Mass

Spectrometry
Theory, Instrumenta0on and
Techniques

Week 12 Lecture 1
Dr. Taitusi Taufa
 Mass Spectrometry: Basic Principle
•  A charged par0cle moving in a magne0c field is deflected

Ionisa1on
Accelera1on
Deflec1on
Detec1on
 Mass Spectrometry
•  MS is very useful for
–  Determining a compound`s molecular weight
(molecular formula)
–  DetecDng the presence elements such as Br, Cl, N, S, Si
atoms in a molecule
–  Structure determinaDon (structural informa0on –
fragmentaDon, MS/MS)

Mass Spectrometry: Basic Principle
A mass spectrometer needs to perform three funcDons:

•  CreaDon of ions
– the sample molecules are subjected to a high energy beam of
electrons, converDng some of them to ions

•  SeparaDon of ions
– as they are accelerated in an electric field, the ions are
separated according to mass-to-charge ra1o (m/z)

•  DetecDon of ions
– as each separated populaDon of ions is generated, the
spectrometer needs to qualify and quanDfy them
The differences in mass spectrometer types are in the
different means to carry out these three functions
 Mass Spectrometry: Basic Principle
Electrically charged par0cles are affected by a magne0c field although electrically
neutral ones aren't.

1. Ioniza1on
–  The atom or molecule is ionized by knocking one or more
electrons off to give a posiDve ion. (nowadays you can also use
nega0ve mode)

–  Most mass spectrometers work with posiDve ions.

 Mass Spectrometry: Basic Principle
Electrically charged par0cles are affected by a magne0c field although electrically
neutral ones aren't.

2.  Accelera1on

–  The posiDvely charged ions are repelled from the ioniza0on
chamber (which is posi1vely charged), and pass through
nega1vely charged slits which focus and accelerate this into a
beam.

–  The ions are accelerated so that they ALL have the same kine0c
energy.

 Mass Spectrometry: Basic Principle
Electrically charged par0cles are affected by a magne0c field although electrically
neutral ones aren't.

3. Deflec1on
–  The ions are then deflected by a magneDc field according to
their masses

–  The lighter they are, the more they are deflected.

–  The amount of deflecDon also depends on the number of
posi1ve charges on the ion - in other words, on how many
electrons were knocked off in the first stage.

–  The more charged is the ion, the more it gets deflected.
 Mass Spectrometry: Basic Principle
Electrically charged par0cles are affected by a magne0c field although electrically
neutral ones aren't.

4. Detec1on
–  The beam of ions passing through the machine is detected
electrically.

 Mass Spectrometry: Basic Principle
•  The need for a vacuum

–  It's important that the ions produced in the ionizaDon chamber
have a free run through the machine without hiWng air
molecules.
 Mass Spectrometry: Basic Principle
1. Ioniza1on







–  The vaporized sample passes into the ioniza0on chamber.

–  The electrically heated metal coil gives off electrons which are

aXracted to the electron trap which is a posiDvely charged
plate.
 Mass Spectrometry: Basic Principle
1. Ioniza1on

–  The parDcles in the sample (atoms or molecules) are therefore

bombarded with a stream of electrons

–  Some of the collisions are energeDc enough to knock one or

more electrons out of the sample parDcles to make posi0ve
ions.
 Mass Spectrometry: Basic Principle
1. Ioniza1on

–  Most of the posiDve ions formed will carry a charge of +1

•  because it is much more difficult to remove further electrons from an
already posiDve ion.

–  These posiDve ions are persuaded out into the rest of the
machine by the ion repeller which is another metal plate
carrying a slight posi0ve charge.
 Mass Spectrometry: Basic Principle
2. Accelera1on






–  The posiDve ions are repelled away from the very posiDve
ionizaDon chamber and pass through three slits, the final one of
which is at 0 volts.

–  The middle slit carries some intermediate voltage.

–  All the ions are accelerated into a finely focused beam.

 Mass Spectrometry: Basic Principle
3. Deflec1on






•  Different ions are deflected by the magne0c field by different
amounts.

•  The amount of deflecDon depends on:

–  the mass of the ion. Lighter ions are deflected more than heavier ones.
–  the charge on the ion. Ions with 2 (or more) posiDve charges are deflected
more than ones with only 1 posiDve charge.
 Mass Spectrometry: Basic Principle
3. Deflec1on

•  These two factors are combined into the mass/charge ra/o.

•  Mass/charge raDo is given the symbol m/z (or someDmes m/e).

Example
–  If an ion had a mass of 28 and a charge of 1+, its mass/charge
raDo would be 28

–  An ion with a mass of 56 and a charge of 2+ would also have a

mass/charge raDo of 28


 Mass Spectrometry: Basic Principle
3. Deflec1on

Example
•  Ion stream A is most deflected - it will contain ions with the
smallest mass/charge ra0o.

•  Ion stream C is the least deflected - it contains ions with the

greatest mass/charge ra0o.
–  It makes it simpler to talk about this if we assume that the charge on all the
ions is 1+.

–  Most of the ions passing through the mass spectrometer will have a charge
of 1+, so that the mass/charge raDo will be the same as the mass of the ion.
 Mass Spectrometry: Basic Principle
4. Detec1on

 Mass Spectrometry: Basic Principle
4. Detec1on

•  Only ion stream B makes it right through the machine to the ion
detector.

•  When an ion hits the detector, the charge is neutralised, and this
generates an electrical current.
 Mass Spectrometry: Basic Principle
4. Detec1on

•  This current is propor0onal to the abundance of the ion, these are
sent to a computer for analysis.

•  A mass spectrum is generated, which shows the different m/z

values of ions present, and their rela/ve abundance.
 Mass Spectrometry: Basic Principle
4. Detec1on

•  The other ions collide with the walls where they will pick up
electrons and be neutralised.

•  Eventually, they get removed from the mass spectrometer by the

vacuum pump.


 Mass Spectrometry: Basic Principle
4. Detec1on

•  When an ion hits the metal box, its charge is neutralised by an

electron jumping from the metal on to the ion

•  That leaves a space amongst the electrons in the metal, and the
electrons in the wire shuffle along to fill it.

 Mass Spectrometry: Basic Principle
4. Detec1on

•  A flow of electrons in the wire is detected as an electric current
which can be amplified and recorded.

•  The more ions arriving, the greater the current.

 Mass Spectrometry: Basic Principle
4. Detec1on Detec/ng the other ions

How might the other ions be detected - those in streams A and C

which have been lost in the machine?

•  Remember that stream A was most deflected - it has the smallest

value of m/z (the lightest ions if the charge is 1+).

•  To bring them on to the detector, you would need to deflect them

less - by using a smaller magneDc field (a smaller sideways force).

•  To bring those with a larger m/z value (the heavier ions if the
charge is +1) on to the detector you would have to deflect them
more by using a larger magneDc field.
 Mass Spectrometry: Basic Principle
4. Detec1on Detec/ng the other ions

•  If you vary the magneDc field, you can bring each ion stream in
turn on to the detector to produce a current which is proporDonal
to the number of ions arriving.

•  The mass of each ion being detected is related to the size of the
magneDc field used to bring it on to the detector.

•  The machine can be calibrated to record current (which is a

measure of the number of ions) against m/z directly.

•  The mass is measured on the 12C scale – is a scale on which the 12C
isotope weighs exactly 12 units.
 Mass Spectrometry: Basic Principle
What the mass spectrometer output looks like
–  The output from the chart recorder is usually simplified into a
"s0ck diagram".
–  This shows the relaDve current produced by ions of varying
mass/charge raDo.
The ‘’s0ck diagram’’ for molybdenum looks like this:

 Mass Spectrometry: Basic Principle
•  The verDcal axis is labelled as either "rela0ve abundance" or
"rela0ve intensity".

•  It means the same thing.

•  The verDcal scale is related to the current received by the chart

recorder - and so to the number of ions arriving at the detector:
the greater the current, the more abundant the ion.

 Mass Spectrometry: Basic Principle

•  As you will see from the diagram, the commonest ion has a mass/
charge raDo of 98.

•  Other ions have mass/charge raDos of 92, 94, 95, 96, 97 and 100.

 Mass Spectrometry: Basic Principle

•  That means that molybdenum consists of 7 different isotopes.

•  Assuming that the ions all have a charge of 1+, that means that the
masses of the 7 isotopes on the carbon-12 scale are 92, 94, 95, 96,
97, 98 and 100.
 Mass Spectrometry: Basic Principle
•  Mass spectrometry is a vacuum technique that creates, separates,
and detects mass ions (mass to charge raDo) from molecules of
interest

•  Mass Spectrometry (MS) is not true ‘’spectroscopy’’ because it

does not involve the absorpDon of electromagneDc radiaDon to
form an excited state.

•  Mass spectrometry is an extremely sensi0ve and specific analyDcal

technique
–  capable of providing qualitaDve and quanDtaDve analyDcal data
on nanomolar to aRomolar amounts of analyte.
 Mass Spectrometry: Basic Principle
MS is a very sensi0ve analy0cal method

Many organic compounds can be idenDfied:

–  PharmaceuDcal drug discovery and drug metabolism
–  Organic Synthesis reacDon monitoring, product characterizaDon
–  Biotech amino acid sequencing, analysis of macromolecules
–  Clinical neonatal screening, hemoglobin analysis
–  Environmental water quality, food contaminaDon analysis
–  Geological evaluaDng oil composiDon
–  Forensic explosives, illegal drugs

•  Many more
 Mass Spectrometry: Basic Principle
•  The most common method of ionizing molecules (ionizaDon) is by
Electron IonizaDon (EI)

•  The sample is bombarded with a beam of high energy electrons

(1600 kcal or 70 eV)

•  Electron impact causes an electron to be ejected from the

molecule

•  A radical caDon is the result

 Mass Spectrometry: Basic Principle
•  The iniDally formed radical ca1on is known as the
molecular ion (M+Ÿ)

•  The mass of the M+Ÿ is the same as the mass of the original
molecule (electron mass is insignificant)

•  The M+Ÿ is generally very unstable and usually undergoes a variety

of fragmentaDon reacDons

 Mass Spectrometry: Basic Principle
•  This reacDon creates the molecular ion so is very useful.

•  However, the excess energy from the electron can cause the
molecular ion to fall apart

•  Excess energy get redistributed throughout ion to cause

fragmenta0on

 Mass Spectrometry: Basic Principle
•  The resulDng fragments may undergo even further fragmentaDon
ooen to radicals and caDons





•  The caDons are accelerated toward an analyzer, which separates
them based on the mass to charge raDo, m/z
–  SeparaDon Methods include using magneDc field, Dme-of-flight, ion
trapping, and quadrapole

•  Neutral fragments are not detected

 Mass Spectrometry: Basic Principle
•  Fragment InformaDon is useful.

•  It can help structural determinaDon.

•  However, many ions produce only fragments with no molecular ion
remaining.

•  Molecular ion ooen very unstable.

 Mass Spectrometry: Basic Principle
Here is the MS of methane (MW =16)

•  The base peak is the tallest peak in the spectrum

•  For methane, the base peak is M+Ÿ

•  For some molecules, the M+Ÿ peak is
not observed in the spectrum
Why?

•  What is the small peak at m/z = 17
 Mass Spectrometry: Basic Principle
•  Peaks with a mass of less than M+Ÿ represent fragments:

•  Subsequent H radicals can be fragmented to give the ions with a

m/z = 12, 13, and 14
 Mass Spectrometry: Basic Principle
•  In this mass spectrum for benzene, the M+Ÿ peak is the base peak

•  The M+Ÿ peak does not easily fragment (MW = 78)

 Mass Spectrometry: Basic Principle
•  Like most compounds, the M+Ÿ peak for pentane (MW = 72) is NOT
the base peak

•  This is because the molecular ion fragments easily

 Mass Spectrometry: Glossary
•  Molecular ion - The ion obtained by the loss of one electron from
the molecule (M+)

•  Base peak – The most intense (tallest) peak in the MS, assigned
100% intensity
–  The most abundant ion formed in ionizaDon

•  Radical caDon - PosiDvely charged species with an odd number of

electrons

•  Fragment ions - Lighter caDons (and radical caDons) formed by the

decomposiDon of the molecular ion.
–  These ooen correspond to stable carbocaDons.

•  m/z - mass to charge raDo

 Mass Spectrum: Presentation of data

•  The mass spectrum is presented in terms of ion abundance vs m/z raDo
(mass)

•  The most abundant ion formed in ionizaDon gives rise to the tallest peak on
the mass spectrum – this is the base peak

base peak, m/z 43

 Mass Spectrum: Presentation of data

•  All other peak intensiDes are relaDve to the base peak as a percentage

•  If a molecule loses only one electron in the ionizaDon process, a molecular
ion is observed that gives its molecular weight – this is designated as M+ on
the spectrum

M+, m/z 114

 Mass Spectrum: Presentation of data

•  In most cases, when a molecule loses a valence electron, bonds are
broken, or the ion formed quickly fragment to lower energy ions

•  The masses of charged ions are recorded as fragment ions by the

spectrometer – neutral fragments are not recorded!

fragment ions
Mass Spectrum: Determination of Molecular Mass

•  When a M+ peak is observed it gives the molecular mass –
assuming that every atom is in its most abundant isotopic form

•  Remember that carbon is a mixture of 98.9% 12C (mass 12), 1.1%
13C (mass 13) and <0.1% 14C (mass 14)

•  We look at a periodic table and see the atomic weight of carbon
as 12.011 – an average molecular weight

•  The mass spectrometer, by its very nature would see a peak at
mass 12 for atomic carbon and a M + 1 peak at 13 that would be
1.1% as high
We will discuss the effects of this later…
Mass Spectrum: Determination of Molecular Mass

•  Some molecules are highly fragile and M+ peaks are not
observed – one method used to confirm the presence of a
proper M+ peak is to lower the ionizing voltage – lower energy
ions do not fragment as readily

•  Three facts must apply for a molecular ion peak:

–  The peak must correspond to the highest mass ion on the spectrum
excluding the isotopic peaks

–  The ion must have an odd number of electrons – usually a radical ca0on

–  The ion must be able to form the other fragments on the spectrum by
loss of logical neutral fragments
Mass Spectrum: Determination of Molecular Mass

•  The Nitrogen Rule is another means of confirming the observance
of a molecular ion peak

•  The Nitrogen Rule
–  If m/z for the M+Ÿ is odd, this usually means that there is a nitrogen atom in
the molecule. (or an odd number of Ns)
–  If m/z for the M+Ÿ is even, there are no nitrogen atoms in the molecule. (or
an even number of Ns)
 Mass Spectrum: High Resolution MS
• 

The molecular mass can be recorded to precise values (6
to 8 significant figures)

•  It is ooen possible to find an exact molecular formula
from High Resolu0on MS

•  Example: HRMS gives you a molecular ion of 98.0372; from

mass 98 data:

C3H6N4 98.0594
C4H4NO2 98.0242
C4H6N2O 98.0480
C4H8N3 98.0719
C5H6O2 98.0368 ß gives us the exact formula
C5H8NO 98.0606
C5H10N2 98.0845
C7H14 98.1096
 Mass Spectrum: Isotopic Effects

•  The ability of a mass spectrometer to disDnguish different
isotopes is one of the reasons why mass spectrometry is such
a powerful technique.

•  The presence of isotopes – a presence that is ubiquitous in

nature – gives each fragment a characterisDc series of peaks
with different intensiDes.

•  These intensiDes can be predicted based on the abundance of

each isotope in nature, and the relaDve peak heights can also
be used to assist in the deducDon of the empirical formula of
the molecule being analyzed.
 Mass Spectrum: Isotopic Effects

•  Almost all elements have a variety of naturally occurring
isotopes – some notable excep0ons are fluorine, phosphorous,
sodium, and iodine.

Example

•  12C has 6 protons with a mass number of 12
•  Carbon has two other naturally occurring isotopes, 13C and 14C

The abundance for secondary isotopes is reported in number of
atoms of secondary isotope for every 100 atoms of the most
abundant isotope
 Mass Spectrum: Isotopic Effects

Abundance of several naturally occurring biologically important
isotopes
 Mass Spectrum: Isotopic Effects

•  Mass spectrometry disDnguishes elements based on a mass to
charge raDo, m/z.

•  Because of this, isotopes play an important role in mass

spectra.

•  Each isotope will show up as a separate line in any mass

spectrum with good enough resolu0on.

•  The Y-axis on a mass spectrum is rela/ve intensity.

•  Therefore, the height of each of the peaks will correspond to

the relaDve abundance of each isotope in the sample.
 Mass Spectrum: Isotopic Effects
• 

12C, with a natural abundance of 98.89%, naturally has a very high

peak.

•  13C, with a natural abundance of only 1.11%, has a very low peak.

•  The radioacDve 14C has such a low natural abundance that it is not
even seen in relaDon to the other two carbon isotopes.

•  The 12C peak in this spectrum would be the base peak, and would be
labeled M+

•  The 13C peak, with an atomic mass of 1 greater than 12C, would be
labeled as [M + 1]+

•  14C, if it were present, would be [M + 2]+

 Mass Spectrum: Isotopic Effects
Remember this slide
Here is the MS of methane (MW =16)

•  The base peak is the tallest peak in the spectrum (m/z =16)

•  For methane, the base peak is M+Ÿ

•  For some molecules, the M+Ÿ peak is
not observed in the spectrum
Why?

•  What is the small peak at m/z = 17
 Mass Spectrum: Isotopic Effects

You can also use the use the M + 1 peak to calculate the number of
carbons in a molecule
relative intensity of M + 1 peak
# of carbon atoms =
0.011 X (relative intensity of M+ peak)

0.042
# of carbon atoms = ≃4
0.011 X 1
 Mass Spectrum: Isotopic Effects

•  For molecules that contain Br or Cl, the isotopic peaks are
diagnosDc

•  In both cases the [M + 2] isotope is prevalent:

–  79Br is 50.52% and 81Br is 49.48% of naturally occurring bromine

atoms
–  35Cl is 75.77% and 37Cl is 24.23% of naturally occurring chlorine
atoms
 Mass Spectrum: Isotopic Effects

Bromine
•  Bromine has two isotopes, 79Br and 81Br in an approximately 1:1
raDo (50.5 : 49.5 if you want to be fussy!)

•  That means that a compound containing 1 bromine atom will have

two peaks in the molecular ion region
 Mass Spectrum: Isotopic Effects

Bromine

•  The carbons and hydrogens add up to 29. The M+ and [M + 2]+ peaks are
therefore at m/z values given by:

•  29 + 79 = 108
•  29 + 81 = 110

•  So . . . if you have two lines in the molecular ion region with a gap of 2 m/
z units between them and with almost equal heights, this shows the
presence of a bromine atom in the molecule.


 Mass Spectrum: Isotopic Effects

2 X Bromines

•  The carbons and hydrogens add up to 42 - so the various possible molecular ions
could be:

•  42 + 79 + 79 = 200
•  42 + 79 + 81 = 202
•  42 + 81 + 81 = 204

•  You get M+ : [M + 2]+ : [M + 4]+ peaks in the raDo of 1 : 2 : 1

 Mass Spectrum: Isotopic Effects

Chlorine
•  Chlorine in nature is 75.77% 35Cl and 24.23% 37Cl

•  A raDo of M+ to [M + 2]+ of approximately 3 : 1 indicated the

presence of a single chlorine in a compound
–  as seen in the MS for 2-chloropropane
 Mass Spectrum: Isotopic Effects

2 X Chlorines

•  The carbons and hydrogens add up to 28 - so the various possible molecular ions
could be:

•  28 + 35 + 35 = 98
•  28 + 35 + 37 = 100
•  28 + 37 + 37 = 102

•  You get M+ : [M + 2]+ : [M + 4]+ peaks in the raDo of 9 : 6 : 1

 Mass Spectrum: Isotopic Effects

2 X Chlorines

•  The carbons and hydrogens add up to 28 - so the various possible molecular ions
could be:

•  28 + 35 + 35 = 98
•  28 + 35 + 37 = 100 sulfur and silicon can also give prominent M+2s
•  28 + 37 + 37 = 102

•  You get M+ : (M + 2)+ : (M + 4)+ peaks in the raDo of 9 : 6 : 1

 Mass Spectrum: Isotopic Effects
Problem

An unknown hydrocarbon has a molecular ion peak at m/z = 84, with
a rela0ve intensity of 31.3. The[ M + 1] peak has a rela0ve intensity of
6.58, and the [M + 2] peak has a rela0ve intensity of 0.08. What is the
molecular formula for this substance?
 Mass Spectrum: Isotopic Effects
Problem

An unknown hydrocarbon has a molecular ion peak at m/z = 84, with
a rela0ve intensity of 31.3. The[ M + 1] peak has a rela0ve intensity of
6.58, and the [M + 2] peak has a rela0ve intensity of 0.08. What is the
molecular formula for this substance?

M+ at m/z = 84 implies an even # of N or no N

M+ peak it is not the base peak, recalculaDon necessary
M + 1 peak 6.65% 6.58/1.1 = 5.98 ~ 6 carbons
M + 2 peak 0.80% no S, Cl, Br

84 – 6(12) = 12

Therefore the molecular formula is C6H12
 Mass Spectrum: Isotopic Effects
Problem

An unknown substance has a molecular ion peak at m/z = 107, with a
rela0ve intensity of 100. The rela0ve intensity of the [M + 1] peak is
8.00, and the rela0ve intensity of the [M + 2] peak is 0.30. What is
the molecular formula for this unknown?
 Mass Spectrum: Isotopic Effects
Problem

An unknown substance has a molecular ion peak at m/z = 107, with a
rela0ve intensity of 100. The rela0ve intensity of the [M + 1] peak is
8.00, and the rela0ve intensity of the [M + 2] peak is 0.30. What is
the molecular formula for this unknown?

M+ at m/z = 107 implies an odd # of N or 1 N present

M+ peak it is the base peak, no recalculaDon necessary
M + 1 peak 8.00% 8.00/1.1 = 7.3 ~ 7 carbons
M + 2 peak 0.80% no S, Cl, Br

107 – 1(14) – 7(12) = 9

Therefore the molecular formula is C7H9N