Mass Spectroscopy

Dr Adel Mohamed Ahmed

Mass Spectroscopy
Mass Spectrometry (MS or “mass spec”) – a method of separating and analyzing ions by
their mass-to-charge ratio
MS does not involve a specific region of the electromagnetic spectrum (because it is not
directly interested in the energies of emitted photons, electronic or vibrational transitions,
nuclear spin transitions, etc…)
MS spectrometers are used in industry and academia for both routine and research
purposes.
The following list is just a brief summary of the major mass spectrometric applications:
• Biotechnology: the analysis of proteins, peptides, oligonucleotides
• Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug
metabolism
• Clinical: neonatal screening, haemoglobin analysis, drug testing
• Environmental: PAHs, PCBs, water quality, food contamination
• Geological: oil composition
• General Schematic
1. A mass spectrometer needs to perform three functions:
• Creation of ions – the sample molecules are subjected to a high energy beam of electrons,
converting some of them to ions
• Separation of ions – as they are accelerated in an electric field, the ions are separated
according to mass-to-charge ratio (m/z)
• Detection of ions – as each separated population of ions is generated, the spectrometer
needs to qualify and quantify them
2. The differences in mass spectrometer types are in the different means to carry out these
three functions
3. Common to all is the need for very high vacuum (~ 10-6 torr), while still allowing the
introduction of the sample
1. A small quantity of sample is injected and vaporized under high vacuum

2. The sample is then bombarded with electrons having 25-80 eV of energy

3. A valence electron is dislodged from the molecule, and an ion is formed

Principles of Electron-Impact Mass Spectrometry
1. Atom or molecule is hit by high-energy electron
2. electron is deflected but transfers much of its energy to the molecule
3. This energy-rich species ejects an electron.
4. forming a positively charged, odd-electron species called the molecular ion
5. Molecular ion passes between poles of a magnet and is deflected by magnetic
field
6. amount of deflection depends on mass-to-charge ratio
7. highest m/z deflected least
8. lowest m/z deflected most
9. If the only ion that is present is the molecular ion, mass spectrometry provides a
way to measure the molecular weight of a compound and is often used for this
purpose. However, the molecular ion often fragments to a mixture of species of
lower m/z.
 The molecular ion dissociates to a cation
and a radical.

+ •

Usually several fragmentation pathways are

available and a mixture of ions is produced.
mixture of ions of
different mass
gives separate peak
for each m/z
intensity of peak
proportional to
percentage of each + +
ion of different
mass in mixture + +
+
separation of peaks +
depends on relative
mass
mixture of ions of
different mass
gives separate peak
for each m/z
+ + + +
intensity of peak
proportional to
percentage of each + +
atom of different
mass in mixture
separation of peaks
depends on relative
mass
Introduction

bonding electrons

e e
e
C—H
e e
e e

Non-bonding electrons
Introduction

Electron beam

e e
e
C—H
e e
e e
Introduction
Molecular ion
Radical cation
.
[CH3CH2CH3] +
• CH3CH2CH3 Electron beam
Fm=Fc
Fm=magnetic force
Fc =Centripetal force
2
Magnet qνB=mν /r
qB=mν/r
𝒎𝒗
𝒓 =
𝒒𝑩

Detector
Mass Spectrum
• Mass spectrum: A plot of the relative abundance of ions
versus their mass-to-charge ratio (m/z).

• Base peak: The most abundant peak.

– Assigned an arbitrary intensity of 100.

• The relative abundance of all other ions is reported as a %

of abundance of the base peak.
Mass spectrum
𝒎𝒗
𝒓 =
Relative 𝒒𝑩
abundance MW= 44 g/mole
[CH3CH2CH3].+

0 m/q 44
Introduction

.
[CH3CH2CH3] +
• CH3CH2CH3 Electron beam

0 m/q 44
Mass spectrum
𝒎𝒗
𝒓 =
Relative 𝒒𝑩
abundance MW= 44 g/mole
[CH3CH2CH3].+

0 m/q 44
Introduction

Electron beam

e e
e
C —H
e e
e
+
e

Carbocation
Introduction

*H
• CH3CH2CH3 [CH3CHCH3]
+ Electron beam

Magnet MW= 43 g/mole

Detector
Introduction
CH3 — CH *+—+*CH3

—
H

Electron beam

[CH3CHCH3] [CH3CH] [CH3]

+ + +
MW= 43 g/mole MW= 28 g/mole MW= 15 g/mole
Fragment Fragment Fragment 0
Mass spectrum

[CH3CHCH3]
2ο +
MW= 43 g/mole
Relative
abundance fragment Base peak
ion peaks
[CH3CH]
Molecular +
ion peak MW= 28 g/mole

[CH3]
+
0 15 28 43 44 MW= 15 g/mole

m/q
 Molecular Ion

• Molecular ion (M): A radical cation formed by removal of a single electron

from a parent molecule in a mass spectrometer = MW.

• For our purposes, it does not matter which electron is lost; radical cation
character is delocalized throughout the molecule; therefore, we write the
molecular formula of the parent molecule in brackets with:

• A plus sign to show that it is a cation.

• A dot to show that it has an odd number of electrons.

MS of dopamine
• A partial MS of dopamine showing all peaks with
intensity equal to or greater than 0.5% of base
peak.
MASS SPECTROMETER

AN INSTRUMENT THAT GENERATES IONS FROM MOLECULES AND MEASURES

THEIR MASSES

THE ESSENTIAL COMPONENTS OF A MASS SPECTROMETER:

SAMPLE ION ION ION

INLET SOURCE ACCELERATOR ANALYSER

signal

MASS SPECTRUM COMPUTER ION

DETECTOR

DATABASE
 Diagram of a simple mass spectrometer

Illustration of the basic components of a mass spectrometry system.

Ionization Mass
Detector
Source Analzyer

selected Data
Inlet all ions ions
System
WAYS TO PRODUCE IONS (Ionization Sources)

Electron Ionization (EI)

Gas Phase
Chemical Ionization (CI/APCI)
Photo-ionization (APPI)
Electrospray (ESI)
Matrix-assisted Laser Desorption (MALDI)
Field Desorption (FD)
Plasma Desorption (PD) Desorption
Fast atom bombardment (FAB)
High-temperature Plasma (ICP)
EI: Electron Ionization/Electron Impact
 The electron ionization
(EI) source is designed Heated Incandescent
to produce gaseous ions Tungsten/Rhenium Filament
for analysis.
e-

 EI, which was one of the Accelerate!

earliest sources in wide
use for MS, usually 70 eV
operates on vapors
Vaporized
(such as those eluting Molecules Ions To
Mass
from a GC) Analyzer
EI: Electron Ionization/Electron Impact
Advantages
• inexpensive, versatile and reproducible
• fragmentation gives structural information
• large databases if EI spectra exist and are searchable

Disadvantages
• fragmentation at expense of molecular ion
• sample must be relatively volatile
 CI: Chemical Ionization

 Vaporized sample reacts with pre-ionized reagent gas via proton transfer, charge
exchange, electron capture, adduct formation, etc.
 Common CI reagents:
 methane, ammonia, isobutane, hydrogen, methanol

 “soft” ionization gives little fragmentation

 selective ionization-only exothermic or thermoneutral ion-molecule reactions will
occur
 choice of reagent allows tuning of ionization
CI: Hard and Soft Sources

 The energy difference

between EI and CI is
apparent from the
spectra:
 Electrospray Ionization (ESI)
 The ESI process:
– Electrospray ionization (ESI) is accomplished by flowing a solution through an
electrically-conductive capillary held at high voltage (several keV DC).
– The capillary faces a grid/plate held at 0 VDC.
– The solution flows out of the capillary and feels the voltage – charges build up on
nebulized droplets, which then begin to evaporate
– Coulombic explosions occur when the repulsion of the charges overcomes the surface
tension of the solution (holding the drop together) – known as the Rayleigh limit.
– Depending on whose theory you believe
 the analyte ion is eventually the only ion left
 or…the analyte ion is evaporated from a small enough droplet
Electrospray Ionization (ESI)
 A picture of two ideas for the electrospray process:

Note – ions which

are surface-active
will be preferentially
ionized – this can
lead to ion
suppression!

 The Taylor cone – the shape of the

cone that shoots from the needle
when surface tension is overcome by
electrostatic forces, and forms a jet
Typical ESI Spectra
 An ESI mass spectrum of a 14.4 kDa enzyme:
MALDI: Matrix-Assisted Laser
Desorption/Ionization
 A method for desorbing a
sample with a laser,
while preventing thermal
degradation

 A sample is mixed with a

radiation-absorbing
“matrix” used to help it
ionize

 MALDI is heavily used

for large biomolecules
and polymers.
 MALDI: Matrix Effects

 The role of the matrix

– Must absorb strongly at the laser
wavelength
– The analyte should preferably not
absorb at this wavelength
 Common matrices include
nicotinic acid and many other
organic acids
MALDI at Atmospheric Pressure
 Advantages: fast, easy and sensitive
 Disadvantages: no LC, matrix still needed
Atomic mass unit

• amu, atomic mass units (uma??)

• “Da” or Dalton=amu
• kD (kiloDalton for macromolecules)
• 1 amu = 1.66056*10-27 kg.
• proton, mp = 1.67265*10-27 kg,
• neutron, mn = 1.67495*10-27 kg.
Resolution

• Resolution: A measure of how well a mass spectrometer separates ions of

different mass.

• low resolution: Refers to instruments capable of separating

only ions that differ in nominal mass; that is ions that differ by at
least 1 or more atomic mass units.
• high resolution: Refers to instruments capable of separating
ions that differ in mass by as little as 0.0001 atomic mass unit.
Resolving Power Example

RP= 3,000 RP= 5,000 RP= 7,000

100 100 100

80 80 80
In ten sity (%)

In ten sity (%)

In ten sity (%)

60 60 60

40 40 40
C6H5Cl C6H5OF

20 20 20

0 0 0
111.95 112.00 112.05 112.10 111.95 112.00 112.05 112.10 111.95 112.00 112.05 112.10
Mass [amu] Mass [amu] Mass [amu]

All resolving powers are FWHM

High Resolution MS
• High resolution data reports include ppm estimate
• ppm = parts per million (1 ppm = 0.0001%)
• 5 ppm @ m/z 300 = 300 * (5/106) = ±0.0015 Da
• 5 ppm @ m/z 3,000 = 3,000 * (5/106) = ±0.015 Da

• A molecule with mass of 44 could be C3H8, C2H4O, CO2, or CN2H4.

• If a more exact mass is 44.029, pick the correct structure from the
table:

C3H8 C2H4O CO2 CN2H4

44.06260 44.02620 43.98983 44.03740
Resolution
• C3H6O and C3H8O have nominal masses of 58 and 60, and can be
distinguished by low-resolution MS.
• C3H8O and C2H4O2 both have nominal masses of 60.

• Distinguish between them by high-resolution MS.

Molecular Nominal Precise

Formula Mass Mass
C3 H8 O 60 60.05754
C2 H4 O2 60 60.02112

– High resolution MS can replace elemental analysis for

chemical formula confirmation
Glossary
• Molecular ion - The ion obtained by the loss of one
electron from the molecule (m+)
• Base peak - The most intense peak in the MS, assigned
100% intensity
• Radical cation - positively charged species with an odd
number of electrons
• Fragment ions - Lighter cations (and radical cations)
formed by the decomposition of the molecular
ion. These often correspond to stable carbcations.
• m/z - mass to charge ratio
Methanol
Octane, m+ = 114
m-71
-71 (base)
Base peak
-57
-43
-29
-15
CH3CH2CH2CH2CH2CH2CH2CH3

m+ = 114
m-57
m-29
m-43

m+
 Isotopes
• Mass spectrometers are capable of separating and detecting individual ions
even those that only differ by a single atomic mass unit.
• As a result molecules containing different isotopes can be distinguished.
• This is most apparent when atoms such as bromine or chlorine are present (79Br
: 81Br, intensity 1:1 and 35Cl : 37Cl, intensity 3:1) where peaks at "M" and "M+2"
are obtained.
• The intensity ratios in the isotope patterns are due to the natural abundance of
the isotopes.
• "M+1" peaks are seen due the the presence of 13C in the sample.
Bromomethane
1-Bromopropane
2-Chloropropane
(3-Chloropropyl)benzene

CH2 CH2CH2Cl

base peak m/e 91 (m-63)

Chloroacetone
O +
CH3CCH2 m/z = 57
.
- Cl
O +
O .
- CH3 m/z = 77,79
CH3CCH2Cl CCH2Cl
.
m+ = 92 - CH2Cl
m+2 = 94 O +
(3:1 ratio) m/z = 43 (base)
CH3C
 +.
O
. loss of +
CH2CH3
3-Pentanone CH3CH2
C
CH2CH3
CH3CH2C=O

m/z = 86 m/z = 57

m-29
base

m+
 +
.
O . loss of
CH2CH2CH3 +
2-Pentanone CH3
C
CH2CH2CH3
CH3C=O

m/z = 86 m/z = 43

m-43
base

m+
m-15
Mass Spectroscopy