CHEM 3320

T5. Molecular Mass Spectrometry

Reading: Chapter 20, pp 501-534

HW problems: 20-1, 20-2, 20-5, 20-7, 20-10

Prof. Yu, Jianzhen

Chemistry Dept.
Spring 2024 1
 Outline

• Molecular mass spectra

• Ion sources for molecular mass spectrometry
• Mass Analyzers for molecular mass spectrometry
• Applications of molecular mass spectrometry

2
 Molecular mass spectra (1)

Molecular ion M+.

a radical ion that has the
same molecular mass as the
neutral molecule.

Fragment ions
Ions that are formed via
fragmentation of part of the
molecular ions and have
mass lower than the
molecular mass.
FIGURE 20-1 Mass spectrum of ethyl benzene
obtained with electron ionization (EI).

3
 Molecular mass spectra (2)
Base peak
• The highest intensity m/z
peak.
• The base peak is assigned
a value of 100 and the
remaining m/z peaks are
shown as a percentage of
the base-peak

FIGURE 20-1 Mass spectrum of ethyl benzene

obtained with electron ionization (EI).

4
Components of a mass spectrometer: Ion sources

Ion Sources

5
Ion Sources for Molecular Mass Spectrometry (1)

• Solid or liquid  gaseous ions

• Applicable to nonvolatile and thermally unstable samples

Ambient sources allow desorption ionization with minimal sample

6
pretreatment and without the enclosures of typical ionization sources.
 Ion Sources for Molecular Mass Spectrometry (2)

• Hard sources
– Hard ionization sources impart enough energy to analyte molecules
to leave them in a highly excited energy state.  Subsequent
rupture of bonds produces fragment ions that have m/z less than
that of the molecular ion.
• Soft sources
– Soft ionization sources cause little fragmentation.  the mass
spectrum often consists of the molecular ion peak and only a few, if
any, other peaks.
• Both hard- and soft-source spectra are useful for analysis.
– Peaks in a hard-source spectrum provide useful information about
functional groups and thus structural information about analytes.
– Soft-source spectra accurate information about the molecular mass
of the analyte molecules.

7
Comparison of mass spectra by a hard and a soft ionization source
(a) a hard ionization source (EI)

(b) a soft ionization source (chemical ionization [CI])

FIGURE 20-2 Mass spectrum of 1-decanol from (a) a hard ionization source (EI) 8
and (b) a soft ionization source (chemical ionization [CI])
Electron Ionization
Electrons are emitted from a heated tungsten or rhenium
filament, accelerated by a potential ~70V that is impressed
between the filament and the anode.

Figure 20-3 Diagram of an EI source.

9
 Self-study
EXAMPLE 20-1
(a) Calculate the kinetic energy that a singly charged ion (z = 1) will
acquire if it is accelerated through a potential of 103 V in an EI source.
(b) Does the kinetic energy of the ion depend on its mass? (c) Does
the velocity of the ion depend on its mass?

To form a significant number of gaseous

ions at a reproducible
rate, electrons from the filament in the
source must be accelerated
by a voltage of greater than about 50 V.

10
EI: Ionization energies of valence electrons

− +• − −
M +e → M +e +e
70 eV Molecular ~55 eV 0.1 eV
ion
The molecular ion usually has enough
residual internal energy (~ 1 eV) to
break into fragments.
The energy of 70eV is customarily used
because it gives reproducible
fragmentation patterns that can be
compared with library spectra.

Ionization energies of valence electrons in

formaldehyde. (Source: Harris book, pp 519)
11
EI: Typical reactions in EI source

Fragmentation

p-bromoacetophenone
12
 EI mass spectrum of
methylene chloride
Isotope peaks for methylene
chloride
• 12C1H235Cl2 (M = 84)
• 13C1H235Cl2 (M = 85)
• 12C1H235Cl37Cl (M = 86)
• 13C1H235Cl37Cl (M = 87)
• 12C1H237Cl2 (M = 88)
where M is molecular mass.

It is not always possible to

identify the molecular ion
peak with electron impact
ionization, certain molecules
yield no molecular ion peak.

Figure 20-4 EI mass spectra of (a) methylene chloride and 13

(b) 1-pentanol.
 EI sources: advantages and disadvantages
Advantages
• Convenient to use
• high ion currents good
sensitivities.
• Fragment ions helps
structural identification
Disadvantages
• Extensive fragmentation
makes less easy in
identifying molecular ion.
• Need to volatilize the sample
 thermal degradation of
some analytes before
ionization can occur.
• applicable only to analytes
having molecular masses
smaller than about 103 Da.
14
Chemical ionization (CI)
• Chemical ionization evolves as a means to ionize compounds too energetically labile for
traditional EI analysis.
• CI can be thought of as EI performed in the presence of a large excess of a reagent gas
(e.g., methane, isobutane, ammonium).
• The excess reagent gas is more likely to interact with the EI electron beam than the
analyte.

• The gas-phase chemical reactions between

the reagent gas ions and the analyte result in
the removal or donation of a charged species
(generally H+) from the reagent gas ion to
the analyte to yield a pseudo-molecular ion
(positively or negatively charged).

• The reaction energies of the gas-phase acid/base reactions are approximately

5 to 10 eV, which is significantly lower than EI. The lower energy ionization
associated with CI results in more abundant molecular ions.
15
CI Schematic

Source: http://www.noble.org/PlantBio/MS/iontech.ci.html

16
 Comparison of EI and CI Mass spectra
(a) EI mass spectrum of 1-decanol

(b) CI mass spectrum of 1-decanol

FIGURE 20-2 Mass spectrum of 1-decanol from (a) a hard ionization source (EI) 17
and (b) a soft ionization source (chemical ionization [CI])
 Field Ionization

• Field Ionization: gaseous sample from a batch inlet system is

allowed to diffuse into the high-field area around the electrode
(usually, a carbon microneedle). Ions are formed under the
influence of the large electric field (108V/cm)
• FI is a soft ionization technique, little vibrational or rotational
energy is imparted to the analyte; thus, little fragmentation
occurs.

18
Field Desorption

• Field Desorption: Sample is directly loaded on the

electrode, ionization takes place by the application of a
high potential to the electrode. For some sample, heating
is also applied to the electrode.
• FD is a mild ionization technique, normally does not
cause any fragmentation.

19
 Glutamic acid

EI
• In the EI spectrum, the molecular ion peak
at m/z 5 147 is not detectable.
• The base peak at m/z 5 84 arises from a loss
• of water and a —COOH group

FI• The FI spectrum is relatively simple, with an

easily distinguished (M+1)+ peak at m/z = 148.
• A limitation to FI is its lower sensitivity, which is
at least an order of magnitude less than that of EI
sources.

FD The FD spectrum consists of only the

molecular ion or the protonated molecular
ion.

20
Figure 20-6 Mass spectra of glutamic acid
 Desorption Sources
• A number of desorption ionization methods have been developed for
dealing with non-volatile or thermally unstable samples.
• Desorption methods dispense with volatilization followed by
subsequent ionization of the gaseous analyte molecules.
• Energy in various forms is introduced into the solid or liquid sample in
such a way as to cause direct formation of gaseous ions.

Field desorption (FD)

Electrospray ionization (ESI)
Matrix-assisted laser desorption (MALDI)
Fast atom bombardment (FAB)
Thermospray ionization (TSI)
Plasma desorption (PD)
Secondary ion mass spectrometry (SIMS)

21
Matrix-assisted Laser Desorption Ionization
• A low concentration of the
analyte is uniformly dispersed
in a solid or liquid matrix
deposited on the end of a
stainless steel probe or placed
on a metal plate.
• A pulsed laser beam strikes
the sample causing desorption
of a plume of matrix, analyte,
and other ions.
• The analyte can be
protonated, be deprotonated,
or form adducts before
entering the mass analyzer.

Figure 20-7 Diagram of the MAL DI process. 22

MALDI Ionization Mechanism

• MALDI utilizes photons to desorb analyte molecular ions, [M+H]+, from a

matrix.
• The primary role of the matrix is to absorb the incident laser radiation.
•This process results in desorption or transfer to the gas phase of matrix
and intact analyte ions. The analyte is thought to desorb as neutral
molecules and then to be ionized by photon-transfer reactions with
prontonated matrix ions in a dense phase over the surface containing the
matrix.
• MALDI, utilized in conjunction with TOFMS, has the ability to generate
molecular ions from pmol to fmol quantities of species with molecular
weights in excess of 100 kDa.
•The capability of MALDI-TOF mass spectrometry to analyze such large
molecules has found a high degree of usefulness in the analysis of high
mass biomolecules.
23
 • Low background
noise
• Complete absence
of fragmentation
• Multiplied charged
ions are possible
• Dimer and trimer
ions are possible

Figure 20-8 MALDI-TOF spectrum from nicotinic acid

matrix irradiated with a 266-nm laser beam.

https://www.youtube.com/watch?v=8R1Oyqx5KfE
8:37
24
Atmospheric Pressure Ionization Methods

Several ionization techniques that operate at

atmospheric pressure:
• Electrospray ionization (ESI)
• Atmospheric pressure chemical ionization (APCI),
• Atmospheric pressure photoionization (APPI).

25
Electrospray Ionization (ESI)

Process:
API-ES is a process of ionization
followed by evaporation. It
occurs in three basic steps:
(1) Nebulization and charging
(2) Desolvation
26
(3) Ion evaporation Figure 20-9 Apparatus for electrospray ionization
 ESI: principle
• A high electric field (3-5 kV/cm) is used to produce a fine mist of highly
charged droplets.
• The droplets gradually decrease in size by evaporation of neutral solvent
molecules until the droplet reaches a size at which the charge repulsion
forces overcome the cohesive forces of the droplet.
• Subsequent Coulomb explosions result in droplets containing a single solute
molecule that accumulates charge as the remaining solvent is evaporated
• Another view of the process is the "ion evaporation" model, in which the
analyte ion is ejected from the droplet to alleviate the high electrical
potential produced as the solvent evaporates.
• Both positive or negative ions can be produced.
• Little fragmentation of large and thermally fragile molecules.
• ESI is readily adapted to direct sample introduction to HPLC and CE
columns.

https://www.youtube.com/watch?v=r6TGvG7RUyo 27
 ESI mass spectra of
protein and peptides
(The numbers above
the peaks represent
the molecular charge
associated with each
peak.)

FIGURE 20-10 Typical electrospray mass spectra of proteins and peptides. The numbers
above the peaks represent the molecular charge associated with each peak.

In general, electrospray ionization produces single or doubly charged

inorganic or organic ions and multiply charged macro-ions. The ions formed
multiply charged so that their m/z are small enough to make them detectable
with a quadrupole instrument with a range of 1500 dalton or less.
28
https://www.youtube.com/watch?v=Sbxda9ipc_s
Molecular Weight Determination from a ESI Spectrum

This distribution of ions permits the calculation of the molecular mass of the original analyte from any
two neighboring ions at m/z values m1 and m2, with n1 and n2 charges, respectively, if m1<m2, and n2=n1-
1, then
M=n1(m1-mA)=n2(m2-mA)
n2=(m1-mA)/(m2-m1)
Where M represents the mass of the uncharged molecule and mA the mass of the charged adduct A
(mostly H+, or sometimes Na+, K+ or NH4+)
29
 Electrospray of protein

ESI is well suited for the study of charge

macromolecules such as proteins.

ro 30
 135.4
-1.008 110.9
92.4
78.6
67.4
58.1
51.0
44.9
40.0
35.6

mass M + n × 1.008 M
mn = = = + 1.008
ch arg e n n
M
MHnn+: mn − 1.008 =
n
M
mn +1 − 1.008 =
n +1
m − 1.008 Step 1
n = n +1 Step 3
mn − mn +1 Step 2
31
M = n × ( mn − 1.008) Step 4
If high resolution capability is available, i.e., with FT-MS or
double focus sector instrument, one can resolve the
individual carbon isotope peaks of a given charge state.

* This figure shows the high resolution ESI ion

distribution of a single charge state of horse
myoglobin. The distribution is caused by
isotopes. Since these peaks are approximately
0.06 m/z units apart, then the charge state
should be 1/0.06=17. The mass of protein is
then easily calculated.

32
 Atmospheric pressure chemical ionization (APCI)

• In APCI, ionization is brought about by low-energy electrons emitted by a

radioactive beta source or corona discharge.
• Usually a reagent gas (N2, O2, H2O) is ionized by the low-energy electrons,
which leads to subsequent ionization of the analyte by means of several
complex ion-molecule reactions.
• Because of its operation at atmospheric pressure, molecules of the
reagent gas and the analyte undergo frequent collisions, which lead to
high ionization efficiencies.
• Just as with electrospray ionization, the mass spectrometer used with APCI
must be designed to input ions at atmospheric pressure. This requires
“skimmer” interfaces similar to that shown in Figure 20-9. Atmospheric
pressure CI is readily adaptable to samples separated by liquid
chromatography (LC).

33
 Atmospheric pressure photoionization (APPI)

• APPI uses an intense ultraviolet light source instead of low-

energy electrons to ionize either the analyte directly or a
dopant gas.
• With the latter, a series of reactions then leads to the
formation of a nalyte ions. Many of the same compounds
ionized by APCI can be ionized by APPI, but the range of APPI
extends to less polar species.

34
 Ambient Ionization Methods

The two most popular forms of ambient mass

spectrometry are desorption electrospray
ionization (DESI) and direct analysis in real
time (DART).

35
 Mass Analyzers

• Quadrupole Mass Analyzers

Atomic Mass
• Time-of-Flight Mass Analyzers Spectrometry
• Double-focusing Analyzers Molecular Mass
Spectrometry
• Magnetic Sector Analyzers
• Ion-trap Analyzers
• Fourier Transform Spectrometers
• Separate channel mass spectrometers

36
 Ion Trap MS Analyzer
• A ion trap mass analyzer consists a central
doughnut -shaped ring electrode and a pair of
separated end cap electrodes.
• A variable radio-frequency voltage is applied to
the ring electrode while the two end-cap
electrodes are grounded.
• Ions with an appropriate m/z value circulate in a
stable orbit within the cavity surrounded by the
ring.
• As the RF voltage is increased, the orbits of
heavier ions become stabilized, while those for
Figure 20-17 Ion-trap mass spectrometer lighter ions become destablized, causing them
End caps to collide with the wall of the ring electrode.
• In operating this device, a burst of analyte ions
from source is admitted through a grid in the
upper end cap. The RF voltage is then scanned,
and the trapped ions, as they become
destablized, leave the ring electrode cavity via
openings in the lower end cap and then pass
Ring into a transducer. 37
electrode https://www.youtube.com/watch?v=3uUwa1DDoHQ
 Ion Trap Analyzers
Ion trap are rugged, compact and less costly
than sector or quadrupole instruments.
It provides unit mass resolution and mass
limit is usually less then 2000 dalton.
The Ion Trap is based on the same
principle with the Quadrupole mass
filter, except that the rf and DC
resonance field is generated within a
three-dimensional trap. The Ion Trap is
often called the Quadrupole Ion Trap
• Ion traps are ion trapping devices that
make use of a three-dimensional
quadrupole field to trap and mass-analyze
ions
• invented by Wolfgang Paul (Nobel
Prize1989)
• Offer good mass resolving power, and even
MSn capability.
38
http://www.youtube.com/watch?v=3uUwa1DDoHQ
http://www.youtube.com/watch?v=PYpbKSmOnNc
 Orbitrap Mass Analyzer
The orbitrap analyzer is an electrostatic trap consisting of an inner electrode that is
wide in the middle and tapered at both ends (spindle-shaped) and an outer coaxial
split electrode.

• When ions are injected into the

analyzer, they follow a complex
path composed of rotary motion
around the inner electrode with
an axial oscillation that is
proportional to the m/z ratio of
the injected ions.
• Prior to injection, the ions are
collected in a C-trap that tightly
Figure 20-18 Orbitrap analyzer.
focuses them in time and space.

Orbitraps can have high resolution (>200,000) and a high dynamic range. Mass
measurements can be made with high accuracy. The orbitrap analyzer is small
and less expensive than many other high resolution analyzers.
39
Fourier Transform Ion Cyclotron Resonance Mass
Spectrometers (FT-ICR-MS)
Ions entering a chamber are trapped in circular
orbits by magnetic fields. The angular frequency
(i.e., cyclotron frequency) of this motion is:

v zeB
m eB
ωc = = =
r m z ωc

• A measurement of ωc provides an accurate

indication of m/z of the ion.
• Ions of different m/z circles at different ωc.

• Uses powerful magnet (5-10 Tesla) to create miniature cyclotron

• Originally developed in Canada (UBC) by A.G. Marshal in 1974
• FT approach allows many ion masses to be determined
simultaneously (efficient)
• Has higher mass resolution than any other MS analyzer available
• Will revolutionize proteomics studies 40
ICR-MS: measurement of ωc
• When an AC of a frequency ωc is applied,
ions circling at ωc is capable of absorbing
energy from the electric field.  v increases
 r increases proportionally.  Ion circling at
ωc is separated in space from other ions.
• Ions traveling at an angular frequency
different from ωc are unaffected by the ac field.
 Ions circling at ωc is separated in space
from other ions.
• When the excitation electrical field is
stopped, the radius r becomes constant. An
AC current is observed that decreases
exponentially with time.
•The frequency of the current is the same as
the cyclotron resonance frequency. The
magnitude of the AC current depends on the
number of ions with the corresponding m/z.
•This time-domain signal is converted using Figure 20-20. A trapped-ion analyzer cell
Fourier transform into orbital frequencies of
the ions which correspond to their mass-to
charge ratios. 41
http://www.youtube.com/watch?v=GSYueQzo2n8&feature=related
 Time-domain signal

• Mass resolution is limited by the

precision of the frequency
measurement rather than slits or field
measurements.  extremely high
mass resolution is possible (>106)
• The resolution and mass range also
depend on the magnitude and
stability of the magnetic field.

Frequency- or mass-domain signal

Figure 20-22. Time-domain (a) and (b) frequency- or

mass-domain spectrum for 1,1,1,2-tetrachloroethane 42
Fourier Transform
amplitude
N −1
f (t ) = ∑ [ai cos(2πν i t )]
i =0
frequency
FT

Single
frequency

Double
frequency

43
 Separative-Channel Mass spectrometers

With the analysis of mixtures, ideally its components are separated in

advance so that they can be presented one at a time for measurement. 
With other components eliminated, these individual analytes will suffer
minimal matrix effects during measurements.

• Sample channel: a train of modules to prepare a sample for measurement.

• State–of-the-art separative sample channels: MS-MS, GC-MS, LC-MS.
• Interface is often necessary to couple a sample channel to a signal channel:
• A flow of matter in a sample channel may not be compatible for a signal
channel.

44
 Characteristic modules of a tandem mass spectrometer
(MS/MS)
• One mass spectrometer can be coupled to a second (maybe even third). In this
method, the first spectrometer serves as a separative channel.
• MS I isolates the ion that is interested and then further fragmented, the
fragments transmitted to another mass spectrometer for analysis.
• Any ion selected in the first mass analyzer is called parent ion, the fragment
ions that pass to the second mass analyzer called product ion or daughter ion.
• Daughter ions are generated through collisions between the fast-moving parent
ions and collision gas (e.g., He) molecules.
(selected parent (Daughter
ion, Mi) ions)

Ion source Mass Analyzer 1 Collision Chamber Mass Analyzer 2 Detector

(Soft Ionization) (usually Q or B) (often rf only quadrupole) (Q, E, or B)

sample MS-I collision gas MS-II

Source: Strobel & Heineman, Chemical Instrumentation: A systematic Approach,

pp.709
45
 Diagram of a tandem mass spectrometer

Figure 20-23 A block diagram of a tandem mass spectrometer

46
 Dissociative Interactions in the Interaction Cell

Several types of interactions can be used to produce

fragmentation in the interaction cell.
• Metastable ions decompose into fragments after a certain time
• collisionally activated dissociation (CAD) or alternatively collision-induced
dissociation (CID): Fragmentation is induced by adding a collision gas to
the interaction cell so that interaction with precursor ions occurs, leading
to decomposition into product ions.
• surface-induced dissociation (SID): precursor ions interact with a surface
to induce dissociation.
• electron-capture dissociation (ECD): precursor ions capture a low-energy
electron to produce an intermediate that rapidly dissociates
• Photo-induced dissociation (PID): an intense laser beam is used in the
interaction cell to promote the dissociation.

47
 Example product-ion mass spectra
These two
compounds
have identical
whole-number
[M+H]+
masses of 278.

[M+H]+

FIGURE 20-24 Product-ion spectra for dibutylphthalate and Sulfamethazine

obtained after the protonated precursor ion peaks at 279 Da were isolated by the
first mass analyzer of an MS/MS instrument. 48
Types of MS/MS spectrometers

• Tandem in space
– The best choice for MS-I is a high resolution mass analyzer.
MS-II may be a device of lower resolution, since simpler
ions are to be resolved by MS-II.
– e.g. (MS-I: magnetic sector MS or double focusing MS; MS-
II: quadrupole MS)
– Numerous combinations of mass analyzers are
commercially available. e.g. EBEB, EBqQ, B-TOF or Q-TOF.
The most common one is triple stage quadrupole (QqQ)
• Tandem in time (Ion trap, FTMS)

49
Various combinations of mass analyzers to perform tandem MS
analysis

50
Triple-stage quadrupoles
(TSQ)

• MS-I serves as the initial mass separator to select an ion

• MS-II contains reagent gas and through chemical ionization
provides soft fragmentation of the selected ion.
– It is set up as an rf-only quadrupole, and transmit all ions.
• The third MS resolves the daughter ions
• TSQ is less expensive than tandem sector MS/MS analyzers

https://www.youtube.com/watch?v=LFB14D8pkoc
https://www.youtube.com/watch?v=EuD2LnAypBs 51
 Schematic of a triple quadrupole mass spectrometer

Figure 20-25 Schematic of a tandem quadrupole MS/MS instrument.

CID: collision-induced dissociation in which ions break apart

as a result of collision with other molecules. 52
TSQ: an example
The parent ion was obtained by a soft ionization technique, then recorded on the
Q1 of a Triple-Stage- Quadrupole (QqQ) mass spectrometer. The collision of the
precursor ion m/z 368Da in the q can induce dissociation (CID). These product
ions can be analyzed on the third quadrupole Q3 mass analyser.

MS spectrum obtained on
Q1

MS/MS spectrum obtained

on Q3, precursor ion is M/Z
of 368Da

53
 Tandem in time
ICR (FT) or ion trap mass analyzer has ability to hold the ions in the
trap allows high yields of product ions for MS/MS or even MSn

Ions inside a 3D ion trap

mass analyzer can be
analyzed to produce a
mass spectrum, or a
particular ion can be
trapped inside and made
to undergo collisions to
produce fragmentation
information.

Source: http://masspec.scripps.edu/MSHistory/whatisms.php
54
http://www.youtube.com/watch?v=3uUwa1DDoHQ
Tandem-in-Time
Tandem Mass Spectrometric
Analysis for Oleanolic Acid by
Using an Ion-trap Mass
Spectrometer:

LCQR sequential MS6

analysis of an oleanolic
acid glycoconjugate shows
dramatic control over loss
of the sugar monomers,
allowing simple, rapid
elucidation of a complex
structure.

55
Applications of Molecular Mass Spectrometry

56
Applications of Molecular Mass Spectrometry

A. Identification of Pure Compounds

Molecular weight determination from mass spectra
Molecular Formula determination from exact mass measurement
Molecular formula determination from isotopic ratios.
Structure information from fragmentation patterns.
Compound identification from comparison spectra.

B. Analysis of Mixtures by Hyphenated Methods

Chromatography/Mass Spectrometry
Tandem Mass Spectrometry.

57
 Molecular Weight Determination from Mass Spectra

Molecular Formula: C21H25N5 Average Measured Mass (6 data sets)

Molecular Weight: 347.2110 (monoisotopic) M+: 347.2106 + 0.0020
Theoretical MH+: 348.2188 MH+: 348.2171 + 0.0018

Single Mass Measurement 348.2168

12000 Calib
379.093
347.2094
349.2098
172.031
Counts

8000
190.045
344 348 352

4000

212.025 335.103
164.061 227.077 294.071

0
150 200 250 300 350 400
Mass (m/z)

58
 Molecular formulas from exact molecular masses

Example:
Measured mass of the molecular ion: 120.070 ± 0.005
Candidate compounds:
• purine C5H4N4 120.044
• benzamidine C7H8N2 120.069 √
• ethyltoluene C9H12 120.096
•Acetophenone C8H8O 120.058

59
Molecular formulas determination by isotope ratios

Example 20-5
Calculated the ratios of the (M+1)+ to M+ peak heights for the following two
compounds: dinitrobenzene, C6H4N2O4 (m=168) and an olefin, C12H24
(m=168)
From the table 20-3, we see the relative abundance of heavy isotopes for
each element.
C6H4N2O4
----------------------------------------------------
13C 6x1.08=6.48%
2H 4x0.015=0.06%
15N 2x0.37=0.74%
17O 4x0.04=0.16%
(M+1)+/ M+ = 7.44%
C12H24
----------------------------------------------------
13C 12x1.08=12.96%
2H 24x0.015=0.36% (M+1)+/ M+ = 13.32%
60
Structural Information from Fragmentation Pattern

Source:
http://oncampus.richmond.edu/academics/a&s/chemistry/CMoR/info/ModAdd/MS/spectrum.htm
61
GC/MS
Sample: air sample collected during the
combustion of cloth treated with a fire-
retarding chemical.
Mass spectrum of peak 12 (benzene)
Typical output from a GC/MS
instrument.
The upper curve is a computer-
reconstructed chromatogram. The
peaks correspond to: (1) air, (2) water,
(3) hydrogen cyanide, (4) unknown, (5)
acetaldehyde, (6) ethanol, (7)
acetonitrile, (8) acetone, (8b)
unknown, (9) carbon disulfide, (10)
unknown, (11) unknown, (12)
benzene, (13) toluene, (14) xylene.
The lower plot is the computer-
reconstructed mass spectrum for peak
12 (benzene).
62
 LC/MS • LC/MS combines the resolving power
of liquid chromatography with the
detection specificity of mass
spectrometry to "see inside" the
chromatographic peak and to resolve
co-eluting compounds of different
molecular weights.
•LC/MS data may be used to provide
information about the molecular
weight, structure, identity and quantity
of specific sample components.
•In LC/MS-based methods, little or no
heat is imparted to the analyte. 
suitable for polar, ionic, thermally
unstable and involatile compounds.
• applicable to most organic
compounds, ranging from small
pharmaceutical compounds to large
protein.
Applications of various LC/MS techniques
Source: Basics of LC/MS, Agilent 63
Applications of LC/MS: Molecular weight determination

The Figure shows the spectra

of two peptides whose mass-to-
charge ratios differ by only 1
m/z. The only difference in the
sequence is at the C-terminus
where one peptide has
threonine and the other has
threonine amide. The smaller
fragments are identical in the
two spectra, indicating that
large portions of the two
peptides are very similar. The
larger fragments contain the
differentiating peptides.

Mass spectra differentiating two very

similar octapeptides
64
 Applications of LC/MS: Structural determination

Full scan product

ion (MS/MS)
spectrum from the
Full scan mass spectrum
sodium adduct at
of ginsenoside Rb1
m/z 1131.7
showing primarily sodium
adduct ions

Subsequent full scan

product ion spectrum
(MS3) from the ion at
m/z 789.7
65