Mass Spectrometry
Objectives
• Explain the basic principles of mass spectrometry.
• Explain the significance of the (M+1) peak in a mass spectrum
• Use mass spectral data to:
• Determine relative isotopic masses and relative isotopic abundance.
• Distinguish between molecules of similar relative molecular mass.
• Predict possible identities of simple organic molecules based on their
fragmentation pattern.
Introduction
• Mass spectrometry (MS) is an analytical technique that
separates components of a sample based on their mass to
charge (m/z) ratios.
• Mass Spectroscopy can be used to determine:
• Relative atomic mass of an element
• Molecular mass of a molecule
• Structure elucidation of organic compounds
Background
• Mass spectrometry (Mass Spec or MS) uses high energy electrons to
break a molecule into fragments.
• Separation and analysis of the fragments provides information about:
• Molecular weight
• Structure
Background
• The impact of a stream of high energy electrons causes the
molecule to lose an electron forming a radical cation.
• A species with a positive charge and one unpaired electron
H H
- -
H C H + e H C H + 2e
H H
Molecular ion (M+)
m/z = 16
Background
• The impact of the stream of high energy electrons
can also break the molecule or the radical cation
into fragments.
H H
+
H C C H molecular ion (M ) m/z = 30
H H
H H H H
-
H C C H + e + H
H C C
H H H H
m/z = 29
H H
m/z = 15 H C + C H (not detected by MS)
H H
Background
• Molecular ion (parent ion):
• The radical cation corresponding to the mass of the
original molecule
H H H
H C H H C C H
H H H
• The molecular ion is usually the highest mass in the
spectrum
• Some exceptions w/specific isotopes
• Some molecular ion peaks are absent.
Schematic of a Mass Spectrometer
The cations that are formed are
separated by magnetic deflection.
SUBSTANCE IS
VAPORISED
Vapourisation
• Sample is vapourised after it is injected into the mass spectrometer,
by gentle heating.
Ionisation
• The next step in the operation of this instrument is to IONISE the
sample.
• A beam of the vapourised sample is subjected to an electric discharge.
• This causes an electron to be dislodged from the sample and so produce a
positive ion.
•
M g M g e
Acceleration
• The positive ions are repelled away from the very positive
ionisation chamber.
• As the ions pass through the acceleration chamber, they are
made to accelerate with the same velocity.
• The ions are accelerated into a finely focused beam.
Deflection
• At this stage, the ions with the
same mass/charge (m/z) ratio,
travel in the same path
• but in a different path to ions with
a different m/z ratio.
• The ions are deflected by a
magnetic field according to their
masses. The lighter they are, the
more they are deflected.
• The strength of the magnetic
field increases until all the ions
of interest get detected.
Detection
• The beam of ions passing through the machine is detected
electrically.
• Ions with a particular mass reach the detector. Different masses can
be collected by changing the magnetic or electric field.
• The electrometer measures the
charge carried by each beam,
which is then recorded graphically.
Detection
• Only cations are detected.
• Radicals are “invisible” in MS.
• The amount of deflection observed depends on the mass to charge
ratio (m/z).
• Most cations formed have a charge of +1 so the amount of deflection
observed is usually dependent on the mass of the ion.
Background
• The resulting mass spectrum is a graph of the mass of each cation vs.
its relative abundance.
• The peaks are assigned an abundance as a percentage of the base
peak.
• the most intense peak in the spectrum
• The base peak is not necessarily the same as the parent ion peak.
Review
• The following is a diagram of a mass spectrometer.
• Identify the parts labelled A, B, and C.
• State and explain which one of the following ions
will undergo the greatest deflection, under
the same conditions, in a mass spectrometer.
6Li+ , 6Li2+ , 7Li+ , 7Li 2+
Isotopes and Relative Atomic Mass
Isotopes
Atoms of an element that have same atomic number but different mass
number.
Examples: 1H, 2H, 3H & 12C, 13C
Determining Relative Atomic Mass
Exercise
1) Copper exists as a mixture of two isotopes. Copper-63 is 69.17% abundant and it has a mass of
62.9296 amu. Copper-65 is 30.83% abundant and it has a mass of 64.9278 amu. Calculate the
atomic mass of copper.
2) Calculate the atomic mass of silicon. The three silicon isotopes have atomic masses and relative
abundances of 27.9769 amu (92.2297%), 28.9765 amu (4.6832%) and 29.9738 amu (3.0872%).
3) Gallium has two naturally occurring isotopes. The mass of gallium-69 is 68.9256 amu and it is
60.108% abundant. The mass of gallium-71 is 70.9247 amu and it is 39.892% abundant. Calculate
the atomic mass of gallium.
4) Bromine has two naturally occurring isotopes. Bromine-79 has a mass of 78.918 amu and is
50.69% abundant. Using the atomic mass reported on the periodic table, determine the mass of
bromine-81, the other isotope of bromine.
5) Calculate the atomic mass of lead. The four lead isotopes have atomic masses and relative
abundances of 203.973 amu (1.4%), 205.974 amu (24.1%), 206.976 amu (22.1%) and 207.977 amu
(52.4%).
6) Antimony has two naturally occurring isotopes. The mass of antimony-121 is 120.904 amu and
the mass of antimony-123 is 122.904 amu. Using the average mass from the periodic table,
calculate the abundance of each isotope.
Mass Spectrum of Magnesium
Based on the mass spectrum
results, magnesium has 3
isotopes of atomic masses 24,
25 and 26 amu.
The relative abundances of
these isotopes are 78.9%, 10.1%
and 11.3% respectively.
Isotopes and Relative Atomic Mass (RAM)
• Based on the mass spectra of magnesium, the relative atomic mass of
Mg can be calculated as:
78 .1 10.1 11.3
• RAM = ( 24 ) ( 25 ) ( 26 ) 24 .2 amu
100 100 100
An element’s atomic weight is a weighted average of the isotopic
masses of the element’s naturally occurring isotopes.
Chlorine has two stable isotopes:
chlorine – 35 and chorine – 37.
The atomic weight of chlorine given on
the periodic table is 35.47
Different isotopes have different relative
abundances ,chlorine – 35 has a
relative abundance of 75.76% ,whereas
chlorine – 37 has a relative abundance
of 24.24%.
Chlorine -35 is about 3 times more
abundant than chlorine – 37, the
weighted average is closer to 35 than
37.
Chlorine is such an element which contain more than one atom per molecule. It
has two isotopes Cl-35 and Cl-37, so it contain 3 atoms of Cl-35 and 1 atom of
Cl-37. It consist of molecules so when it passed into the ionization chamber, the
electrons are knocked off, and give molecular ion, Cl2+. The ions are not stable
so some will form chlorine atom and a Cl+ ion. This process is called
fragmentation.
• The mass spectrum of chlorine is good example
of molecular element. Chlorine has two stable
isotopes; chlorine-37 (25%) and chlorine -35
(75%).There are five main peaks of isotopes of
chlorine of various isotopic monatomic ions.
• [37 Cl2]+ m\z = 74 ( molecular ion)
• [37 Cl 35 Cl]+ m\z = 72
• [35 Cl2]+ m/z= 70 (molecular ion)
• [37 Cl]+ m/z = 37 (monatomic ion)
• [35 Cl]+ m/z = 35 (monoatomic ion)
• The presence of five peaks for chlorine shows the
ratio of heights for peaks 1 and 2 is 3: 1. For
bimolecular ions, ¾ of the chlorine isotopes are
Cl 35 and ¼ of the isotope of chlorine is cl37.
• The mass spectrum of an organic compound
having chlorine atoms also show different mass
spectrum. Organic compound having one
chlorine atom show molecular peaks M+ and
M+2.the molecular ion containing 35Cl isotope
has relative atomic mass 78 whereas molecular
ion containing 37Cl has relative atomic mass 80.
Mass spectra of organic compounds
• The mass spectrum of an organic sample is obtained in the same way
described earlier:
• Vapourisation, Ionisation, Acceleration, Deflection and Detection. (V.I.A.D.D.)
• The positive organic ion of the molecule that is formed is called the
molecular ion – or the parent ion.
• The molecular ion is often given the symbol M+.
Difference between mass spectrum of a
compound and that of an element.
• In the mass spectrum of an element, each line represents a different
isotope of that element.
• For a compound, each line represents a different fragment produced
when the molecular ion breaks up.
Mass spectra of organic compounds
• The vapourised organic molecules are energetically unstable, and
break up into smaller charged fragments.
• The charged fragments will travel through the mass spectrometer like
any other positive ion until it gets to the detector.
Background
• Mass spectrum of ethanol (MW = 46)
M+
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/1/09)
Background
The mass spectrum of ethanol
base peak
M+
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/1/09)
Easily Recognized Elements in MS
• Nitrogen:
• Odd number of N = odd MW
+
M = 41
CH3CN
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/2/09)
Easily Recognized Elements in MS
Bromine:
M+ ~ M+2 (50.5% 79Br/49.5% 81Br)
2-bromopropane Peaks for Br
isotopes are in
1:1 ratio
M+ ~ M+2
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial
Science and Technology, 11/1/09)
Easily Recognized Elements in MS
• Chlorine: (~75% 35Cl and 25% 37Cl)
• M+ and M+2 peak heights are in 3:1ratio
Cl M+
M+2
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/2/09)
Easily Recognized Elements in MS
• Iodine
• I+ at 127 M+
Large gap
• Large gap
ICH2CN I+
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/2/09)
Typical Mass Spectrum
Molecular
Ion Peak..
M+ peak
Mass spectrum of pentane
m/z Fragment
72 +CH CH CH CH CH
3 2 2 2 3
57 +CH CH CH CH
2 2 2 3
43 +CH CH CH
2 2 3
29 +CH CH
2 3
[CH 3CH 2 CH 2 CH 2 CH 3 ] CH 3CH 2 CH 2 CH 2 CH 3
Background
• Most elements occur naturally as a mixture of isotopes.
• The presence of significant amounts of heavier isotopes leads to small peaks
that have masses that are higher than the parent ion peak.
• M+1 = a peak that is one mass unit higher than M+
• M+2 = a peak that is two mass units higher than M+
Some common fragments and their (m/z
ratio)
* C-C single bond
m/z Fragment fragments with m/z
ratio of 43 depends
15 +CH
3 on the intensity of
29 +CH CH or +CHO this peak, since at
* 3
2
this point, the
+
*43 * +CH2CH2CH3 or or C CH3
fragment can be
O
isomeric.
Difference in mass of fragment and Fragment lost
mass of molecular ion
14 .CH
2
17 .OH
26 .CN
31 .OCH
3
35 .Cl
45 .OCH
2CH3
Peak heights and the stability of ions
• The most intense peak on the mass spectrum is the most stable ion
formed.
• The positive fragments formed from organic compounds are called
“carbocations”
• A carbocation is a carbon atom with a +ve charge.
Carbocations
The (M+1) peak
A complete mass
spectrum, will have
an M+1 peak
M+1 peak is one m/z
unit to the right of
the main molecular
ion peak.
The (M+1) peak is
caused by the
presence of the 13C
isotope in the
molecule
13C is a stable isotope of carbon and
Carbon-13 accounts not to be confused with the radioactive
for approximately1%
of all carbon atoms 14C
Fragmentation Patterns
• The impact of the stream of high energy electrons often breaks the
molecule into fragments, commonly a cation and a radical
Bonds break to give the most stable cation
Fragmentation Patterns
Alkanes
• Fragmentation often splits off simple alkyl groups:
Loss of methyl M+ - 15
Loss of ethyl M+ - 29
Loss of propyl M+ - 43
Loss of butyl M+ - 57
• Branched alkanes tend to fragment forming the most stable carbocations.
Fragmentation Patterns
Mass spectrum of 2-methylpentane
Fragmentation Patterns
Aromatics may have a peak at m/z = 77 for the benzene ring
77
NO2
77 M+ = 123
Fragmentation Patterns
Mass Spectrum for 1-propanol
M+-18 M+
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/28/09)
Fragmentation Patterns
86
CH3CH2 CH2 N CH2 CH2CH2CH3
H
72
Fragmentation Patterns
Mass Spectrum for hydrocinnamaldehyde
105 91
H H O
C C C H
M+ = 134
H H
133 105
91
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/28/09)
Frgamentation Patterns
77 105
O
C O CH3
77
M+ = 136
105
SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of
Advanced Industrial Science and Technology, 11/28/09)
Where are mass spectrometers used?
• Mass spectrometers are used in industry and academia for both routine and research
purposes. The following list is just a brief summary of some of the major mass spectrometric
applications:
• Biotechnology: The analysis of proteins, peptides, oligonucleotides
• Pharmaceutical: Drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism
• Clinical: Haemoglobin analysis, drug testing
• Environmental: Water quality, food contamination
• Geological: Oil composition
• Forensic Science:
Question 1
• Compound Q has the molecular formula C4H7ClO and
does not produce misty fumes when added to water.
The mass spectrum of Q contains two molecular ion
peaks at m/z = 106 and m/z = 108. It also has a major
peak at m/z = 43.
• Suggest why there are two molecular ion peaks.
• A fragment ion produced from Q has m/z = 43 and
contains atoms of three different elements.
• Identify this fragment ion and write an equation showing
its formation from the molecular ion of Q.
Answer 1
• For a m/z = 43, three possible fragments should come to mind.
• or or
H3C CH2 H 3C CH
+
H3C C
+
+
CH2 CH3 O
• Can rule out the first two because the question told you, the fragment has 3
different atoms….
Answer 1 cont’d
• Molecular ion is therefore:
or
Question 2
• A low resolution mass spectrum of 2-methybutan-2-ol shows peaks at
m/z values of 73, 71 and 59. The molecular ion peak is absent.
Propose a structural formula for EACH of the given peaks.
• m/z = 73
• m/z = 71
• m/z = 59.
• The mass spectrum of pentan-2-ol which is isomeric to 2-
methybutan-2-ol shows a molecular ion peak. Account for this
difference between the two isomers.
• Give TWO other uses of mass spectrometry as an analytical tool.
Answer 2 CH3
• 2-methybutan-2-ol H 3C C CH2
RMM =88 OH CH3
CH3
+
• m/z = 73 or C CH2
OH CH3
CH 3
• m/z = 71 H 3C C
+
CH 2
CH 3
CH3
+
H 3C C
• m/z = 59
OH
