussing Technique:
to determine precursor ions of a chosen product
2.Linked scanning:
E2/V are varied to determine fragment ions
of a precursor ion
B/E to determine all product ions of a chosen
precursor ion
B2/E to determine precursor ions of a daughter
ion

What do we mean by molecular weight?

Nominal molecular weight: Calculated using the
nominal (non fractional) atomic weight for the most
abundant isotope (which for many elements is also
the lowest mass isotope; H = 1, C = 12, O = 16,
Cl = 35).
Monoisotopic exact molecular weight: calculated
using the exact atomic weight for the most abundant
isotope. (H = 1.0078, C = 12.000, O = 15.995,
Cl = 34.969)

 For example, C2 H6 , CH2 O, and NO all

have a nominal mass of 30 u. Because they
have the same nominal mass, a mass
spectrometer with unit mass resolution can
not distinguish these three ions. However,
the exact masses for C2 H6 (30.04695039),
CH2O
(30.01056487)
and
NO
(29.99798882) are different and a high
resolution
mass
spectrometer
can
distinguish these three compounds.

FIRST LECTURE (SUMMARY)

Why you should learn mass spectrometry.
What information you can get from a mass spectrum
of an unknown compound to solve its structure.
How a mass spectrum is produced (with a
conventional EI mode of ionization).
Basic functions of Mass Spectrometer
Mass spectrum formation (Fragmentation Pathway)
Relative Abundance of ions
TIC
Multiply Charged ions
Isotope Peaks
Metastable ions
Low Resolution/high Resolution of Mass Peaks

FIRST LECTURE (Questions)

1. What are the basic functions of a Mass
Spectrometer.
2. What information you can get from a mass
spectrum of an unknown compound to solve its
structure?
3. How a mass spectrum is produced? (with a
conventional EI mode of ionization).
4. Which property of the ions determine the
fragmentation pathway).
5. What does the relative abundance of ions tell you?
6. How the abundant isotope peaks help in mass
spectrum interpretation?
7. In which techniques the metastable ions are used?

MASS SPECTROMETRY
1. Sample introduction
2. Methods of sample ionization
3. Analysis and separation of sample
ions
4. Detection and recording of sample
ions.

MASS SPECTROMETER

1. Sample introduction
The method of sample introduction to the ionisation
source often depends on the ionisation method being
used, as well as the type and complexity of the sample.
The sample can be inserted directly into the ionisation
source, or can undergo some type of chromatography en
route to the ionisation source. This latter method of
sample introduction usually involves the mass
spectrometer being coupled directly to a high pressure
liquid chromatography (HPLC), gas chromatography
(GC) or capillary electrophoresis (CE) separation
column, and hence the sample is separated into a series
of components which then enter the mass spectrometer
sequentially for individual analysis.

(EI Source)
At pressure not more than 10-5 torr to 10-7 torr to avoid:
1) the collision of the ions with each other and with the molecules,
as this would produce inter-ionic intermolecular or ion molecular
reactions,
ii) the erosion of the electron producing filament.
Methods:
i)-a) Direct insertion

(Direct probe inlet)

i)-b) in-beam or direct ionization
ii) Chromatographic eluent insertion
iii) Molecular Leak (cold inlet) for highly volatile solids and liquids
and gaseous sample

2. Methods of sample ionization

The ionisation method to be used should depend on the type of sample under
investigation and the mass spectrometer available.
Major Ionisation methods include the following:
Electron Impact (EI)
Chemical Ionisation (CI)
Electrospray Ionisation (ESI)
Fast Atom Bombardment (FAB)
Field Desorption / Field Ionisation (FD/FI)
Matrix Assisted Laser Desorption Ionisation (MALDI)
Thermospray Ionisation (TSP)
The ionisation methods used for the majority of biochemical analyses are
Electrospray Ionisation (ESI) and Matrix Assisted Laser Desorption
Ionisation (MALDI)

 Electron Ionization. Electron Ionization (EI) is the

most common ionization technique used for
mass spectrometry. EI works well for many gas
phase molecules, but it does have some
limitations. Although the mass spectra are very
reproducible and are widely used for spectral
libraries, EI causes extensive fragmentation so
that the molecular ion is not observed for many
compounds. Fragmentation is useful because it
provides structural information for interpreting
unknown spectra.

 The electrons used for ionization are produced

by passing a current through a wire filament.
(The amount of current controls the number of
electrons emitted by the filament). An electric
field accelerates these electrons across the
source region to produce a beam of high energy
electrons. When an analyte molecule passes
through this electron beam, a valence shell
electron can be removed from the molecule to
produce an ion.

Electron Ionization Source

 Ionization does not occur by electron capture, which is

highly dependent upon molecular structure. Instead, EI
produces positive ions by knocking a valence electron off
the analyte molecule. As the electron passes close to the
molecule the negative charge of the electron repels and
distorts the electron cloud surrounding the molecule.
This distortion transfers kinetic energy from the fastmoving electron to the electron cloud of the molecule. If
enough energy is transferred by the process, the
molecule will eject a valence electron and form a radical
cation (M+.).

Since the ionization is produced by a single electron that is accelerated to

70 V, this is commonly referred to as 70 eV EI. This is enough energy to
cause extensive fragmentation. The amount of energy transferred during
this process depends upon how fast the electron is traveling and how close
it passes to the molecule. In most 70 eV EI experiments, approximately 15
eV of energy is transferred during the ionization process. There is, however,
a distribution of energy and as much as 30 eV is transferred to some
molecules. Since approximately 10 eV is required to ionize most organic
compounds and a typical chemical bond energy is 3 eV, extensive
fragmentation is often observed in 70 eV EI mass spectra. The distribution
of energy transferred during ionization and the large number of
fragmentation pathways results in a variety of products for a given molecule.
Other electron voltages may be used to vary the amount of fragmentation
produced during ionization. For most organic compounds the threshold
energy for EI is about 10 eV. Because a mass spectrum is produced by
ionizing many molecules, the spectrum is a distribution of the possible
product ions. Intact molecular ions are observed from ions produced with
little excess energy. Other molecular ions have more energy and undergo
fragmentation in the source region. Changing the ionization energy changes
the observed distribution of fragment ions. This distribution provides the
structural information for interpreting mass spectra.

SOFT IONIZATION TECHNIQUES

FIELD IONISATION
An organic compound in the gas phase can be ionized when
the molecules pass near a sharp metal anode carrying a high
electric field. Electrons are sucked from the sample molecules into
incomplete orbitals in the metal, and the resulting molecular ions are
then propelled towards a slit cathode. Primary focusing takes place
at the cathode slit before the ions pass through the entrance slit of
the mass spectrometer to be focused magnetically and
electrostatically as in electron-impact studies.
The principal advantages of field ionization from an organic
chemists point of view are the increased abundance of molecular
ions and the minimization of complex fragmentations and
rearrangements. Disadvantages are the lower sensitivity and
resolution obtained.

2. FIELD IONIZATION:
A very high local electric field
( 2x108 V/cm or 2V/Ao)

On the tip of a very fine metal wire

Molecule (as vapour)

+vely charged conductor

-e

M+.

FIELD DESORPTION (FD)

Outstanding advantages can be achieved by a

modification of the technique in which the
sample is deposited directly onto the anode, and
the high field produces not only ionisation but
desorption. Unstable and involatile material can
be handled in this way and molecular ion peaks
have thus been produced from complex
naturally occurring compounds (notably the
carbohydrates) that do not show M+ peaks on
electron impact.

MAIN FEATURERS OF FI AND FD

1.

Intense peaks related to the original

molecule.
2.
With HR power instrument ____ precise ms
measurements.
3.
A few frag. Ions are produced, hence the
spectrum differs from that of EI
4.
Contain major frags. hence components are
complementary.
5.
FD avoids decompn.

APPLICATION OF FI/FD
FI has the advantage over CI that only M+ + (M+H)+ ions are formed;
adduct ions, (M+X)+ in which the nature of X may not be certain do
not usually occur.
FD is the method of choice for determining the Mol.wt. of thermally
labile molecules e.g:

CARBOHYDRATES
Monosaccharides
Methyl derivs
Disaccharides

Nucleosides
Porphyrins
Triglycerides
Hydrocarbons
Chloroalkanes
Pesticide molecules

Abundant (M+1)+
more abundant (M+1)+
(With FI sometime decompn) but
Methyl derivatives
M+ abundant frags. In FI due to
fission of the glycoside Bond.

 Fast Atom Bombardment and Secondary Ion Mass

Spectrometry
Fast Atom Bombardment (FAB) and Secondary Ion Mass
Spectrometry (SIMS) both use high energy atoms/ions to sputter
and ionize the sample in a single step. In these techniques, a beam
of rare gas neutrals (FAB) or ions (SIMS) is focused on the liquid or
solid sample. The impact of this high energy beam causes the
analyte molecules to sputter into the gas phase and ionize in a
single step (Figure 6). The exact mechanism of this process is not
well understood, but these techniques work well for compounds with
molecular weights up to a few thousand dalton. Since no heating is
required, sputtering techniques (especially FAB) are useful for
studying thermally labile compounds that decompose in
conventional inlets.

 The most significant difference between FAB and SIMS is

the sample preparation. In FAB the analyte is dissolved in
a liquid matrix. A drop of the sample/matrix mixture is
placed at the end of an insertion probe and introduced to
the source region. The fast atom beam is focused on this
droplet to produce analyte ions. Glycerol or similar low
vapor pressure liquids are typically used for the matrix.
Ideally, the analyte is soluble in the liquid matrix and a
monolayer of analyte forms on the surface of the droplet.
According to one theory, this monolayer concentrates the
analyte while the dissolved sample provides a reservoir to
replenish the monolayer as the analyte is depleted.
Without this constant replenishment from the bulk
solution, the ionizing beam will rapidly deplete the analyte
and the signal is difficult to observe.

 SIMS experiments are used to study surface species

and solid samples. No matrix is used and the ionizing
beam is focused directly on the sample. Although this
makes sampling more difficult, it is useful for studying
surface chemistry. High resolution chemical maps are
produced by scanning a tightly focused ionizing beam
across the surface and depth profiles are produced by
probing a single location(9, 10). Although SIMS is a very
sensitive and powerful technique for surface chemistry
and materials analysis, the results are often difficult to
quantitate.

3. Fast atom Bombardment (FAB) Mass Spectrometry

High energy particles
To desorb charged molecular species from the surface of a
liquid in which it is dissolved.
The ions in the gaseous phase are analysed in the usual
manner.
Eg:

Xenon atoms
ionized Xe+. Ions
Accelerated Fast Ions
Xe+. + Xe (thermal)
(Fast) (gas chamber)

Xe
(Fast)

Xe+.
(Thermal)
access
is
deflected.

Beam of fast atom

bombards a metal plate
coated with the sample.
The large amount of K.E. in the atoms volatilizes
and ionizes the sample.
Either-ve or +ve ions may be produced and
analyzed.
Other gases: He, Ar.
In-volatile polar liquids e.g. glycerol, triethyl amine,
1,1,3,3-tetramethyl urea, thioglycerol, crown
ethers.

Desorption takes place by a combination of:

i)
ii)

Thermal Vaporization:
due to the localized heating caused by the impact
of a single H.E. particle.
Sputtering:
Molecules interact with the impacting particles as
hard spheres and desorption occurs during a very
short time interval following impact and prior to
equilibration of energy.

Common Matrices
Glycerol MW 92
1-S Glycerol MW 108
m-Nitrobenzyl Alcohol MW 153
Sample must be soluble
In the matrix
Matrix must have low volatility

Despite much effort in several

laboratories, substantially
better matrices have not been
reported

Additives can
spectra

be used to enhance

Most effective for non-ionic polar

compound 0.1-1% by weight usually
works well
Additive

Pos Ion

Neg Ion

NaCl

MNa+

MCl-

HCl

MH+

MCl-

NH4OH

MNH4+

M-H-

Fast Atom Bombardment Ionization (FAB)

Liquid Secondary lon Mass Spectrometry (LSIMS)
First reported ~1980
Major extension in role of MS for analyses of biological materials
Use for compounds with MW less than 1000-1500
Peptides, drugs, natural products, pollutants, metalorganics
Samples should be easily charged in solution
Acids, bases, highly polar compounds
Samples must be soluble in a liquid matrix such as glycerol
Use with solids probe
No HPLC; No automation
Samples should be relatively pure and free of additional salts, buffers, etc.
Often used to confirm MW of compounds analyzed by El or Cl
May give some fragments;
No heating required.

Main Features of FAB:

High molecular wt., low volatility subs. e.g.
oligonucleotides
containing
upto
10
nucleotides.
(M++H) as well as adduct and frag. Ions
(Like in CI)
(M+-H) ions
Peptides upto 3,000 daltons
Complete amino acid sequence from the key
fragment ions
Phospholipids
Carbohydrates
Glycosides

Chemical Ionization
For organic chemists, Chemical Ionization (CI) is especially
useful technique when no molecular ion is observed in EI mass
spectrum, and also in the case of confirming the mass to charge
ratio of the molecular ion. Chemical ionization technique uses
virtually the same ion source device as in electron impact,
except, CI uses tight ion source, and reagent gas. Reagent gas
(e.g. ammonia) is first subjected to electron impact. Sample ions
are formed by the interaction of reagent gas ions and sample
molecules. This phenomenon is called ion-molecule reactions.
Reagent gas molecules are present in the ratio of about 100:1
with respect to sample molecules. Positive ions and negative
ions are formed in the CI process. Depending on the setup of the
instrument (source voltages, detector, etc...) only positive ions or
only negative ions are recorded.
Unlike molecular ions obtained in EI method, MH + and [M-H]detection occurs in high yield and less fragment ions are
observed.

Chemical Ionization (CI) is a soft ionization technique that produces

ions with little excess energy. As a result, less fragmentation is
observed in the mass spectrum. Since this increases the abundance
of the molecular ion, the technique is complimentary to 70 eV EI. CI is
often used to verify the molecular mass of an unknown. Only slight
modifications of an EI source region are required for CI experiments.
In Chemical Ionization the source is enclosed in a small cell with
openings for the electron beam, the reagent gas and the sample. The
reagent gas is added to this cell at approximately 10 Pa (0.1 torr)
pressure. This is higher than the 10-3 Pa (10-5 torr) pressure typical for
a mass spectrometer source. At 10-3 Pa the mean free path between
collisions is approximately 2 meters and ion-molecule reactions are
unlikely. In the CI source, however, the mean free path between
collisions is only 10-4 meters and analyte molecules undergo many
collisions with the reagent gas. The reagent gas in the CI source is
ionized with an electron beam to produce a cloud of ions. The
reagent gas ions in this cloud react and produce adduct ions like
CH5+, which are excellent proton donors.

When analyte molecules (M) are introduced to a source region with

this cloud of ions, the reagent gas ions donate a proton to the
analyte molecule and produce MH+ ions. The most common reagent
gases are methane, isobutane and ammonia. Methane is the
strongest proton donor commonly used with a proton affinity (PA) of
5.7 eV. For softer ionization, isobutane (PA 8.5 eV) and ammonia
(PA 9.0 eV) are frequently used. Acid base chemistry is frequently
used to describe the chemical ionization reactions. The reagent gas
must be a strong enough Brnsted acid to transfer a proton to the
analyte. Fragmentation is minimized in CI by reducing the amount of
excess energy produced by the reaction. Because the adduct ions
have little excess energy and are relatively stable, CI is very useful
for molecular mass determination.

Positive ion mode:

GH+ + M ------> MH+ + G
Negative ion mode:
[G-H]- + M ------> [M-H]- + G
These simple proton transfer reactions are true gas-phase Acid-Base
processes in the Bronsted-Lowrey sense.
A "tight" ion source (pressure=0.1-2 torr) is used to maximize
collisions which results in increasing sensitivity.
Proton transfer is one of the simple processes observed in positive CI:
RH+ + M -----> MH+ + R
One of the decisive parameter in this reaction is the proton
affinity. For the reaction to occur, the proton affinity of the molecule
M must be higher than that of the gas molecule.
Choice of reagent gas affect the extent of fragmentation of the
quasi-molecular ion.

Positive chemical ionization:

Methane:
CH4 + e -----> CH4+. + 2e
CH4+. + CH4 -----> CH5+ + CH3.
CH4+. + CH4 -----> C2H5+ + H2 + H.
Isobutane:
i-C4H10 + e -----> i-C4H10+. + 2e
i-C4H10+. + i-C4H10 ------> i-C4H9+ + C4H9 +H2
Ammonia:
NH3 + e -----> NH3+. + 2e
NH3+. + NH3 ------> NH4+ + NH2.
NH4+ + NH3 ---------> N2H7+
In methane positive ion mode CI the relevant peaks observed are MH+,
[M+CH5]+, and [M+C2H5]+; but mainly MH+

Choice of reagent gas:

Two factors determine the choice of the gas to be used:
Proton affinity (PA): H+ + M -------MH+
(Energy transfer)
Excitation energy of MH+ = PA (M) PA (R)
Excitation energy in MH+ facilitates fragmentation
NH3 (ammonia) is the most used reagent gas in CI because of
the low energy transfer of NH4+ compare to CH5+ for example.
With NH3 as reagent gas, usually MH+ and MNH4+ (17 mass units
difference) are observed.

1.CHEMICAL IONIZATION:
CH4 + e

CH4+. + 2e

CH4 + CH4+.

CH5+ + CH3.

Reagent gas

[~ 10-6 tor; 70 eV]

(in EI)

1Torr; eV 100-500
ion chamber

Sample gas
R+e

10-3 Torr
[R+.]*
excited
primary reactant
gas ions
.

collisional
H-transfer

(R+H)
equilibrated sec
reactant gas ion

(R+H)+
reactant
gas ion

(M+H)+ + R

molecule
under
investigation

(M-H)+
quasi-mol
(tertiary)
ions

CH4

affords

principally

Isobutane

NH3

H2

R:M should be atleast 103 at a 1 tor

+RH2
neutral
species

CH5+
C4H9+ Sec.
ions
NH4+
H 3+

Negative Ion Chemical Ionization:

Three mechanisms can be underlined:
1- Electron capture reaction due to attainment of slow
moving, low energy "thermalized" electrons which may be
transfered more efficiently to sample molecules.
2- Electron transfer from ionized reagent gas (e.g. NH 2may transfer an electron to a molecule having a greater
electron affinity than NH2).
3- Reagent gas ions participate in true CI reactions (e.g.
proton abstraction, according to relative acidities).
4- Molecular ions observed in negative ion chemical
ionization mass spectra are usually M - or [M-H]-

Chemical Ionization
Similar to EI (Electron Ionization)
Used to analyze same types of compounds (must be volatile)
Use same sample inlets (solids probe or GC)
Requires only minor changes to the ion source
Different from EI
Gives less fragmentation, charge-induced fragmentation only
Can determine the number of labile hydrogens
Can selectively ionize amines

SUMMARY ( SECOND LECTURE)

Sample introduction
Methods of sample ionization
Comparison b/w Cl and El; source modifications required to
increase pressure
Why is high reagent gas pressure required?
Reagent gas; common gases; reagent ions; common reactions
Proton transfer; hydride abstraction; adduct formation
Proton Affinity; how does it relate to ion excitation energy and
fragmentation
Use proton affinity to predict PT or A in ammonia Cl
Use ammonia Cl to determine the number of exchangeable H
Explain apparent M+ in ammonia Cl spectra
Fast Atom Bombardment Ionization (FAB) and
Liquid Secondary lon Mass Spectrometry (LSIMS)

6.Electrospray ionisation

Electrospray Ionisation (ESI) is one of the Atmospheric Pressure

Ionisation (API) techniques and is well-suited to the analysis of
polar molecules ranging from less than 100 Da to more than
1,000,000 Da in molecular mass.

It has undergone remarkable growth in recent years and is

frequently used for LC/MS of thermally labile and high molecular
weight compounds. The electrospray is created by applying a large
potential between the metal inlet needle and the first skimmer in an
API source (Figure 7). The mechanism for the ionization process is
not well understood and there are several different theories that
explain this ionization process. One theory is that as the liquid
leaves the nozzle, the electric field induces a net charge on the
small droplets. As the solvent evaporates, the droplet shrinks and
the charge density at the surface of the droplet increases. The
droplet finally reaches a point where the coulombic repulsion from
this electric charge is greater than the surface tension holding it
together. This causes the droplet to explode and produce multiply
charged analyte ions. A typical ESI spectrum shows a distribution of
molecular ions with different charge numbers.

 Because electrospray produces multiply

charged ions, high molecular weight
compounds are observed at lower m/z
value. This increases the mass range of
the analyzer so that higher molecular
weight compounds may be analyzed. An
ion with a mass of 5000 u and a charge of
+10 is observed at m/z 500 and is easily
analyzed.

Standard electrospray ionisation source

During standard electrospray ionisation the sample is
dissolved in a polar, volatile solvent and pumped through
a narrow, stainless steel capillary (75 - 150 micrometers
i.d.) at a flow rate of between 1L/min and 10L/min. A
high voltage of 3 or 4 kV is applied to the tip of the
capillary, which is situated within the ionisation source of
the mass spectrometer, and as a consequence of this
strong electric field, the sample emerging from the tip is
dispersed into an aerosol of highly charged droplets, a
process that is aided by a co-axially introduced
nebulising gas flowing around the outside of the capillary.
(REF: J. Fenn, J. Phys. Chem., 1984, 88, 4451).

This gas, usually nitrogen, helps to direct the spray emerging from
the capillary tip towards the mass spectrometer. The charged
droplets diminish in size by solvent evaporation, assisted by a
warm flow of nitrogen known as the drying gas which passes across
the front of the ionisation source. Eventually charged sample ions,
free from solvent, are released from the droplets, some of which pass
through a sampling cone or orifice into an intermediate vacuum
region, and from there through a small aperture into the analyser of
the mass spectrometer, which is held under high vacuum.

Electrospray ionisation
In ESI, samples (M) with molecular
masses up to ca. 1200 Da give rise to
singly charged molecular-related ions,
usually protonated molecular ions of the
formula (M+H)+ in positive ionisation
mode, and deprotonated molecular ions
of the formula (M-H)- in negative
ionisation mode.
4

ESI
n1 charge of m/z1
n2 charge of m/z2
n 2 = n1 + 1
n1 = m/z2 - mH
m/z1 m/z2
Suppose 1st peak is at m/z 1001
2nd peak is at m/z 501
n1 = (501-1)/1001-501
= 500/500 = 1
Mr = 1x (1001-1) = 1000
And doubly protonated ion is at m/z 501 b/c
15

1000+2/2 = 501

Calculation of MW in ESI

Mass of Ist peak = 949.66

Mass of 2nd peak = 712.5
Mass of 3rd peak = 570.2
Mass of 4th peak = 475.33

Using formula:
n1 = m/z2 - mH
m/z1 m/z2
712.5-1
949.66-712.5

We know,
MW = (m/z x n) n
MW = (949.66 x 3) 3 = 2846.06

17

= 3.00 i.e. n1 = 3

Electrospray Ionization (ESI)

Used to analyze polar, neutral and ionic molecules
Natural products, drug and drug metabolites, proteins, nucleic acids etc.
Mol. Wt. range 100,000 and higher
Can be directly coupled with HPLC to analyze complicated mixtures
Environmental samples, drug metabolites, protein digests etc.
Often produces multiply charged ions
High MW compounds can be analyzed on low m/z analyzers
Easily automated to run samples 24 hrs/day
One person can operate multiple instruments
High throughput allows running 1 sample per minute
No fragmentation
Most frequently used ionization method
Courtesy: Dr. David Smith

5. Matrix assisted laser

desorption ionisation
Matrix Assisted Laser Desorption Ionisation (MALDI)
deals well with thermolabile, non-volatile organic
compounds especially those of high molecular mass and
is used successfully in biochemical areas for the analysis
of proteins, peptides, glycoproteins, oligosaccharides,
and oligonucleotides. It is relatively straightforward to
use and reasonably tolerant to buffers and other additives.
The mass accuracy depends on the type and performance
of the analyser of the mass spectrometer, but most
modern instruments should be capable of measuring
masses to within 0.01% of the molecular mass of the
sample, at least up to ca. 40,000 Da.
(REF: F. Hillenkamp, M. Karas, R. C. Beavis, B. T. Chait,
Anal. Chem., 1991, 63, 1193)
10

MALDI is based on the bombardment of sample molecules

with a laser light to bring about sample ionisation. The
sample is pre-mixed with a highly absorbing matrix
compound for the most consistent and reliable results, and a
low concentration of sample to matrix works best. The matrix
transforms the laser energy into excitation energy for the
sample, which leads to sputtering of analyte and matrix ions
from the surface of the mixture. In this way energy transfer is
efficient and also the analyte molecules are spared excessive
direct energy that may otherwise cause decomposition. Most
commercially available MALDI mass spectrometers now have
a pulsed nitrogen laser of wavelength 337 nm.

11

The sample to be analysed is

dissolved in an appropriate volatile
solvent, usually with a trace of
trifluoroacetic
acid
if
positive
ionisation is being used, at a
concentration of ca. 10 pmol/L and
an aliquot (1-2 L) of this is removed
and mixed with an equal volume of a
solution containing a vast excess of
a matrix. A range of compounds is
suitable for use as matrices:
sinapinic acid is a common one for
protein analysis
while alphacyano-4-hydroxycinnamic acid is
often used for peptide analysis. An
aliquot (1-2 L) of the final solution
is applied to the sample target which
is allowed to dry prior to insertion
into the high vacuum of the mass
spectrometer.
12

The laser is fired, the energy arriving at the

sample/matrix
surface
optimized,
and
data
accumulated until a m/z spectrum of reasonable
intensity has been amassed. The time-of-flight analyser
separates ions according to their mass(m)-tocharge(z) (m/z) ratios by measuring the time it takes
for ions to travel through a field free region known as
the flight, or drift, tube. The heavier ions are slower
than the lighter ones.

13

Matrix assisted laser desorption ionisation

MALDI is also a "soft" ionisation method and so results
predominantly in the generation of singly charged
molecular-related ions regardless of the molecular
mass, hence the spectra are relatively easy to interpret.
Fragmentation of the sample ions does not usually
occur.
In positive ionisation mode the protonated molecular
ions [M+H]+ are usually the dominant species, although
they can be accompanied by salt adducts, a trace of the
doubly charged molecular ion at approximately half the
m/z value, and/or a trace of a dimeric species at
approximately twice the m/z value. Positive ionisation is
used in general for protein and peptide analyses.
14

Positive or negative ionisation?

If the sample has functional groups that readily
accept a proton (H+) then positive ion detection
is used e.g. amines R-NH2 + H+ = R-NH3+ as in
proteins or peptides.
If the sample has functional groups that readily
lose a proton then negative ion detection is used
e.g. carboxylic acids R-CO2H = R-CO2- and
alcohols R-OH = R-O- as in saccharides or
oligonucleotides
15

 In
negative
ionisation
mode
the
deprotonated molecular ions (M-H)- are
usually the most abundant species,
accompanied by some salt adducts and
possibly traces of dimeric or doubly charged
materials. Negative ionisation can be used
for the analysis of oligonucleotides and
oligosaccharides.

16

SOFT IONIZATION TECHNIQUES

These techniques attempt to overcome the main
drawbacks of EI, namely.
Absence of M+ in many cases and hence Mol.Wt.
and Mol.Formula.
The presence of rearrangement reactions.
The possibility of pyrolysis during vapn.
Particularly of less volatile substances.

17

13

14

18

19

11

12

37

What ionization technique would be appropriate for analyzing the

following compounds: a) gasoline fractions, b) pesticide residue, c)
ibuprofen and acetaminophen, d) insulin, e) tripeptides, f) heavy
metals in water

10

Analysis and Separation of Sample Ions

The main function of the mass analyser is
to separate , or resolve the ions formed
in the ionisation source of the mass
spectrometer according to their mass-tocharge (m/z) ratios. There are a number
of mass analysers currently available, the
better
known
of
which
include
quadrupoles , time-of-flight (TOF)
analysers, magnetic sectors , and both
Fourier transform and quadrupole ion
18
traps .

SEPARATION OR ANALYSIS OF IONS

19

Magnetic Sector Mass Spectrometers

Quadrupole Mass Spectrometers
Time-of-Flight Mass Analyzers
Tandem (MS-MS) mass spectrometry

 The m/z scale of the mass spectrometer is

calibrated with a known sample that can
either be analysed independently (external
calibration) or pre-mixed with the sample
and matrix (internal calibration).

20

Magnetic-Sector Mass
Spectrometry

Magnetic-Sector Mass Spectrometry

THEORY:
The ion source accelerates ions to a kinetic energy given by:
KE = mv2 = eV
Where m is the mass of the ion, v is its velocity, e is the charge on the
ion, and V is the applied voltage of the ion optics.

Magnetic-Sector Mass Spectrometry

The ions enter the flight tube and are deflected by the magnetic
field, B.
Only ions of mass-to-charge ratio that have equal centripetal and
centrifugal forces pass through the flight tube:
mv2 /r = BeV, where r is the radius of curvature

Magnetic-Sector Mass Spectrometry

mv2 /r = BeV
By rearranging the equation and eliminating the velocity term using
the previous equations, r = mv/eB = 1/B(2Vm/e)1/2
Therefore, m/e = B2r2/(2V)
This equation shows that the m/e ratio of the ions that reach the
detector can be varied by changing either the magnetic field (B) or
the applied voltage of the ion optics (V).

Magnetic-Sector Mass Spectrometry

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.

Time of Flight Mass Spectrometry (TOF-MS)

INTRODUCTION:

Separates ions based on flight time

Operates in pulsed mode
Ions accelerated by an electric field
Lighter ions reach the detector first

Time of Flight Mass Spectrometry (TOF-MS)

THEORY:
KE=eV when electrons are accelerated through an
electric field
KE of ion is mv2, so eV= mv2 and velocity is
inversely proportional to mass
Transit time (t) is L/v, where L is drift tube length
and v is velocity
So t=L/(2V/m/e) can be solved for charge-mass ratio

Time of Flight Mass Spectrometry (TOF-MS)

HOW ITS DONE:

Reflectron is series of rings or grids that serves to focus

ions to improve resolution
Exact values of L and V do not need to be known if two or
more ions of known mass are used as mass calibration points
Produces a mass spectrum as a function of time (can be
measured every 10 nsec)

Quadrupole Mass Analyzers

Basis of Quadrupole Mass Filter

consists of 4 parallel metal
rods, or electrodes
opposite electrodes have
potentials of the same sign
one
set
of
opposite
electrodes
has
applied
potential of [U+Vcos(t)]
other set has potential of
- [U+Vcost]
U= DC voltage, V=AC
voltage, = angular velocity
of alternating voltage

Operation of Quadrupole Mass Filter

voltages applied to electrodes
affect trajectory of ions with the
m/e ratio of interest as they travel
down the center of the four rods
these ions pass through the
electrode system
ions with other m/z ratios are
thrown out of their original path
these ions are filtered out or lost
to the walls of the quadrupole,
and then ejected as waste by a
vacuum system
in this manner the ions of interest
are separated

Background Info
Quadrupole Mass Analyzers
are in use since the 1950s
most commonly used mass spec today
sometimes referred to as mass filters because
ions of only a single mass to charge (m/z) ratio pass
through the apparatus
separate ions based on oscillations in an electric
field (the quadrupole field) using AC and DC
currents

Benefits
Quadrupole Mass Analyzers

easy to use
simple construction
fast
low cost
can achieve unit to 0.1 m/q resolution

 tolerant of relatively poor vacuums (~5 x 10-5torr),

which make them well suited to electrospray ionization
(because these ions are produced under atmospheric
conditions)
quadrupoles are now capable of routinely analyzing up
to a m/q ratio of 3000, which is useful in electrospray
ionization of biomolecules, which commonly produces a
charge distribution below m/z 3000

Obtaining A Spectrum
Quadrupole Mass Analyzers
a mass spectrum is obtained by varying the voltages on the
rods and monitoring which ions pass through the filter
two methods for varying rod voltages:
vary while holding U and V constant
vary U and V but keep the ratio U/V fixed
remember: U= DC voltage, V=AC voltage, = angular
velocity of alternating voltage
The resolution is determined by the magnitude of U/V ratio
beam currents for individual m/e ratios may be as low as
10-13 A and so electron multipliers are usually used

Applications
Quadrupole Mass Analyzers

partial pressure analyzers

GC/MS
upper atmosphere and space research
separation of proteins and other
biomolecules with electrospray
plasma diagnostics
multielement and isotopic analyses

Summary

Magnetic Mass Spectroscopy shows the relationship between

voltage and mass to charge ratio:
m/e = B2r2/(2V)
Time of Flight Mass Spectroscopy shows the relationship
between tube time and mass to charge ratio:
m/e = 2Vt2/L2
Electric Quadrupole shows the relationship between AC/DC
currents and mass to ratios.

Tandem (MS-MS) mass spectrometry

Tandem (MS-MS) mass spectrometers are
instruments that have more than one analyser and
so can be used for structural and sequencing
studies. Two, three and four analysers have all been
incorporated into commercially available tandem
instruments, and the analysers do not necessarily
have to be of the same type, in which case the
instrument is a hybrid one. More popular tandem
mass spectrometers include those of the
quadrupole-quadrupole,
magnetic
sectorquadrupole and more recently, the quadrupoletime-of-flight geometries.
30

Tandem (MS-MS) mass spectrometry

Tandem mass spectrometry (MS-MS) is used
to produce structural information about a
compound by fragmenting specific sample ions
inside the mass spectrometer and identifying the
resulting fragment ions. This information can
then be pieced together to generate structural
information regarding the intact molecule.
Tandem mass spectrometry also enables
specific compounds to be detected in complex
mixtures on account of their specific and
characteristic fragmentation patterns.
31

32

SUMMARY

33

Electrospray Ionization (ESI)

ESI Versus EI (Advantages)
ESI Versus EI (Difficulties)
Applications
MALDI
Negative and Positive Ions
Analyzers
Magnetic Sector Analyzers
Benefits of Magnetic Sector Analyzers
Limitations of Magnetic Sector Analyzers
Quadrupole Analyzers
Time of Flight Analyzers
Tandem MS-MS
Detectors

EXAMINATION OF THE MASS SPECTRUM

A.

Recognition of the Molecular ion

a)
Often (M-1)+ and sometime (M-2)+, (M-3)+ etc. are
observed due to losses of H atoms from M+.

b) It is accompanied by isotope ions. For Cl,

Br, S, M+2 are more abundant compared
with the (M+2)+ ions of similar compounds
without these heteroatoms. [Isotope
abundance].

c)

In nitrogen compounds most abundant

ion is (M-1)+ and not the M+

In case, (a) and (c) it is worthwhile

examining the spectrum by a soft
ionization technique.

d)

Absence of molecular ions (or extremely weak M+)

suggests highly branched molecules. [Soft ionization
techniques]

 e) Organic compounds usually have C,H,N,O,S, Cl, Br,

etc. and it is very unlikely that M+ give rise to losses
of 5-14 and 21-25 units. If M+ shows such losses it
is most unlikely to be correctly assigned.
f) losses of H & CH3 (1 & 15 mass units) are
commonly observed.
g) M+ ions must contain all the elements that are
evident elsewhere in the spectrum.

B)
If metastable ions which implicate the candidate peak as being due to product ions of
some higher mass species, then it can not be the true M+ peak.
Metastable ions in the routine spectrum; recall that the position of the metastable ion
(m) is given by,
m22
m = ______
m1

where,

m1 = mass of the precursor

m2 = mass of the product
10

C)

It is useful to remember that:

i)

An odd molecular weight indicates an odd No. of N

atoms.

ii)

Abundant (M+2)+ ions would suggest the presence of

Cl or Br and less abundant ones the presence of S.

iii)

An approximate No. of C atoms can be gained from

the relative heights of M+ (h) and (M+1)+ (h) peaks
since 13C is present in 1.1% natural abundance in
organic compounds., the approx. No. of C atoms is
given by 100 h/1.1h
(e.g. ms of naphthalene C10H10 :

12

100x10.9 = 10
1.1x100

D)

to obtain the mol.formula _____ it is necessary to

measure the accurate mass of M+ ions and
obtain molecular formula from table or by
calculation.

E)

The molecular formula obtained may then be

used to calculate the number of double bond
equivalents.

F)

The general appearance of the spectrum.

i)

A spectrum exhibiting many frag.ions increasing

in abundance towards low m/z values indicates
a predominantly aliphatic structure.

ii)

A spectrum with a few frag.ions, abundant mol.ions

& doubly charged ions will suggest an aromatic
structure.
13

14

 G) Next step is to note the major frag. ions and

attempt to elucidate the main fragmentation
pathways.
H) In the absence of N atoms, frag.ions occurring
at odd m/z values are even-electron species
resulting from simple bond fission; frag.ions at
even m/z values are odd electron speciesproduced
by multiple bond cleavage suggesting that
rearrangement may have occurred.

15

STRUCTURE ELUCIDATION
Without rationalization
Mass spectra of unknown structure may be compared with the
published spectra compilations either:
i) Manually
tedious ; or
ii) Through a computer and automatic library searching
Possible only if the mass spectrum has already been recorded and
available in DATA.
By rationalization
(Most in use):
i) Functional-group approach
M+-18
M+-60
M+-31
26

(loss of water)
(loss of C2H4O2)
(loss of OCH3 or-CH2OH)

(Lists are available in text books)

ii) Accurate mass measurements
For example m/z 27 = HCN or C2H3 can be determined by obtaining
exact mass of the ion at m/z 27.
iii)Characteristic m/z values
Peaks characteristic of a certain feature of molecular structure
e.g. m/z 43 (CH3CO+, C3H7+)
91

(C7H7+)

m/z 105

C6H5CO (OR ??)

17

iv) Fragmentation Processes

Many simple fragmentation reactions of heteroatom molecular ions
may be summarized in the following scheme in which X represents
the heteroatom and R1, R2 and R3 are substituents; when X is doubly
bonded to the adjacent carbon atom R3 is not present.
R2
C+

R1
C1

R2

C2
R1

C
(R3)

C1

(R3)

X
C2
R1

18

X+

or

R2
or

C
(R3)

+
X

Type C1 cleavage is breaking of the C-X bond and is observed with

saturated heteroatom substituents (X=Cl, Br, I, OH, SH, NH2). The
C1 cleavage is not observed with unsaturated heteroatom
substituents.
Type C2 cleavage is commonly observed for both saturated and
unsaturated heteroatom substituents of alkanes. The extent to which
C1 or C2 bond breaking occurs is very dependent on the nature of
the heteroatom and the structure of the molecule. Either or both
fragments from C1 and C2 reactions may appear as even-electron
charged species in the mass spectrum as shown in the scheme.

19

CH3

CH3

20

CH

CH2

O +

CH

CH3

CH2

+
HC

+
CH3 + CH2

HC

CH

CH2

+
O

CH2

CH

+
CH2

The other common process observed in aliphatic heteroatom

compounds is rearrangement of hydrogen accompanying elimination
of a neutral species to give odd-electron fragment ions. Again the
process can be represented in a common formalism by two reaction
types (RE1 and 2). With saturated substituents (X), the
rearrangement elimination reaction (REI) is general except for
amines, but for unsaturated substituents, the alternative process
(RE2) is general. The processes shown below have been drawn with
the six-membered ring transition state, but it should be recalled that
the results of deuterium labeling are not unequivocal and some
hydrogen transfer occurs through smaller and larger cyclic transition
states. For halogen compounds, a similar five membered state
appears to be more important.

21

R1

H
CH

(R )
CH2

C
CH2

+
HX

R1

H
CH

+ R1CHCH2CH2CR2
(R3)

RE1

+
X

(R )
CH2

C
CH2

XH
RE2

R1CH

CH2 + CH2

CR2

A double bond or an aromatic ring can be considered here as an

unsaturated substituent in alkanes. The allylic or benzylic types of
cleavage adjacent to these unsaturated centres are comparable with
for example the -cleavage (C2) of carbonyl compounds if the
unsaturated centres take the place of the heteroatom.
22

Aromatic compounds exhibit many of the features found in the mass

spectra of aliphatic compounds but these are usually considerably
modified because of:
a) The increased stability of the molecular ions
b) The aromatic ring system itself behaving as a substituent (X) in an
alkane
c) Substituents on the aromatic ring changing fragmentation behavior
and
a) Heteroatoms in the aromatic ring influencing the fragmentation of
the side chain depending on its position.

23

SUMMARY

24

How to recognize the molecular ion.

(isotope peaks, rel.abundance, M+-H peaks, soft ionization for confirmation.
Examination of mass spectrum
(Aromatic, aliphatic, straight chain or branched)
Odd molecular ion indicates odd no. of N.
Look at major fragments
(Odd m/z values, in absence of N are even electron species ---------simple
bond fission)
(Even m/z values are odd electron species -------multiple bond cleavages.
Irrational approach------------data search.
Rational approach.
Use of accurate mass to differentiate b/w two or more elemental
compositions.
Fragmentation processes.

Elimination by multiple -bond rupture. Elimination

by multiple -bond rupture may occur, leading to the
extrusion of a neutral molecule such as CO, C2H4,
C2H2, etc. A well known example is the retro-DielsAlder reaction of cyclo-hexenes, which can be
represented as in (a). Highly stabilized ene fragments
may cause charge retention to be in part reversed as in
(b).

(a)

one-electron
mechanism

(b)

e
+

two-electron
mechanism

Rearrangements. These are common, the most frequently encountered

having been described by F.W. McLafferty and named after him. It is
exemplified in the case of a carbonyl compound (I) by the extrusion of
an alkene, but is also exhibited by ions such as II, III, IV, etc.
H

O+

O+

O+

H2N

HO

RO
ester
II

+
X

H
+

amide
IV

carboxylic acid
III

X
R

The even-electron rule. The even electron rule is a rule-of-thumb

interpretation of sound thermodynamic principles: in essence it
states that an even-electron species (an ion, as opposed to a radical
ion) will not normally fragment to two odd-electron species (that is,
it will not degrade to a radical and a radical ion), since the total
energy of this product mixture would be too high.
A+

even

B+

odd

odd

In preference, an ion will degrade to another ion and a neutral molecule

A+

even

even

even

Radical ions, being odd-electron species, can extrude a neutral molecule, leaving a radical
ion as coproduct.
.

A+

B+

odd

odd

even

Radical ions can also degrade to a radical and an ion

A+

odd

B+

even

C (or B + C+)

odd odd

even

A)

Fragmentation of odd-electron cations

or radical cations

The fragmentation of these ions without any

rearrangement or without any cleavage of an
even number of bonds such as occurs in rings
necessarily leads to an even ion and to a
radical.

R2
+

R1

C
(R3)

C+

R1

X+

or

C1

R2

C2

C1

(R3)

X
C2
R1

R2
or

C
(R3)

+
X

1. Cleavage of the sigma bond adjacent to a

heteroatom (Direct dissociation)
In radical cations, the charge is
delocalized over the whole molecule.
However , heteroatoms with weak IP carry
it preferentially.

The predominant cation is the one that corresponds to the radical

with the lower IP. This is Stevensons rule.

The cleavage of the bond adjacent to the heteroatom is similar to

direct dissociation
OR
C-1 cleavage =direct dissociation = cleavage of bond adjacent to
heteroatom

10

11

2. Cleavage of the alpha bond

The bond alpha to the radical cation site
can be broken by a radical site initiated
reaction, i.e. by a transfer of an electron
from this bond.
If several alkyl chains can be lost as
radicals, the loss of the longest chain is
favored.

12

13

Alpha-Cleavage
-Cleavage of Acetone Molecular Ion
The EI mass spectrum of acetone is comparatively
simple. It basically shows three important peaks at m/z
58, 43, and 15. According to the formula C3H6O, the
peak at m/z 58 corresponds to the molecular ion. The
base peak at m/z 43 is related to this signal by a
difference of 15 u, a neutral loss which can almost
always be assigned to loss of a methyl radical, CH3. The
m/z 15 peak may then be expected to correspond to the
ionic counterpart of the methyl radical, i.e., to the CH3+
carbonium ion. The question remains, as to whether this
mass spectrum can be rationalized in terms of ion
chemistry. Let us therefore consider the steps of electron
ionization and subsequent fragmentation in greater detail
14

-Cleavage - A Radical-Site Initiated Fragmentation

15

The acetone molecule possesses two non-bonding electron pairs at

the oxygen that will atleast formally be the preferred source of the
ejected electron. The excited molecular ion may then cleave off a
methyl radical by simply shifting one electron (single-barbed arrow
or "fishhook") from the CO-CH3 bond to the radical site at the
oxygen atom allowing the products to drift apart. This homolytic
bond cleavage is a radical-site initiated process with charge
retention, i.e., the ionic charge resides within the moiety where it
was initially located. The process is also known as -cleavage. The
neutral fragment, CH3. is not detected by the mass spectrometer,
whereas 'the charged fragment, C2H3O+, gives rise to the base
peak m/z 43.

16

-Cleavage of Non-Symmetrical Aliphatic

Ketones

17

When a ketone grows larger it does not necessarily imply that it has
two identical alkyl groups at the carbonyl. In case of different alkyls
at the carbonyl, Stevenson's rule may also be applied to decide
which of them will dominantly be detected as part of the acylium ion
and which should preferably give rise to a carbenium ion. Overall, a
nonsymmetrical ketone will yield four primary fragment ions in its EI
mass spectrum.
For example the 70 e V EI mass spectrum of butanone shows an
ethyl loss, m/z 43, which is largely preferred over methyl loss, m/z
57. Furthermore, the C2H5+ ion, m/z 29, is more abundant than the
less stable CH3+ ion, mlz 15. If the alkyls become larger than ethyl,
another pathway of ion dissociation will occur in addition.

18

3. COMPETITION BETWEEN THE CLEAVAGE OF THE

ADJACENT BOND AND THE CLEAVAGE OF THE ALPHA BOND

The cleavage of the adjacent bond (C-1 cleavage) occurs more

easily if the heteroatom is a large atom. In the case of neighboring
atoms, the more electronegative one leads more easily to the
cleavage of the adjacent bond. The cleavage becomes
predominant for electron donors. The following order is observed:
Br, Cl < R; bond, S, O < N
Thus the halogens preferentially cause the loss of the radical X
through a cleavage of the adjacent bond, whereas the amines
preferentially lose a radical through a cleavage of the alpha bond.

19

As an example compare the spectra of butylamine and of butanethiol.

In the case of butylamine, the ion that is most intense corresponds to a
radical-initiated a cleavage.

NH2

+
CH2=NH2
m/z=30

However, in the case of butanethiol, the main fragmentation

corresponds to the loss of an HS- radical and the most intense ion is the
butyl cation at 57. The cleavage leading to the CH2=SH+ ion at 47 is
also observed but is clearly less intense.

20

21

22

SUMMARY
Fragmentation Processes

23

Single bond rupture

Multiple bond rupture
RDA
McLafferty Rearrangement
Even Electron rule
Fragmentation of odd electron cations (radical cations)
Fragmentation of even electron cations (cations)
C-1 cleavage = direct dissociation = cleavage of bond adjacent to
heteroatom
C-2 cleavage = cleavage of bond
Radical-site initiated cleavage
Charge initiated cleavage
Competition between the cleavage of the adjacent bond and the
cleavage of the alpha bond

4. Fragmentation of radical cations with

rearrangement

McLafferty Rearrangement

Figure: Typical appearance of mass spectra (a) with three mirror galvanometers
scanning simultaneously and (b) with a logarithmic galvanometer. Note the
metastable ions at m/z 43.4 and m/z 60.2 in (a)

Interpretation of Mass Spectra

OC2H5

+
O

77

O
C +

105
8

C2H5O

51

C2H5OC

McLafferty Rearrangement
+

OH

Cl

+
O

OH

Cl

+
m/z 56

Characteristics of the various ionization methods used in the

mass spectrometric analysis of peptides and proteins.
Method

18

Detection
limit (pmol)

Common
application
field (Da)

Precision
(%)

Analyzer

FABMS

1-50

6000

0.05

Magnetic or
quadrupole

ESIMS

0.01-5

>130000

0.01

Magnetic or
quadrupole

MALDI-MS

0.001-1

>300000

0.05

Time-offlight

Mass Spectrometry
Points to remember:
1. Ions are detected at a certain m/z value, and therefore only ionic species may
be correlated with peaks. Neutral losses are exclusively indicated by the mass
difference between peaks.
2. Even-electron rule: Odd-electron ions (such as molecular ions and fragment
ions formed by rearrangements) may eliminate either a radical or an evenelectron neutral species, but even electron ions (such as protonated molecules
or fragments formed by a single bond cleavage) will not usually lose a
radical to form an odd-electron cation. In other words, the successive loss of
radicals is forbidden.
3. It is important to assign the correct charge and radical state to all species
encountered and to carefully track them through a fragmentation scheme.
Otherwise, impossible fragmentation pathway may be formulated, thereby
misleading the assignment of elemental composition and molecular
constitution.
4. The detection of the positive halogen ion and a less intensive peak due to the
hydrogen halogenide is a generally observed characteristic of halogenated
compounds. In accordance with the relative electronegativities of the
halogens their intensities follow the order I+ > Br+ > CI+ > F+. In case of Br
21and CI the isotopic patterns give additional evidence for the presence of the
respective halogen.

The term -cleavage for the widespread radical-site

initiated process with charge retention can be misleading,
because the bond cleaved is not directly attached to the
radical site, but to the next neighboring atom.
6. Stevensons rule: When a fragmentation takes place, the
positive charge remains on the fragment with the lowest
ionization energy.
7. The homolytic dissociation of a C-C bond always
proceeds to yield both product pairs, their relative
abundances being basically governed by Stevensons rule.
8. The acylium ions and the saturated carbenium ions are
isobaric, i.e., they have the same nominal mass. However,
their exact masses are different, i.e., CH3OC+, m/z
43.0178, and C3H7+, m/z 43.0542.
5.

22

23

24

9. Nitrogen rule: If a compound contains an even umber of nitrogen

atoms (0,2,4,), its monoisotopic molecular ion will be detected at an
even numbered m/z (integer value). Vice versa, an odd number of
nitrogens (1,3,5) is indicated by an odd-numbered m/z value.
10. Rule: Cleaving off a radical (that contains no nitrogen) from any ion
changes the integer m/z value from odd to even or vice versa. Loss of a
molecule (that contains no nitrogen) from an ion produces even mass
fragments from even mass ions and odd mass fragments from odd mass
ions.

25

11. A distonic ion is a positive radical ion, which would formally arise by
ionization of a zwitterion or a diradical by isomerization or fragmentation of
classical molecular ion, or by ion-molecule reactions. Consequently, distonic
ions have charge and radical at separate atoms in a conventional valence bond
description.
12. Note: The mere occurrence of the [C7H7]+ ion especially when it is of low
intensity is not sufficient to prove that the analyte belongs to the
phenylalkanes. This ion and its characteristic fragments may also be observed
if there is some way at all to generate a [C7H7]+ fragment ion.

26

13. Note: Extensive isomerization prior to or between single steps of fragmentation

makes the mass spectra of isomeric alkenes become very similar.
14. Rule of thumb: The stability of molecular ions roughly decrease in the
following order: aromatic compounds > conjugated alkenes > alkenes > alicylic
compounds > carbonyl compounds > linear lkanes > ethers > esters > amines >
carboxylic acids > alcohols > branched alkanes.
15. Even for the softest ionization method, there is no guarantee that the highest m/z
ion must correspond to molecular mass.

27

16.Requirements for the McLafferty rearrangement in a broad sense: i) the atoms

A,B, and D can be carbons or heteroatoms, ii) A and B must be connected by
double bond, iii) at least one -hydrogen is available, iv) -hydrogen is
selectively transferred to B via six membered transition state, v) alkene loss
occurs upon cleavage of the -bond.
17. The McLafferty rearrangement and the RDA reaction have several features in
common: i) both belong to the rearrangement type of fragmentations, although
the name conceals this fact in case of the latter, ii) both represent pathways for
alkene loss from molecular ions, and iii) both are highly versatile in structure
elucidation.

18. Phthalates are commonly used plasticizers in synthetic polymers. Especially di2-ethyl hexylphthalate (also known as dioctyl phthalate, DOP) accounts for 45%
of all plasticizer usage. Unfortunately, they are extracted from the polymer by
elongated exposure to solvents such as dichloromethane, chloroform or toluene,
e.g. from syringes, tubing, vials etc. Therefore, they are often detected as
impurities. They are easily recognized from their typical peaks at m/z 149 (often
base peak), 167 and [M-(R-2H)]+ (m/z 279 for DOP). The molecular ions are
often absent in their EI spectra.

28

SUMMARY (LECTURE -6)

Fragmentation of radical cations with rearrangement
Examples:
McLafferty rearrangemet
1,5- H radical rearrangement
Fragmentation of aromatic systems
Interpretation of Ms spectra
Examples of CI, FD/FI, FAB and ESI spectra
Calculation of molecular weights in ESI
Peak matching
Mass spectrometry----- important points to
remember.
29

 Applications on Natural Products

11

FATTY ACIDS
(Derivatives for MS Structure Determinations)

The abundances of the molecular ions

decrease with increasing chain length
of the acid and M+. is absent. In such
cases derivatives can afford important
informations.
Methyl Esters:
Increment of 14 u
12

Benzyl Esters
Fragments characteristic of the acylium cations
are present at m/z (M-107) formed by loss of
benzyloxy radicals and at m/z (M-91) (loss of the
benzyl radical) and m/z (M-91-18) (loss of the
benzyl radical and water molecule).
The McLafferty-rearranged ion at m/z 150 is
observed only in low abundance, whereas the
corresponding ions at m/z 74 usually form the
base peak in spectra of straight-chain methyl
esters.
Generally, spectra of benzyl esters appear to give
less information about the hydrocarbon skeleton
of the acid than those of the corresponding methyl
esters. As a typical example, the mass spectrum of
benzyl
dodecanoate
is
shown
(fig.8-23).
13

14

 Trimethyl Silyl Esters

A typical mass spectrum of TMS ester (of an unsaturated
acid, eicosenoic acid) is shown in Fig. 8-24.
The largest peaks are seen at m/z 73 (CH3)3Si)+ and m/z 75,
((CH3)2Si = OH)+.
Other prominent ions are at m/z 117, 129, 132, and 145.
Those of m/z 132 corresponding to the McLafferty
rearranged ions.
Of particular interest for molecular-weight determinations
is the observed comparatively large M+. accompanied by an
ion representing loss of a methyl group.
The TMS esters appear to have certain advantages over the
methyl esters. Trimethylsilylation allows complete
derivatization of free fatty acids.
The TMS esters yield proportionally larger amounts of high
mass fragments including the parent ions. This should be
important for the characterization of polyunsaturated fatty
15
acids.

16

Chemical Ionization of Fatty Acids

In the CI spectrum of methyl stearate (Fig.8-30a)
fragments at m/z 297 (M-H), form the parent peak, the
(M+H) ions at m/z 299 representing about 60%.
The CI spectra indicate that in long-chain esters, the
probability of attack on the alkyl chain has become as
important as attack at the methoxy carbonyl group.
Other ions of significant abundance in the CI (H2)
spectrum are observed at m/z 271, (M+H-C2H4); m/z
267, (M-CH3OH); and m/z 283, 269.
Also in the corresponding CI (CH4) spectrum (Fig.830b) (M-H) ions form the base peak.
Here the (M+H) fragments represent about 87% of the
parent peak. Apart from the (M+C2H5) and (M+C3H5)
clusters, the spectrum is similar to that obtained with
H2 as reactant regarding characteristic peaks.
17

THE STEROIDAL SKELETON

CI Spectrum

20

21

22

23

24

Pentacyclic Triterpenoids (12)

25

Pentacyclic Triterpenoids (12)

The RDA cleavage of ring C is a characteristic and

diagnostically valuable feature in the mass
spectra of most 12-oleanene and 12-ursene
derivatives. Peaks associated with the 12,13double bond cleavage appear at m/z 248, 233, 207,
203, 189 and 133.

26

Oleananes and ursanes

189

70

-18
-H
207

HO

COOH

248

-45

203
-70
133

27

28

However, the introduction of a functional

group may induce other fragmentation
reactions
that
offer
complementary
structural information, Thus, in addition to
the peaks associated with the 12,13-double
bond (noted above), 3-methoxy-2,12olean-dien-1-one gives rise to an abundant
m/z 167 ion comprising ring A with
substituents.

COOH

CH3O
+H
167

29


The presence of an oxo or hydroxyl substituent at
C-6 as in methyl 6-oxo-3-hydroxy-12-oleanen-28- oate
and methyl sumaresinolate), respectively, leads to
scission of the 7,8-and 9,10-bonds with
formation of
an ion of mass 302 consisting of rings
C, D, and E)

30

Besides the well-documented peaks due to the

RDA and McLafferty rearrangements, the spectra
of 11-oxo-12-oleanene and 11-oxo-12-ursene
derivatives contain a prominent m/z 135 peak,
which is reported to be of equal diagnostic
importance. The mechanism for the formation of the
corresponding ion from 3-acetoxy-12-oleanen-11one which involves methyl
migration from
C-14
to C-13 in the
intermediate McLafferty fragment as
a crucial
step is shown.

32

33

 Flavonoids

34

3'
2'
8

6'

and/or

5'
2

4'

HC

B1+, m/z 102

A1+, m/z 120

M+; m/z 222

Basic flavone skeleton

+O

+
+ O

A
O

Basic flavone skeleton

35

B2+, m/z 105

The CIMS of perdeuteromethylated flavonoid glycosides

(perdeuteromethyl = PDM) showed much less
fragmentation than that observed with EIMS. Using
isobutene at 175oC, for example, Mabry and co-workers
(unpublished) found that PDM-rutin (perdeuteroquercetin
3-O-rutinoside) gave two base peaks, (M+H)+ and
(aglycone + H)+ (Fig.35-1). Other peaks were observed for
the sequential loss of the sugars. PDM-myricetin 3-Oglycosides under the same conditions gave an (M+H)+ ion
at 20% relative abudance while (aglycone +H)+ was the
base peak. PDM-naringin, in which ring opened to the
chalcone on derivatization, gave as base peak (M+H) +,
with the only notable fragments resulting from the
sequential loss of the sugars.
36

 Flavonols

38

39

40

SUMMARY (LECTURE -7)

Type of Mass Spectrometers
MS/MS experiments
Principles of Charged particle
Analysis
Analyzers Electric sector, magnetic
sector, two sectors
Linked Scans
Applications on Natural Products
41

RESOLUTION
Magnetic instruments function at constant resolution
R = m/m
or
m m
As a result, d m varies proportionality to m.
In the low-mass range m is small.
In the high-mass range m is large.
Suppose the resolution is adjusted to 1000, then for an ion with mass exactly
100.00,
m = m/R
= 100/1000 = 0.1
For an ion with mass 1000,
m = 1000/1000 = 1
i.e. for ion with mass100, the instrument is scanning from mass 99.95 to 100.05
for ion with mass 1000, this ranges from 999.5 to 1000.5.

-Bond Cleavage in Small NonFunctionalized Molecules

The most intensive peaks in the EI mass
spectrum of methane are the molecular ion, m/z
16 and the fragment ion at m/z 15. Explicitly
writing the electrons helps to understand the
subsequent dissociations of CH4 +. to yield CH3
+, m/z 15, by H. loss (1) or H+ by CH3. loss
(2), respectively. In general, it is more
convenient to write the molecular ion in one of
the equivalent forms. The charge and radical
state are then attached to the brackets (often
abbreviated as ) enclosing the molecule.

Time-of-Flight Mass Analyzers

New Terms and Concepts

Compare Cl to El; source modifications required to increase pressure
Why is high reagent gas pressure required?
Reagent gas; common gases; reagent ions; common reactions
Proton transfer; hydride abstraction; adduct formation
Proton Affinity; how does it relate to ion excitation energy and
fragmentation
Use proton affinity to predict PT or A in ammonia Cl
Use ammonia Cl to determine the number of exchangeable H
Explain apparent M+ in ammonia Cl spectra

DETECTORS

Photographic plates
The ions impinge on a metal plate connected to
earth
through
a
resistor.
The
resulting
neutralization of the charge on the ions leads to a
flow of current through the resistor, which can be
detected and affords a measure of ion abundance
(disadvantage lack of sensitivity).
Electron multiplier
Consists of a series of 10 electrodes; provides gain
of the order of 106.

2. Fragmentation of cations with an even

number of electrons (EE+)
The production of a radical cation from an even-electron ion is necessarily
accompanied by that of a radical corresponding to the homolytic cleavage of a
bond. This process is highly endothermic and thus improbable.

A+

even

B+

odd

odd

In preference, an ion will degrade to another ion and a neutral molecule

A+

even

even

even

INTERNATIONAL CENTER FOR CHEMICAL AND

BIOLOGICAL SCIENCES
H.E.J. RESEARCH INSTITUTE OF CHEMISTRY
UNIVERSITY OF KARACHI
Mass Spectrometry (June 07, 2008)
(Assignment)

Name:

Q.1.

Q.2.

Q.3.

Q.4.
EI.
Q.5.
Q.6.
Q.7.

(Total Marks = 10)

Why different fragment ions have different relative abundances in the mass spectrum of any
compound. What are the three basic factors on which the appearance of mass spectrum
depends.
Explain the relative abundances of the peaks at m/z 146, 147, 148, 149, 150 and 151 in the
mass spectrum of dichlorobenzene.
Calculate monoisotopic exact molecular weight of a compound with molecular formula
C30H48O3 (H = 1.0078, C = 12.000, O = 15.995).
What is the eV and source pressure normally used in EI. Why a low pressure is required in

Write down basic principles of CI and ESI.

Why conventional FAB can not be coupled with HPLC.
What is the principle of linked scans and what information do we get on performing B/E
linked scanning. Suggest an example.
Q.8. Why Quadrupole mass analyzers are also called mass filters.
Q.9. In the electric sector, the ions are not separated according to their m/e values. Comment,
and support by the equation applied for this sector ( only mention the relevant equation).
Q.10. Why the soft ionization techniques give only molecular ion (or pseudo-molecular ion) and

Figure 1.6 contributions of carbon and chlorine isotopes to the pattern of peaks in the
molecular ion region of a dichlorobenzene. The molecular ion (M+*) is usually
considered to be the peak having contributions from the isotopes of lowest mass but
strictly all six ions are molecular ions.

REFERENCES

Mass Spectrometry for Chemists and Biochemists

M.E. Rose
Mass Spectrometry Principles and Applications
Edmond de Hoffmann and Vincent Stroobant
Mass Spectrometry
William Kemp
Mass Spectrometry/Mass Spectrometry Techniques and Applications of Tandem
Mass Spectrometry
Kenneth L. Busch, Gary L. Glish and Scott A. McLuckey
Biochemical Applications of Mass Spectrometry
(First supplementary volume)
George R. Waller and Otis C. Dermer

3'
2'
8
7

5'
2

4'

6'

and/or

A
C

HC

M+; m/z 254

Basic flavone skeleton

35

A1+, m/z 136

B1+, m/z 118

3'
2'
8

6'

and/or

5'
2

4'

HC

B1+, m/z 102

A1+, m/z 120

M+; m/z 222

Basic flavone skeleton

+O

+
+ O

A
O

Basic flavone skeleton

35

B2+, m/z 105

1)

4+

2)

5+

3)

6+

4)

7+

5)

8+

6)

9+

= Mr + 4/4
= 5068/4
= 1267
[1267 x 4 = 5068 - 4 = 5064]
= 5064 + 5/5
= 1013.8
[1013.8 x 5 = 5069 - 5 = 5064]
= 5064 + 6/6
= 845
[845 x 6 = 5070 - 6 = 5064]
= 5064 + 7/7
= 724.428
[724.43 x 7 = 5071 - 7 = 5064]
= 5064 + 8/8
= 634
[634 x 8 = 5072 - 8 = 5064]
= 5064 + 9/9
= 563.666
[563.666 x 9 = 5073 - 9 = 5064]

INTERNATIONAL CENTER FOR CHEMICAL AND

BIOLOGICAL SCIENCES
H.E.J. RESEARCH INSTITUTE OF CHEMISTRY
UNIVERSITY OF KARACHI
Exam Mass Spectrometry (June 14, 2008)
Name:

P.1 of 8

(Total Questions 07)

(Total Marks = 15)

Q.1. (b) and (c) are the spectra of two isomeric hydrocarbons with molecular mass 150
(not observed in the spectra). Explain the ions at m/z 43, 57 and 71 with respect to their
relative abundances for the two compounds (2 Marks).

Q.2. Draw the McLafferty rearrangement of the following ester and

calculate the m/z of the ion produced (2 Marks).

O
CH3

CH2 C

O CH2 CH2 CH2 CH2 CH3

Q.3. Explain the ions at m/z 49, 51, 56, 57, 77 and 79 in the following
spectrum. Draw the formation of ion at m/z 56 showing electron
transfer (2.5 Marks).

Q.4. The following compound has two hydroxyl substituents. Place these
in rings A/B in the light of fragment ions at m/z 136 and 118 (2 Marks).

3'
2'
8

5'
2

4'

6'

and/or

A
C

B
HC

C15H10O4

M ; m/z 254

A1+, m/z 136

B1+, m/z 118

Q.5. The following compound has the molecular ion peak at m/z 488
and besides the OH at C-3 and a COOH at C-18, you have to place two
more OH groups. The mass spectrum has significant ions at m/z 248,
203, 133, 239, 221 and 203. Suggest in which of the rings A/B, C, D or E,
these OH groups should be placed (2 Marks).

E
18
D

COOH

+ 2 x OH

HO

C30H48O5 ; M+ 488

Q.6. Following is the spectrum of human parathyroid hormone. Calculate the

m/z values of the ions carrying 4, 5, 6 and 7 protons (2 Marks).

Q.7. The FAB mass spectra of glucose are recorded in:

i) positive mode in glycerol,
ii) negative mode in glycerol and,
iii) with NaCl in glycerol in +ve mode.
Predict the m/z of the molecular ion (pseudo-molecular ion or adduct molecular ion)
peaks in each case. [Molecular weight of glucose = 180; mass of Na = 23] (2.5 Marks)

11

Interpreting Electrospray Mass Spectra

"an IonSource.Com tutorial"
page 1 The Mass Spectrum (Intro)
"The first time I saw electrospray data"
(a personal story)
-The focus of this tutorial-This tutorial mainly focuses on
the interpretation of mass spectra containing multiply
charged molecules generated by the electrospray
ionization process. In this tutorial we use peptides to
demonstrate the complexity of ESI spectra. While small
molecule pharmaceutical drugs (mw 200-500) in most
instances ionize quite well in electrospray they
routinely display only a single charge state [M+H]1+
making interpretation fairly straight forward. Don't be
discouraged, if your business is low molecular weight
analysis the majority of the principles discussed in this
tutorial still apply in full scan mass analysis of small
molecules. In the near future we will present a tutorial
that focuses on small molecule quantification in
pharmacokinetic analysis, so check back often.

A The Y axis is labeled relative intensity. This is the intensity

relative to the tallest peak in the spectrum with the tallest
peak set to 100%.
B The X axis is mass divided by charge, m/z. For example if the
mass of a molecule is 2000 u and the molecule posses two
proton adducts its m/z value is equal to(2000+2)/ 2, the m/z
value read on the spectrum is 1001.
C This is the tallest peak in the spectrum also known as the "base
peak"
D A spectrum will have a certain number of counts associated
with the tallest peak in the spectrum. This number can be
used to gauge the relative intensity or concentration of the
analyte. One should be forewarned that the count number is

1. It is common to overhear people talking about the "molecular ion"

while pointing at an electrospray peak. Molecular ions are not
generally observed in the electospray ionization process. A
molecular ion is formed by the loss of an electron. In the
electrospray process, ionization is accomplished by the loss or gain
of a proton (or other adduct), some refer to this ion as the "pseudo
molecular ion."
2.The electrospray process usually produces a population of
multiply charged molecules and this population is accurately
reflected in the intensity of the peaks in the spectrum.
3.In positive ion mode the number of charged species normally
observed in an electospray spectrum is reflected in the number of
basic sites on a molecule that can be protonated at low pH.

Electrospray Ion Generation

From Gas Phase Ions to Peaks on a Mass Spectrum-Try MassQC Before Making Your Next Injection
Interpreting
Electrospray
Mass
Spectra
"an
IonSource.Com
tutorial"
page 4
--From Gas Phase Ions to Peaks on a Mass Spectrum-A population of variably charged ions are generated in the electrospray process. In this example the population will contain
peptides that have 1, 2 and 0 sites of protonation.
- - The resulting spectrum contains singly and doubly charged species. The intensity of the peaks is a reflection of the
population generated in the electrospray process. Some researchers have used peak abundance information in protein
folding studies. The principles that they apply are; the more tightly folded a protein the more difficult it will be to protonate
and it then follows that as a protein unfolds the peak distribution may change across a spectrum possibly favoring the more
highly charged species.
Note: The uncharged peptide is not observed because the mass spectrometer can not influence it and thus it never
reaches the detector.

home | disclaimerCopyright 2000-2009 IonSource All rights reserved. Last updated: Saturday, February 26, 2005
01:43:20 PM

Magnetic Sector Mass

Spectrometers

21

In the ion-deflection technique (single or double focussing) the

ions in the analyzer tube are subjected to a uniform magnetic
field, H (strength about 500-10,000 gauss) which is generated
by an electromagnet and is perpendicular to the direction of the
ionic beam. In the magnetic field, the ions are deflected along a
circular path of radius according to equation (1).

r = mv

(1)

eH

that is,

22

r2 = m2v2
e2H2

(2)

v2 = r2e2H2
m2

(3)

Now given that the mass of the ions is m and their charge e, and that
their initial kinetic energy is negligible then their kinetic energy after
they have been accelerated to velocity v must be equal to the
electrostatic energy acquired in passing through the applied voltage V.
That is,
m2 = eV
or
2 = 2eV
m

(4)
(5)

From Eqs. (3) and (5), we have

2eV = r2 e2 H2
m
m2
or
m
= H2 r2
e
2V
23

(6)

It can be concluded from Eq. (6) that:

1. The radius r of the circular path of the ions (i.e., their
deflection) is dependant on the following factors:
a) V, the accelerating voltage
b) H, the strength of the deflecting magnetic field
c) m/e, the mass-to-charge ratio of the ions
2. For the ions accelerated by a fixed voltage V, the
deflection caused by a fixed magnetic field H is
proportional to the m/e ratio of the ions.

24

Benefits

25

Linked scans, in which the magnetic and electric fields are

scanned together, can be used to perform MS/MS experiments
(product, precursor, and neutral loss) with a double focusing mass
spectrometer.
Classical mass spectra
Very high reproducibility
Best quantitative performance of all mass spectrometer analyzers
High resolution
High sensitivity
High dynamic range
Linked scan MS/MS does not require another analyzer

Limitations
Not well-suited for pulsed ionization
methods (e.g. MALDI)
Usually larger and higher cost than other
mass analyzers
Linked scan MS/MS gives either limited
precursor selectivity with unit product-ion
resolution, or unit precursor selection with
poor product-ion resolution
26

Applications
All organic MS analysis methods
Accurate mass measurements

27

Quadrupole Mass Spectrometers

A constant voltage U and a radio-frequency potential V are applied b/w
opposite pairs. Ions are injected along the x direction and the spectrum
scanned either by varying the amplitude of U and V, while keeping the
ratio U/V constant, or by varying the frequency of the radio-frequency
potential. DC/AC component are such that only ions with a given m/z pass
through at a time.

28

Time-of-Flight Mass Analyzers

29

In order to cover a mass unit within a time t,

the ion with mass 100 is observed during a time equal to 0.1 t
While that with mass 1000 is observed during 1t.
Suppose the No. of ions produced in the ion source during this time t is the same at
mass 100 and 1000,
then the total no. of ions measured at the detector is 10 times smaller at mass 100
than at mass 1000.
i.e. the no. of ions detected does not correspond to the no. of ions produced. (in
this case).
The scanning is therefore made exponential to correct this error. i.e. the time spent
for scanning at mass 100 is 10 times longer than that at mass 1000.
The no. of detected ions is then proportional to the no. produced.

19

Peak Matching
Comparing the masses of two compounds simultaneously ionized in
the source.
One unknown
One known with accurate mass.

b/c
mk

2 Vk

munk
20

r2 B2

r2 B2
2 Vunk

16

Time of Flight Mass

Spectrometry (TOF-MS)
ADVANTAGES:
Good for kinetic studies of fast reactions and for use
with gas chromatography to analyze peaks from
chromatograph
Can register molecular ions that decompose in the flight
tube

Mass Analysis

Type of Mass Spectrometers:

Single focusing
(Low resolution)

Quadrupole mass filters

or
Single magnetic sector

Double focusing
(High resolution)

Electric sector followed b

magnetic sector

MS/MS EXPERIMENTS
These experiments can do more than
that and a diverse type of data can be
obtained instead of only obtaining a
mass-to-charge ratios. This requires
consideration of analyzer parameters
and principles involved in the analysis
of charged particles to understand the
actual physical property on which the
instrument operation is based.
2

Principles of Charged particle

Analysis

The kinetic energy of an ion after it has been fully accelerated from the ion
source is:
(1)
m2 = eV
2
or m2 = 2eV
m = mass of a single ion
= velocity of the ion
e = charge of the ion
V = potential drop though which ion is accelerated

Electric Sectors
The equation of motion of an ion in an electric field is:

m2
r

eE

(2)

r = radius of deflection
E = electric field strength
Rearranging wil give:
m2
= Er
e
In coventional operation at a:

(3)

constant electric field (E = Ep) and

constant accelerating voltage (V = Vp),
all the quantities in equation (3) are fixed and independent of
mass, since from equation (1),
m2 = 2eV
The electric field (Ep) is set such that r = rc where,
rc = central axis of the electric sector

Therefore the ions formed in the ion source travel in a path that
follows this radius of curvature.

If an ion fragments after it has been accelerated, but in a non-accelerating field, its
mass will change

will be same

Now, if the electric field strength remains fixed,

i.e., E = Ep
The daughter ion will experience a different radius of deflection, rx and will not pass
along the central axis rc
In order to pass the daughter ions m+d along the central axis, the electric field must
be changed hence the parent ions will be
DEFOCUSSED
(DEFOCUSSING TECHNIQUE)

Magnetic Sectors
An ion in a magnetic field B (or H) travels in a circular path in a plane normal to the direction of
the magnetic field. The motion is described by:
m2/r = eB

(4)

m/e = Br

(5)

and rearranging would give,

By substituting the expression for the ion velocity from equation (1)
m2/2 = eV

(1)

in equation (5), we get the mass-to-charge ratio as:

m/e = B2r2 / 2V
If an ion fragments after acceleration from the ion source, but prior to entering the magnetic
field, the equation no longer holds and the daughter ion is passed through the analyzer and
appears in a normal mass spectrum at an apparent mass m* given by,
m* = m2d /mp
7

Two-Sector MS/MS Instruments

EB Geometry,
Scanning the accelerating voltage V with E and B held on constant
Results in parent ion scan
The MS is set up so that the desired daughter ion is transmitted to the detector

From eq (1) and (4),

m2
2
and

eV

m2
r

(1)
eB

(4)

From eq (1),
if = const
Vd is increased to Vp, then md must increase to mp
If,
mp fragments to md prior to the first sector,
The mass
ion is determined from the equation:
m V of
/ Vthe=parent
m
8

Linked Scans
Two of the fields are scanned simultaneously.
When an ion fragments in a reaction region and experiences no acceleration or
deceleration, then,
d = p
The trajectory of an ion through a magnetic field and an electric field is a function of both
m and
By maintaining the ratio of the fields such that the ion velocity necessary to pass through
both sectors is constant, a daughter ion scan is performed
Division of eq. (5) by (3) gives,

m/e = Br

(5)

m2/e = Er

(3)

= E/B
or
9

B/E = 1/

Metastable ions are used in:

1.Defocussing Technique
2.Linked scanning

to determine precursor
ions of a chosen product

E2/V are varied

to determine fragment ions

of a precursor ion

B/E

to determine all product

ions of a chosen
precursor ion

B2/E

10

to determine precursor ions

of a daughter ion