Addis Ababa Institute of Technology

School Of Chemical and Bio Engineering

Fundamentals of Analytical Chemistry

(ChEg3218)

Chapter IV

Anteneh Wodaje Bayeh (PhD)

Introduction to Spectroscopic Methods
• Spectroscopy

o The study of interaction between radiation (or other forms of

energy) and matter (a branch of science).
 Spectrometer
o Is an instrument that provides information about the intensity of
radiation as a function of wavelength or frequency.
 Spectrophotometer
o Is a spectrometer equipped with one or more exit slits and
photoelectric transducers that permits the determination of the
ratio of the radiant power of two beams as a function of
wavelength as in absorption spectroscopy.
What Is Electromagnetic Radiation (EMR)
• Electromagnetic radiation (light) is a form of energy whose
behavior is described by the properties of both waves and
particles.
 The optical properties of electromagnetic radiation, such as
diffraction, are explained best by describing light as a wave.
 Many of the interactions between electromagnetic radiation and
matter, such as absorption and emission, however, are better
described by treating light as a particle, or photon.

1. Wave Properties of EMR: EMR consists of oscillating electric

and magnetic fields that propagate through space along a linear
path and with a constant velocity.
• Oscillations in the electric and magnetic fields are perpendicular
to each other, and to the direction of the wave's propagation.

Plane-polarized electromagnetic radiation showing the electric field, the magnetic field,
and the direction of propagation.
• The interaction of electromagnetic radiation with matter can be
explained using either the electric field or the magnetic field.

Electric field component of plane-polarized electromagnetic

radiation.
The electromagnetic spectrum showing the colors of the visible spectrum.

Frequency, n in Hz
~1019 ~1017 ~1015 ~1013 ~1010 ~105

Wavelength, l
~.0001 nm ~0.01 nm 10 nm 1000 nm 0.01 cm 100 m

Energy (kcal/mol)
> 300 300-30 300-30 ~10-4 ~10-6

g-rays X-rays UV IR Microwave Radio

nuclear core electron electronic molecular molecular Nuclear Magnetic
excitation excitation excitation vibration rotation Resonance NMR
(PET) (X-ray cryst.) (π to π*) (MRI)

Visible
Visible
• An electromagnetic wave, is characterized by several
fundamental properties, including:
 Period (p): The time required for one cycle to pass a fixed point in space.
 Frequency (ν): The number of cycles which pass a fixed point in space per
second.
 Amplitude (A): The maximum length of the electric vector in the wave
(Maximum height of a wave).
 Wavelength (λ): The distance between two identical adjacent points in a
wave (usually maxima or minima).
 Wave number ( ν ): The number of waves per cm in units of cm-1.

 Radiant Power ( P ): The amount of energy reaching a given area per

second. Unit in watts (W).
 Intensity ( I ): The radiant power per unit solid angle.
• The relationships between these variables

Speed of light (c ) = Wavelength x Frequency

c = ν, c = 3.00 x 108 m/sec

• The relationship between Wavelength and Frequency

 ∝ 1/ν
• The Higher the Frequency the Shorter the Wavelength

800 nm

Wavelength is inversely
proportional to frequency

Infrared radiation Ultraviolet radiation

V = 3.75 x 1014 s-1 V = 7.50 x 1014 s-1
• Q. In 1859, Gustav Kirchhoff (1824-1887) showed that the D line in
the solar spectrum was due to the absorption of solar radiation by
sodium atoms. The wavelength of the sodium “D” line is 589 nm.
What are the frequency and the wave number for this line?
Ans: ν =5.09x1014s-1  = 1.70x104cm-1
2. Particle Properties of Electromagnetic Radiation: When a sample
absorbs EMR, it undergoes a change in energy.
• Wave theory failed to explain phenomena associated with the

absorption and emission of radiation.

• Thus, EMR is viewed as a stream of discrete particles, or wave

packets, of energy called photons.

• We can relate the energy of photon to its wavelength, frequency and

wavenumber by:

E = hν
ν - frequency

=hc
λ - wavelength

λ
ν - wavenumber

= hcν

h – Planck’s constant =6.63x10-34 J·s

• Q. What is the energy per photon of the sodium “D” line (=589 nm)?

• Ans: 3.37x10-19J
• Types of spectroscopy
• Based on the nature of the radiation that is being absorbed or
emitted, there are different types of spectroscopy. These could be:
 Ultraviolet/visible (UV-Vis) spectroscopy

 Infra-red (IR) spectroscopy

 Nuclear Magnetic Resonance (NMR) spectroscopy

 Mass spectroscopy

 X-ray spectroscopy

• Apart from the above classification, spectroscopy can also be broadly

classified into three categories:
 Electronic spectroscopy

 Vibrational spectroscopy

 Rotational spectroscopy
 The electronic spectroscopy: Involves the movement of electrons
between their various energy levels, due to the absorption or emission of
electromagnetic radiation. The energy required for electronic transition
comes from Ultraviolet or Visible Region.
 Vibrational spectroscopy: Is concerned with the vibrations of
molecules when photons of radiation are absorbed. Molecules can
vibrate with particular amounts of energy whose values are determined
by a quantum number. The energy needed for vibration of molecule
comes from the Infra Red Region.
 Rotational spectroscopy: Is concerned with the energy that causes the
rotation of a molecule. The energy of the rotational level depends on the
rotational quantum numbers. The energy required for the molecular
rotation is provided by the Microwave Region of the Spectrum.
• Types of Atomic & Molecular Transitions
 γ-rays: Nuclear
 X-rays: Core-level electrons
 Ultraviolet (UV)
Valence electrons
 Visible (Vis)
 Infrared (IR): Molecular vibrations
 Microwave: Molecular rotations; electron spin
 Radio waves: Nuclear spin
• Spectroscopy can divide into two broad classes of techniques

1. The first class of techniques there is a transfer of energy between

the photon and the sample.
A. Absorption spectroscopy: In absorption spectroscopy, a photon is
absorbed by an atom or molecule, which undergoes a transition
from a lower-energy state to a higher energy, or excited state. The
type of transition depends on the photon’s energy.
• The absorption of photon of Visible Light promotes one of the
atoms or molecules valence electrons to a higher-energy level.
• When a molecule absorbs Infrared Radiation, one of its chemical
bonds experiences a change in vibrational energy.
Simplified energy diagram showing the absorption and emission of a photon by an atom or a molecule

• When a photon of energy hν strikes the atom or molecule, absorption may occur if the
difference in energy, ΔE, between the ground state and the excited state is equal to the
photon’s energy.
• An atom or molecule in an excited state may emit a photon and return to the ground state.
The photon’s energy, hν, equals the difference in energy, ΔE, between the two states.
• When it absorbs electromagnetic radiation, the number of photons passing
through a sample decreases. The measurement of this decrease in photons, which
we call Absorbance.

• Absorption occurs only when the photon’s energy, hν, matches the difference in
energy, ΔE, between two energy levels. A plot of absorbance as a function of
the photon’s energy is called an Absorbance Spectrum.

B. Emission spectroscopy: When an atom or molecule in an excited state returns

to a lower energy state, the excess energy often is released as a photon, a process
called Emission Spectroscopy.

• There are several ways in which an atom or molecule may end up in an excited
state, including thermal energy, absorption of a photon, or by a chemical reaction.

• Emission following the absorption of a photon is also called photoluminescence,

and that following a chemical reaction is called chemiluminescence.
• Examples of Spectroscopic Techniques Involving an Exchange of Energy Between a
Photon and the Sample.
Type of Energy Region of Spectroscopic Technique
Transfer EM Spectrum
γ-ray Mossbauer spectroscopy

X-ray X-ray absorption spectroscopy

UV/Vis UV/Vis spectroscopy, Atomic Absorption Spectroscopy

Absorption
IR Infrared Spectroscopy, Raman spectroscopy

Microwave Microwave Spectroscopy

Radio wave Electron Spin Resonance Spectroscopy

Nuclear Magnetic Resonance Spectroscopy
Emission UV/Vis Atomic Emission Spectroscopy

X-ray X-ray Fluorescence

Photoluminescence Fluorescence Spectroscopy

UV/Vis Phosphorescence Spectroscopy
Atomic Fluorescence Spectroscopy
2. In the second broad class of spectroscopic techniques, the electromagnetic
radiation undergoes a change in amplitude, phase angle, polarization, or
direction of propagation because of its refraction, reflection, scattering,
diffraction, or dispersion by the sample.
• Examples of spectroscopic techniques that do not involve an exchange of
energy between a photon and the sample.

Region of EMR Type of Interaction Spectroscopic Technique

X-ray diffraction X-ray diffraction

refraction refractometry

scattering nephelometry
UV/Vis
turbidimetry

dispersion optical rotary dispersion

• Basic Components of Spectroscopic Instruments

• The spectroscopic techniques use instruments that share several common basic
components, including a source of energy, a means for isolating a narrow range
of wavelengths, a detector for measuring the signal, and a signal processor that
displays the signal in a form convenient for the analyst.
• General Design of Optical Instruments
• Five Basic Optical Instrument Components

1. Light source: A stable source of radiant energy at the desired wavelength

2. Sample Holder: A transparent container used to hold the sample

(cells, cuvettes, etc.).

3. Wavelength Selector (Diffraction grating): A device that isolates a

restricted region of the EM spectrum used for measurement
(monochromators, prisms, & filters).

4. Photoelectric Transducer (Detector): Converts the radiant energy

into a useable signal (usually electrical).

5. Signal Processor & Readout: Amplifies or attenuates the transduced

signal and sends it to a readout device such as a meter, digital readout,
chart recorder, computer, etc.
• I. Sources of Radiation
• Generate a beam of radiation that is stable and has sufficient power.

• A. Continuum Sources
o Emit radiation over a broad wavelength range and the intensity of the
radiation changes slowly as a function of wavelength.
(refer page 375 of modern analytical chemistry)

Spectrum showing the emission

from a green LED, which provides
continuous emission over a wavelength
range of approximately 530–640 nm.
• This type of source is commonly used in UV, visible and IR
instruments.
 Deuterium lamp: is the most common UV source.
 Tungsten lamp: is the most common visible source.
 Glowing inert solids: are common sources for IR instruments.

• B. Line Sources

o Emit a limited number lines or bands of radiation at specific wavelengths.

o Used in atomic absorption spectroscopy

• Types of line sources:

• Hollow cathode lamps (E.g., Cu emits: 324.8 nm and Zn emits: 213.9
nm)

o Electrodeless discharge lamps

o Lasers-Light ­amplification by stimulated emission of radiation

• II. Wavelength Selectors
• Wavelength selectors output a limited, narrow, continuous group of
wavelengths called a band.
• Two types of wavelength selectors:
o A. Filters

o B. Monochromators

A. Filters

Two types of filters:

1). Interference filters

2). Absorption Filters

(refer page 376-378 of modern analytical chemistry)
• B. Monochromators
• Wavelength selector that can continuously scan a broad range of
wavelengths.
• Used in most scanning spectrometers including UV, visible, and IR
instruments.

• III. Radiation Transducers (Detectors)

• Early detectors in spectroscopic instruments were the human eye,
photographic plates or films. Modern instruments contain devices that
convert the radiation to an electrical signal.
• Two general types of radiation transducers:

a. Photon detectors

b. Thermal detectors
• A. Photon Detectors
• Commonly useful in ultraviolet, visible, and near infrared
instruments.
• Several types of photon detectors are available:
 Vacuum phototubes

 Photomultiplier tubes

 Photovoltaic cells

 Silicon photodiodes

 Diode array transducers

 Photoconductivity transducers
• B. Thermal Detectors
• Used for infrared spectroscopy because photons in the IR region
lack the energy to cause photoemission of electrons.
• Three types of thermal detectors :

1.Thermocouples

2. Bolometers

3. Pyroelectric transducers
• IV. Sample Holder (Container)
• Sample containers, usually called cells or cuvettes must have
windows that are transparent in the spectral region of interest.
• There are few types of cuvettes:
 Quartz or fused silica: required for UV and may be used in
visible region
 Silicate glass: cheaper compared to quartz and used in UV

 Crystalline sodium chloride: used in IR

Cuvettes
• Summary
• Types of source, sample holder and detector for various EM region

Region Source Sample holder Detector

Ultraviole Deuterium lamp Quartz/fused silica Phototube, PM

t tube, diode array

Visible Tungsten lamp Glass/Quartz Phototube, PM

tube, diode array

Nernst glower Salt crystals

Infrared (rare earth oxides e.g., crystalline Thermocouples,

or silicon carbide sodium chloride bolometers

glowers)
Infrared (IR) Spectroscopy
• Infrared spectra for molecules and polyatomic ions
• IR spectroscopy which is concerned with the measurement of
absorption of electromagnetic radiation by molecules due to their
vibrational ability.
• The energy of infrared radiation produces a change in a molecules or
a polyatomic ions vibrational energy but is not sufficient to effect a
change in its electronic energy.
• Absorption of UV and Visible radiation leads to a change in the
analyte’s electronic energy levels and, possibly, a change in
vibrational energy as well.

A change in vibrational energy without

a change in electronic energy levels
occurs with the absorption of infrared
radiation.

Diagram showing two electronic energy levels

(E0 and E1), each with five vibrational energy levels (ν 0–ν4)
• Vibrational energy levels are quantized; that is, a molecule may have only
certain, discrete vibrational energies. The energy for an allowed
vibrational mode, Eν, is:

• μ is the “reduced mass”, given by

Where:
μ: reduced mass
m: atomic mass
ν: vibrational frequency
k: force constant
• Factors affecting vibration frequency
N.B from the relation and that

• The frequency is affected by:

 The masses of the atoms in the bond
 The strength of the bond
• The lower the mass, the higher the vibrational frequency.
 Stretching frequencies for bonds to carbon: C-H > C-D> C-C > C-N > C-O

• The stronger the bond, the higher the vibrational frequency.

 Stretching frequencies of

• C≡C > C=C > C-C

• C≡N > C=N > C-N
• C≡O > C=O > C-O
• In general: Sp C-H > sp2C-H > sp3C-H
• Factors affecting vibration frequency

• Hooke’s Law

• 1) Vibrational frequency is directly proportional to the force constant (Stronger

the bonds, higher is the frequency)
• 2) Frequency is inversely proportional to reduced mass (μ) (when reduced mass is
small, frequency is more)
• Factors affecting vibration frequency
• Factors affecting vibration frequency
• Factors affecting vibration frequency
• Factors affecting vibration frequency
• Factors affecting vibration frequency
• Factors affecting vibration frequency
• Factors affecting vibration frequency
• Example1: Calculation of a Vibrational Frequency for the C-Cl Bond

• We start with our fundamental equation for calculating frequencies

• Assume that the force constant k is 500 N/m

• Where k is the molecular force constant and μ is the reduced mass
given by the following equation:
• The reduced mass will be calculated as:
• The result of this calculation gives us atomic mass units, or amu.
Essentially this means that we are calculating the mass of a single
molecule made up of two atoms.
• To work in our equation, we must have units of mass, that is,
kilograms (kg) so we must convert the reduced mass units (amu) to
kilograms (kg).
• The simplest way to do this is to multiply by a conversion factor,
kg/amu.
• Therefore, for a C-Cl bond, we would put the following into our
equation gives:

• This is not what we really want. The units are wrong. We want
wavenumbers, or cm-1.
• Remember that c = λν, (and use c = 3x cm/sec rather than 3x
m/sec) we can solve for λ, or more specifically, 1/λ,
• Therefore,

• So, we would expect this bond to be found at 972 wavenumbers on

an IR spectrum (assuming that the force constant is 500 N/m).

• Example 2: An 1H35Cl molecule has a force constant of 516 Nm−1.

Calculate the vibrational stretching frequency. Calculate wave no.

Ans: μ=1.63x10-27kg , ν= 299,246m-1= 2992 cm-1

• Example 2: Calculate the vibrational frequency in cm-1 of CO given the following
data: mass of C=12.01 amu, mass of O=16 amu, the force constant
k=1.86×103kg⋅s−2. (1 amu = 1.66054×10−27 kg).
• To convert frequency in Hz to cm-1, divide by the speed of light in cm/s, (C = 3.0×10 10 cm/s)

Convert the masses from amu

• 𝑚𝐶 = 12.01 amu × 1.66054×10−27 kg/amu = 1.994×10−26 kg

to kg:

• 𝑚𝑂 = 16 amu × 1.66054×10−27 kg/amu = 2.6569×10−26 kg

Calculate the reduced mass:
• Modes of molecular vibrations
• Molecules with large number of atoms possess many vibrational
frequencies. For a non-linear molecule with n atoms, the number of
fundamental vibrational modes is (3n-6); for linear molecules have
(3n-5) fundamental vibrational modes.
• In simple molecules, there are two major types of fundamental
vibrations. These are:
 Stretching of bond: a change in the length of a bond

Symmetrical stretching Asymmetrical stretching

  Bending of bond: a change in the angle between two bonds
• Bending vibration is of 4 different types. These are:
 Scissoring: movement of bonding atoms towards (away from) each other

 Rocking: a change in angle between a group of atoms

 Wagging: a change in angle between the plane of a group of atoms

 Twisting: a change in the angle between the planes of two groups of atoms.

Scissoring Rocking Wagging Twisting

 In a rocking, wagging or twisting coordinate the bond lengths within the groups
involved do not change. The angles do.
 Rocking is distinguished from wagging by the fact that the atoms in the group stay in
the same plane.
• Water: a non-linear molecule, theoretically possesses 3
fundamental vibrations, two stretching and one bending.
• Whereas carbon dioxide a linear molecule possess 4 fundamental
absorption bands involving two stretching and two bending modes.

Vibrational modes for water molecule Vibrational modes for carbone dioxide molecule

• The energy required to bend a bond is not great and falls within the range
of 400-1300cm-1. This region is called the fingerprint region. Thus, this
region is used to establish the identity of the chemical compounds.
• IR absorption range
• The typical IR absorption range for covalent bonds is 600 - 4000 cm-1. The
graph shows the regions of the spectrum where the following types of
bonds normally absorb. For example, a sharp band around 2200-2400 cm-1
would indicate the possible presence of a C-N or a C-C triple bond.
• The fingerprint region
• Although the entire IR spectrum can be used as a fingerprint for the purposes of comparing
molecules, the 600 - 1400 cm-1 range is called the fingerprint region. This is normally a
complex area showing many bands, frequently overlapping each other. This complexity
limits its use to that of a fingerprint and should be ignored by beginners when analyzing
the spectrum. As a student, you should focus your analysis on the rest of the spectrum, that
is the region to the left of 1400 cm-1.

Focus your analysis on this region. This is where most Fingerprint region: complex and difficult to
stretching frequencies appear. interpret reliably.
• The energy required to stretch a bond is a little bit higher than bond
bending. This falls within the region of (1300 – 4000cm-1). This signal is
caused by groups such as: OH, NH, C=O, C=C, CHO, etc. These group
frequencies are independent of other parts of the molecule and are used
to detect the functional groups in molecules.

C-H
stretching C-H
bending

C-C
bending

Increasing wavelength

Increasing wavenumber (energy, frequency)

• IR Spectra of chloroform and deuterochloroform
Increasing absorbance

Shift of peak due to

replacement of H with D
(2x mass)

Mode of vibration CHCl3 CHCl3 CDCl3

Calculated* Measured Measured
C-H stretching 3002 3020 2256
C-H bending 1120 1219 912
C-Cl stretching 701 773 737
C-Cl bending 418 671 652
• Example of some group frequencies are given below:
• Modes of IR Spectrum
• The IR spectrum is basically a plot of transmitted (or absorbed)
frequencies vs. intensity of the transmission (or absorption).
• Frequencies appear in the x-axis in units of inverse centimeters
(wavenumbers), and intensities are plotted on the y-axis in
percentage units.
The graph shows a
spectrum in absorption
mode.
• An IR spectrum in transmission mode

The graph shows a spectrum

in transmission mode.

• This is most used representation, and the one found in most

chemistry and spectroscopy books. Therefore, we will use this
representation.
• Classification of IR bands
• IR bands can be classified as strong (s), medium (m), or weak (w),
depending on their relative intensities in the infrared spectrum. A
strong band covers most of the y-axis. A medium band falls to
about half of the y-axis, and a weak band falls to about one third or
less of the y-axis.
• IR band shapes
• Infrared band shapes come in various forms. Two of the most
common are narrow and broad. Narrow bands are thin and pointed,
like a dagger. Broad bands are wide and smoother.
• A typical example of a broad band is that displayed by O-H bonds,
such as those found in alcohols and carboxylic acids, as shown
below.
• Information obtained from IR spectra
• IR is most useful in providing information about the presence or
absence of specific functional groups.
• IR can provide a molecular fingerprint that can be used when
comparing samples. If two pure samples display the same IR spectrum,

it can be argued that they are the same compound.

• IR does not provide detailed information or proof of molecular formula
or structure. It provides information on molecular fragments,
specifically functional groups.
• Therefore, it is very limited in scope and must be used in conjunction
with other techniques to provide a more complete picture of the
molecular structure.
• IR spectrum of alkanes
• Alkanes have no functional groups. Their IR spectrum displays only C-C
and C-H bond vibrations. Of these, the most useful are the C-H bands,
which appear around 3000 cm-1. Since most organic molecules have such
bonds, most organic molecules will display those bands in their spectrum.
• IR spectrum of alkenes

• Besides the presence of C-H bonds, alkenes also show sharp, medium bands
corresponding to the C=C bond stretching vibration at about 1600-1700 cm-1.
Some alkenes might also show a band for the =C-H bond stretch, appearing
around 3080 cm-1 as shown below. However, this band could be obscured by the
broader bands appearing around 3000 cm-1.
• IR spectrum of alkynes
• The most prominent band in alkynes corresponds to the carbon-carbon
triple bond. It shows as a sharp, weak band at about 2100 cm-1. The
reason it’s weak is because the triple bond is not very polar. In some cases,
such as in highly symmetrical alkynes, it may not show at all due to the
low polarity of the triple bond associated with those alkynes.
• Terminal alkynes: those where the triple bond is at the end of a carbon
chain, have C-H bonds involving the sp carbon (the carbon that forms part
of the triple bond). Therefore, they may also show a sharp, weak band at
about 3300 cm-1 corresponding to the C-H stretch.
• Internal alkynes: those where the triple bond is in the middle of a carbon
chain, do not have C-H bonds to the sp carbon and therefore lack the band.
• The following figure shows a comparison between an unsymmetrical terminal
alkyne (1-octyne) and a symmetrical internal alkyne (4-octyne).
• IR spectrum of nitrile
• In a manner very similar to alkynes, nitriles show a prominent band
around 2250 cm-1 caused by the CN triple bond. This band has a
sharp, pointed shape just like the alkyne C-C triple bond, but
because the CN triple bond is more polar, this band is stronger than
in alkynes.
• IR spectrum of an alcohol
• The most prominent band in alcohols is due to the O-H bond, and it
appears as a strong, broad band covering the range of about 3000 -
3700 cm-1. The sheer size and broad shape of the band dominate the
IR spectrum and make it hard to miss.
• IR spectrum of Aldehydes and Ketones
• Carbonyl compounds are those that contain the C=O functional group. In
aldehydes, this group is at the end of a carbon chain, whereas in ketones it’s in the
middle of the chain. As a result, the carbon in the C=O bond of aldehydes is also
bonded to another carbon and a hydrogen, whereas the same carbon in a ketone is
bonded to two other carbons.
• Aldehydes and ketones show a strong, prominent, stake-shaped band around 1710
- 1720 cm-1 (right in the middle of the spectrum). This band is due to the highly
polar C=O bond. Because of its position, shape, and size, it is hard to miss.
• Because aldehydes also contain a C-H bond to the sp2 carbon of the C=O bond,
they also show a pair of medium strength bands positioned about 2700 and 2800
cm-1. These bands are missing in the spectrum of a ketone because the sp2 carbon
of the ketone lacks the C-H bond.
• A spectrum of an aldehyde and a ketone. Study the similarities and the differences
so that you can distinguish between the two.
• IR spectrum of carboxylic acid
• A carboxylic acid functional group combines the features of alcohols and
ketones because it has both the O-H bond and the C=O bond. Therefore,
carboxylic acids show a very strong and broad band covering a wide range
between 2800 and 3500 cm-1 for the O-H stretch. At the same time, they
also show the stake-shaped band in the middle of the spectrum around
1710 cm-1 corresponding to the C=O stretch.
• IR spectra of amines
• The most characteristic band in amines is due to the N-H bond stretch, and it appears as a
weak to medium band. This band is positioned at the left end of the spectrum, in the range
of about 3200 - 3600 cm-1.
• Primary amines have two N-H bonds; therefore, they typically show two spikes that make
this band resemble a molar tooth. Secondary amines have only one N-H bond, which
makes them show only one spike, resembling a canine tooth. Finally, tertiary amines have
no N-H bonds, and therefore this band is absent from the IR spectrum altogether.
• IR spectrum of amides
• The amide functional group combines the features of amines and ketones because
it has both the N-H bond and the C=O bond. Therefore, amides show a very
strong, somewhat broad band at the left end of the spectrum, in the range between
3100 and 3500 cm-1 for the N-H stretch. At the same time, they also show the
stake-shaped band in the middle of the spectrum around 1710 cm-1 for the C=O
stretch. As with amines, primary amides show two spikes, whereas secondary
amides show only one spike.
• Try to associate each spectrum (A - E) with one of the isomers in the
row above it.
Answer
TRY yourself
Answer
Answer
Answer
• Example: Interpret the following IR spectrum by assigning each of
the major peaks. Identify what functional group(s) are present.

3403
cm-1

1604
cm-1
• Example: Interpret the following IR spectrum by assigning each of
the major peaks. Identify what functional group(s) are present.

2733
cm-1
2814
cm-1

1691 1642
cm-1 cm-1
• Example: Which one of the following compounds is the most
reasonable structure for the IR spectrum shown below?
O
OCH3 O OH
O O

1721
• Example: Which of the following compounds is the most
reasonable structure for the IR spectrum shown below?

O OH OH O
OH
NH2 O O OH

1603

1689
Ultraviolet/visible (UV/Vis) spectroscopy
• Ultraviolet/visible (UV/Vis) spectroscopy
• Used to study molecules and their electronic transitions.
• Principle: The energy absorbed corresponds to the amount necessary to
promote an electron from one orbital to another.
• Commonly used to determine the concentration of an absorbing species in
solution (Quantitative Analysis) using Beer-Lambert law.

where:
A = Absorbance
I0 = Intensity of the incident light
I = Intensity of the light transmitted through the sample
ε = Molar absorptivity (L mol-1cm-1)
l = sample path length (cm)
c = Concentration of the solution (mol/L)
• Remember the MOT
• The formation of bands in a conducting solid in the 3rd period and
overlap between the valence and conduction bands.

Single atom
5 atoms in a Multiple of atoms
Close proximity in a close proximity
• The energy band that results from the bonding orbitals of a molecule
is known as the valence band, while the conduction band is because
of the antibonding orbitals of the molecule.
• The valence band (VB) represents the highest occupied molecular
orbital (HOMO), and the conduction band (CB) represents the lowest
unoccupied molecular orbital (LUMO). The gap between the highest
filled energy level and lowest unfilled energy level is called band gap
(Eg).

• The size of the band gap (Eg):

 Determined by the degree of conjugation for alkenes

 Will determine the electronic property of materials i.e., whether

the material is metal, semiconductor or insulator.
 Energy band diagram
demonstrating band gaps

• Band formation during the polymerization of a conjugated monomer into a

conjugated polymer.
• The origin of the absorptions

• Valence electrons can generally be found in one of three types of

electron orbital:
 Single, or σ, bonding orbitals;

 Double or triple bonds (π bonding orbitals); and

 Non-bonding orbitals (lone pair electrons).

• Sigma bonding orbitals tend to be lower in energy than π bonding

orbitals, which in turn are lower in energy than non-bonding orbitals.
• When electromagnetic radiation of the correct frequency is absorbed,
a transition occurs from one of these orbitals to an empty orbital,
usually an antibonding orbital, σ* or π*
Four types of transitions between quantized energy levels account
for most molecular UV/Vis spectra.
• Of these transitions, the most important are n→π∗ and π→π∗ because they
involve important functional groups that are characteristic of many analytes and
because the wavelengths are easily accessible.
• Absorption of ultraviolet and visible radiation in organic molecules is restricted
to certain functional groups (chromophores) that contain valence electrons of
low excitation energy.
UV/VIS region
Vacuum UV or Far UV
(λ<190 nm )
• n ® p* and p ® p* Transitions

• Most absorption spectroscopy of organic compounds is based on transitions of n

or p electrons to the p* excited state.
• These transitions fall in an experimentally convenient region of the spectrum
(200 -700 nm). These transitions need an unsaturated group in the molecule to
provide the p electrons.Absorption characteristics of some common chromophores
• Factors Affecting the Position of λmax
1. Degree of Conjugation
• Conjugation leads to an increase in λ max , and usually an increase in εmax (the hyperchromic
effect).
Effects of Conjugation on λmax

Ultraviolet/visible spectra of the polyenes CH 3(CH=CH)nCH3 Where: n=3, 4 and 5

• As conjugation is increased in a molecule, more delocalization (stability) of the π
electrons results. The effect of this delocalization is to decrease the π* molecular
orbital. The result is a decrease in transition energy from π-π* and thus, a red or
bathochromic shift. The molar absorptivity will increase in this case and better
quantitative analysis will be achieved.

• In general, the longer the conjugated carbon chain in the absorbing system, the
greater the intensity of the absorption.
• β-carotene, a vitamin found in carrots, and used in food coloring,
has eleven conjugated double bonds and its absorption maximum
has shifted out of the ultraviolet and into the blue region of the
visible spectrum, hence it appears bright orange.

β-carotene
• Auxochromes
• These are saturated groups with lone pairs of electrons, which have
the effect of changing λmax when they are adjacent to a chromophore
as shown:
 π→π* transitions show a red, or bathochromic, shift (to longer λ)
 n→π* transitions show a blue, or hypsochromic, shift (to shorter
λ) Effects of Auxochromes on λmax
Compound λmax /nm εmax
Benzene 204 7900
Chlorobenzene 210 7600
Phenol 210 6200
Aniline 234 8600
Thiophenol 236 10000
• 2. Solvent
• Polar solvents usually cause a red shift for π→π* transitions and a
blue shift for n→π* transitions, especially for ketones.
• The excited states of most π→π* transitions are more polar than
their ground states because a greater charge separation is observed
in the excited state.
• If a polar solvent is used the dipole–dipole interaction reduces the
energy of the excited state more than the ground state, hence the
absorption in a polar solvent such as ethanol will be at a longer
wavelength (lower energy, hence lower frequency) than in a non-
polar solvent such as hexane.
 Ultraviolet/visible spectrum of Ultraviolet/visible spectrum of
4-methyl-3-penten-2-one (mesityl 4-methyl-3-penten-2-one (mesityl
oxide) in hexane oxide) in ethanol
3. E/Z (cis/trans) Configurations

• Systems with conjugated double bonds have different λmax values for E (trans) and

Z (cis) configurations. For example, λmax for the π→π* band in the cis and trans
isomers of azobenzene occurs at very different positions. This reflects structural
differences in the molecule:
Cis-azobenzene Trans-azobenzene

λmax (nm) 280 317

• The N=N bond in trans-azobenzene is in the same plane as the benzene ring; in
cis-azobenzene there is an angle of 56° between the N=N bond and the benzene
ring. Overlap of the π orbitals of N=N and benzene will therefore be much poorer
in the cis configuration, reducing conjugation. Other conformational possibilities
will also alter the position of λmax.
Acid-base indicators
• Absorption is advantageous in the acid-base indicators. A small change in the
chemical structure of the indicator molecule can cause a change in the
chromophore and it will absorb in different parts of the visible spectrum. The
spectra of phenolphthalein and litmus illustrate this point.

UV/Vis spectrum of phenolphthalein (p H = 1) UV/Vis spectrum of phenolphthalein (p H = 13)

 Spectral nomenclature
of shifts

 Chromophores: functional groups that give electronic transitions.

 Auxochromes: substituents with unshared pair of electrons, like OH, NH , when
attached to π chromophore they generally move the absorption max. to
longer λ.
 Bathochromic shift: Shift to longer λ, also called red shift.
 Hysochromic shift: Shift to shorter λ, also called blue shift.
 Hyperchromism: Increase in ε of a band.
• Electronic Transitions of Metal Ions

• Many transition metal ions, such as Cu2+ and Co2+, form colorful
solutions because the metal ion absorbs visible light. The transitions
giving rise to this absorption are valence electrons in the metal ion’s
d-orbitals.
• For a free metal ion, the five d-orbitals are of equal energy
(degenerate). When the ligands bonded with the transition metal ion,
there is repulsion between the electrons in the ligands and the
electrons in the d orbitals of the metal ion. That leads to the splitting
of d-orbitals into two groups that differ in energy.

• For example, in an octahedral complex of Cu(H2O)62+ the six water

molecules perturb the d-orbitals into two groups as shown:
 Splitting of the d
orbitals in an
octahedral field.

When white light is passed through a solution

of this ion, some of the energy in the light is
used to promote an electron from the lower set
of orbitals into a space in the upper set.

Suppose that the energy gap in the d orbitals of the

complex ion corresponded to the energy of yellow
light.
Your eye would see the light passing through as a
dark blue, because blue is the complementary color
of yellow.
• Transmittance and Absorbance (Beer-Lambert law)
• As light passes through a sample, its power decreases as some of it is absorbed.
This attenuation of radiation is described quantitatively by two separate, but
related terms: Transmittance and Absorbance.

Light goes through several cuvettes.

• Transmittance is the ratio of the source radiation’s power exiting
the sample, PT, to that incident on the sample, P0.

• An alternative method for expressing the attenuation of

electromagnetic radiation is Absorbance (A) which we define as:

• Example: A sample has a percent transmittance of 50%. What is its

absorbance?
Ans: A= 0.301
• Absorbance and Concentration: Beer’s Law
• When monochromatic electromagnetic radiation passes through an
infinitesimally thin layer of sample of thickness dx, it experiences a
decrease in its power of dP . The fractional decrease in power is
proportional to the sample’s thickness and the analyte’s
concentration, C; thus:
• Where: P is the power incident on the thin layer of sample, and α is
a proportionality constant. Integrating the left side of the above
equation over the entire sample
• converting from ln to log, and substituting gives,
• where a is the analyte’s absorptivity with units of cm –1 conc–1. If we express
the concentration using molarity, then we replace a with the molar
absorptivity, ε, which has units of cm–1 M–1.
• The absorptivity and molar absorptivity are proportional to the probability
that the analyte absorbs a photon of a given energy. As a result, values for
both a and ε depend on the wavelength of the absorbed photon.

Factors used in deriving the Beer-Lambert law.

• Major application of Beers’law: Determination of unknown
concentration by measuring absorbance:

• Example: A 5.00 × 10–4 M solution of an analyte is placed in a

sample cell with a pathlength of 1.00 cm. When measured at a
wavelength of 490 nm, the solution’s absorbance is 0.338. What is
the analyte’s molar absorptivity at this wavelength?
Ans: ε = 676 L mol–1 cm–1
• Quantitative Applications of uv-vis spectroscopy
• The determination of an analyte’s concentration based on its
absorption of UV or Vis radiation is one of the most frequently
encountered quantitative analytical methods. One reason for its
popularity is that many organic and inorganic compounds have
strong absorption bands in the UV-Vis region of the electromagnetic
spectrum.
1. Environmental applications: The analysis of waters and
wastewaters often relies on the absorption of ultraviolet and visible
radiation.
• Examples of the Molecular UV/Vis Analysis of Waters and Wastewaters
Analyte Method λ (nm)
Trace Metals
Aluminum react with Eriochrome cyanide R dye at pH 6; forms red to 535
pink complex
Arsenic reduce to AsH3 using Zn and react with silver diethyldithiocarbamate; 535
forms red complex
Chromium oxidize to Cr(VI) and react with diphenylcarbazide; forms 540
red-violet product
Inorganic Nonmetals
Ammonia reaction with hypochlorite and phenol using a manganous 630
salt catalyst; forms blue indophenol as product
Cyanide react with chloroamine-T to form CNCl and then with a 578
pyridine-barbituric acid; forms a red-blue dye
Organics
Phenol react with 4-aminoantipyrine and K3Fe(CN)6; forms yellow 460
antipyrine dye
Anionic react with cationic methylene blue dye and extract into 652
surfactant CHCl3; forms blue ion pair
2. Clinical applications
• Examples of the Molecular UV/Vis Analysis of Clinical Samples
Analyte Method λ (nm)

Total serum React with NaOH and Cu2+ forms blue-violet complex 540
protein
Serum React with Fe3+ in presence of isopropanol, acetic acid, and 540
cholesterol H2SO4; forms blue-violet complex
Uric acid React with phosphotungstic acid; forms tungsten blue 710

Glucose React with o-toludine at 100 oC; forms blue-green complex 630

3. Industrial analysis
• UV/Vis molecular absorption is used for the analysis of a diverse
array of industrial samples including pharmaceuticals, food, paint,
glass, and metals.
• Many pharmaceutical compounds contain chromophores that make
them suitable for analysis by UV/Vis absorption. Products that have
been analyzed in this fashion include antibiotics, hormones,
vitamins, and analgesics. One example of the use of UV absorption
is in determining the purity of aspirin tablets, for which the active
ingredient is acetylsalicylic acid.

ASA Acetic Acid

SA
• Salicylic acid, which is produced by the hydrolysis of acetylsalicylic
acid, is an undesirable impurity in aspirin tablets, and should not be
present at more than 0.01% w/w. Samples can be screened for
unacceptable levels of salicylic acid by monitoring the absorbance at
a wavelength of 312 nm.
4. Forensic applications
• UV/Vis molecular absorption is routinely used for the analysis of
narcotics and for drug testing.
• One interesting forensic application is the determination of blood alcohol
using the Breathalyzer test. In this test a breath sample is bubbled through
an acidified solution of K2Cr2O7, which oxidizes ethanol to acetic acid.

C2H5OH + K2Cr2O7 + H2SO4 CH3COOH + Cr2(SO4)3 + K2SO4 + H2O

Orange color Green color

• The concentration of ethanol in the breath sample is determined by the

decrease in absorbance at 440 nm where the dichromate ion absorbs. A
blood alcohol content of 0.10%, which is above the legal limit,
corresponds to 0.025 mg of ethanol in the breath sample.
Characterization Applications
• Molecular absorption, particularly in the UV/Vis range, has been
used for a variety of different characterization studies, including:
 Determining the stoichiometry of metal–ligand complexes and
 Determining equilibrium constants.

• Stoichiometry of a Metal -Ligand Complex: The stoichiometry for

a metal–ligand complexation reaction of the following general form

M+yL →
← MLy can be determined by one of three methods:

the method of continuous variations, the mole-ratio method, and the

slope-ratio method.
Atomic Absorption Spectroscopy
（ AAS ）
 Atomic Emission vs. Atomic Absorption

Atomic absorption Atomic emission

• The "ground state" atom absorbs light • The sample is subjected to a high-energy
energy of a specific wavelength as it thermal environment in order to produce
enters the "excited state." As the number excited-state atoms. This environment can
of atoms in the light path increases, the be provided by a flame or, more recently,
amount of light absorbed also increases. a plasma. However, since the excited state
By measuring the amount of light is unstable, the atoms spontaneously
absorbed, a quantitative determination of return to the "ground state" and emit light.
the amount of analyte can be made
Introduction
• What is AAS ?

• In analytical chemistry, Atomic absorption spectroscopy is a

technique for determining the concentration of a particular metal
element in a sample. Atomic absorption spectroscopy can be used to
analyze the concentration of over 62 different metals in a solution.
- Atomic-absorption spectroscopy quantifies the absorption of ground
state atoms in the gaseous state .
- The atoms absorb ultraviolet or visible light and make transitions to
higher electronic energy levels . The analyte concentration is
determined from the amount of absorption.
- Concentration measurements are usually determined from a working curve
after calibrating the instrument with standards of known concentration.
- Atomic absorption is a very common technique for detecting metals and
metalloids in environmental samples.
The Atomic Absorption Spectrometer
• Atomic absorption spectrometers have 4 principal components

1 - A light source ( usually a hollow cathode lamp )

2 – An atom cell ( atomizer )

3 - A monochromator

4 - A detector , and read out device .

Schematic Diagram of an Atomic Absorption Spectrometer

• Atomic Absorption Spectrophotometer

1 – Light Source
• The light source is usually a hollow cathode lamp of the element that
is being measured . It contains a tungsten anode and a hollow
cylindrical cathode made of the element to be determined. These are
sealed in a glass tube filled with an inert gas (neon or argon ) . Each
element has its own unique lamp which must be used for that
analysis .
• Hollow Cathode Lamp

Quartz window

Pyrex body

Anode

Cathode

Anode
How it works
 Applying a potential difference between the anode and the cathode leads to the
ionization of some gas atoms .
 These gaseous ions bombard the cathode and eject metal atoms from the cathode
in a process called sputtering. Some sputtered atoms are in excited states and emit
radiation characteristic of the metal as they fall back to the ground state .

Scheme of a hollow cathode lamp

• The shape of the cathode which is hollow cylindrical concentrates the emitted

radiation into a beam which passes through a quartz window all the way to the
vaporized sample.

• Since atoms of different elements absorb characteristic wavelengths of light.

Analyzing a sample to see if it contains a particular element means using light from
that element.

• For example with lead, a lamp containing lead emits light from excited lead atoms that

produce the right mix of wavelengths to be absorbed by any lead atoms from the
sample.

• A beam of the electromagnetic radiation emitted from excited lead atoms is passed

through the vaporized sample. Some of the radiation is absorbed by the lead atoms in
the sample. The greater the number of atoms there is in the vapor , the more radiation
is absorbed.
2 – Atomizer

• Elements to be analyzed needs to be in atomic sate

• Atomization is separation of particles into individual molecules and

breaking molecules into atoms .This is done by exposing the analyte to
high temperatures in a flame or graphite furnace.

• The role of the atom cell is to primarily dissolvate a liquid sample and then
the solid particles are vaporized into their free gaseous ground state form .
In this form atoms will be available to absorb radiation emitted from the
light source and thus generate a measurable signal proportional to
concentration .

• There are two types of atomization : Flame and Graphite furnace

atomization .
• Desolvating an aqueous solution of CuCl2, for example, leaves us with solid particulates of

CuCl2. Converting the particulate CuCl2 to gas phases atoms of Cu and Cl requires thermal

energy.

CuCl2 (aq) → CuCl2 (s) → Cu (g)+ 2Cl(g)

• Flame- Flame AA can only analyze solutions , where it uses a slot type burner to increase
the path length, and therefore to increase the total absorbance .
• Sample solutions are usually introduced into a nebuliser by being sucked
up a capillary tube .In the nebuliser the sample is dispersed into tiny
droplets , which can be readily broken down in the flame.

• Flame atomizers
 Used in all Atomic Spectroscopic techniques
 Converts analyte into free atoms in the form of vapor phase free atoms
 Heat is required
 Routes for sample introduction

Various flame atomization techniques

• The types of flames used in AAS
Fuel and oxidant Temperature, 0C

Gas/air 1700-1900

Gas/O2 2700-2800

H2/Air 2000-2100

H2/O2 2550-2700

C2H2/Air 2100-2400

C2H2/O2 3050-3150

C2H2/N2O 2600-2800

most commonly used: air / acetylene

• Processes that take place in flame
• Effect of flame temperature on excited state population
*
E* g = 3 Excited state
Absorption

Emission
ΔE

E0,g0 = 3 ground state

Where: N*- No of atoms in Excited state

N- No of atoms in ground state
ΔE- energy difference
K- Boltzmann constant = 1.381 x 10-23 J /K
T- Temperature
• Q. The lowest excited state of a sodium atom lies 3.371x10 -19J/atom above the ground

state. Degeneracy of the excited state is 2 while that of the ground state is 1. Calculate

the fraction of sodium atoms in the excited state in an acetylene-air flame at 2600k.

Ans:

that is, less than 0.02% of the atoms are in the excited state

For Zn: N*/No = 10-15%

• Thus 99.998% of Na atoms are in the ground state

 Atomic emission uses Excited atoms

 Atomic absorption uses Ground state atoms

Q. How would the fraction of atoms in the excited state change if the
temperature where 2610k instead?
• Ans:
• The fraction of atoms in the excited state is still less then 0.02% ,
but that fraction has changed by 100(1.74-1.67)/1.67=4%
• Small changes in flame temperature (~ 10 K) have little effect in
atomic absorption but have significant effects in atomic emission
spectroscopy.
• In atomic emission spectroscopy the control of the flame
temperature is critical!
3- Monochromators
• This is a very important part in an AA spectrometer. It is used to
separate out all of the thousands of lines. Without a good
monochromator, detection limits are severely compromised.
• A monochromator is used to select the specific wavelength of light
which is absorbed by the sample, and to exclude other wavelengths.
The selection of the specific light allows the determination of the
selected element in the presence of others.
4 - Detector and Read out Device
• The light selected by the monochromator is directed onto a detector
that is typically a photomultiplier tube , whose function is to convert
the light signal into an electrical signal proportional to the light
intensity.
• The processing of electrical signal is fulfilled by a signal amplifier .
The signal could be displayed for readout , or further fed into a data
station for printout by the requested format.
Spectroscopy Based on Emission
Spectroscopy Based on Emission

• An analyte in an excited state possesses an energy, E2, that is

greater than that when it is in a lower energy state, E1. When the
analyte returns, or relaxes to a lower energy state the excess energy,
E,

E = E2-E1 must be released

• The lifetime of an analyte in the excited state, A*, is short; typically

10–5–10–9 s for electronic excited states and 10–15 s for vibrational
excited states. Relaxation occurs through collisions between A* and
other species in the sample, by photochemical reactions, and by the
emission of photons.
• Lifetime- The length of time that an analyte stays in an excited state
before returning to a lower-energy state.
• Relaxation- Any process by which an analyte returns to a lower-
energy state from a higher energy state.
• Relaxation may occur by
 releasing excess energy as heat

A* A + heat
 Relaxation by a photochemical reaction may involve a decomposition
reaction in which A* splits apart

A* X+Y
 or a reaction between A* and another species

A* + Z X+Y
• In either case the excess energy is used up in the chemical reaction
or released as heat.
• In the third mechanism excess energy is released as a photon of
electromagnetic radiation.
A* A + h
• The release of a photon following thermal excitation is called
emission, and that following the absorption of a photon is called
photoluminescence.
• In chemiluminescence and bioluminescence, excitation results from
a chemical or biochemical reaction, respectively.
Molecular Photoluminescence Spectroscopy
• Photoluminescence is divided into two categories: fluorescence and
phosphorescence. Absorption of an ultraviolet or visible photon
promotes a valence electron from its ground state to an excited state
with conservation of the electron’s spin.
• a pair of electrons occupying the same electronic ground state have
opposite spins and are said to be in a singlet spin state. Absorbing a
photon promotes one of the electrons to a singlet excited state.
• Emission of a photon from a singlet excited state to a singlet ground
state, or between any two energy levels with the same spin, is called
fluorescence.
• The probability of a fluorescent transition is very high, and the
average lifetime of the electron in the excited state is only 10 –5–10–8
s. Fluorescence, therefore, decays rapidly after the excitation source
is removed.
• In some cases an electron in a singlet excited state is transformed to
a triplet excited state in which its spin is no longer paired with
that of the ground state. Emission between a triplet excited state and
a singlet ground state, or between any two energy levels that differ
in their respective spin states, is called phosphorescence.
 Difference between singlet and triplet states.
• Fluorescence- Emission of a photon when the analyte returns to a
lower-energy state with the same spin as the higher-energy state.
• Phosphorescence- Emission of a photon when the analyte returns to
a lower-energy state with the opposite spin as the higher-energy
state.
Molecular Fluorescence and Phosphorescence Spectra
• Absorption of a photon of correct energy excites the molecule to
one of several vibrational energy levels in the first excited electronic
state, S1, or the second electronic excited state, S2, both of which are
singlet states.
• Relaxation to the ground state from these excited states occurs by a
number of mechanisms that are either radiationless, in that no
photons are emitted, or involve the emission of a photon.
• The most likely pathway by which a molecule relaxes back to its
ground state is that which gives the shortest lifetime for the excited
state.
Radiationless Deactivation
 vibrational relaxation- A form of radiationless relaxation in which an
analyte moves from a higher vibrational energy level to a lower
vibrational energy level in the same electronic level.
 internal conversion- in which a molecule in the ground vibrational level
of an excited electronic state passes directly into a high vibrational
energy level of a lower energy electronic state of the same spin state.
 external conversion -A form of radiationless relaxation in which energy
is transferred to the solvent or sample matrix.
 intersystem crossing- A form of radiationless relaxation in
which the analyte moves from a higher electronic energy level to
a lower electronic energy level with a different spin state.
Energy level diagram for a molecule showing pathways for deactivation of an
excited state: vr is vibrational relaxation; ic is internal conversion; ec is external

conversion, and isc is intersystem crossing. The lowest vibrational energy level for
each electronic state is indicated by the thicker line.
 Fluorescence- Fluorescence occurs when a molecule in the
lowest vibrational energy level of an excited electronic state
returns to a lower energy electronic state by emitting a photon.
 Phosphorescence A molecule in the lowest vibrational energy
level of an excited triplet electronic state normally relaxes to the
ground state by an intersystem crossing to a singlet state or by
external conversion. Phosphorescence is observed when
relaxation occurs by the emission of a photon.
Excitation Versus Emission Spectra
• Photoluminescence spectra are recorded by measuring the intensity
of emitted radiation as a function of either the excitation wavelength
or the emission wavelength.
• excitation spectrum is a fluorescence or phosphorescence spectrum
in which the emission intensity at a fixed wavelength is measured as
a function of the wavelength used for excitation.
• In an emission spectrum a fixed wavelength is used to excite the
molecules, and the intensity of emitted radiation is monitored as a
function of wavelength.
Example of molecular excitation and emission spectra.
Mass Spectrometry
Mass Spectrometry - is an analytical spectroscopic tool primarily
concerned with the separation of molecular (and atomic) species
according to their mass.
• Mass spectrometry (Mass Spec or MS) uses high energy electrons to
convert a molecule into ions and / fragments depending on their
energy and molecular bond.
• From separated ions and the fragments, we can get information
about:
– Molecular weight
– Structure of the molecule/fragments
– the presence/absence isotopes in a molecule
There are four main stages of the process in MS
• Step 1: Ionization- The sample is vaporized, and then ionized by being
bombarded by a beam of high-energy electrons (70 eV).
• The electron beam knocks out an electron from the molecule of the
injected sample, creating a molecular ion (which is also a radical cation
because it has an unpaired electron and a positive charge).
• Losing an electron weakens the bond, while the collision gives it extra
kinetic energy. These factors make it more likely for the molecular ion to
break into fragments as it travels through the mass spectrometer.
• 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 CH + e H CH +2 e
H H
Molecular ion (M+)
m/z = 16
• 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
H C + C H (not detected by MS)
H H

m/z =
15
• Step 2: Acceleration- There is a pair of oppositely charged plates in
the ionization chamber. The positively charged plate causes the
positively charged radical cation to accelerate into an analyzer tube.

The positive ions are repelled away

from the very positive ionization
chamber and pass through three slits,

• Step 3: Deflection (sorted and separated) - The analyzer tube is

surrounded by a curved magnetic field, which causes the path of the
radical cation to be deflected in proportion to its mass-to-charge
ratio (m/z).
• The flight path of the ion depends on its molecular mass, its charge, the
speed of particles (applied voltage) and the strength of the magnetic
field. Thus, at a given magnetic field strength, ions of only one specific
mass collide with the detector and are recorded.
Where:
B= magnetic field strength
r = radius of the analyzer tube
V= voltage (accelerator plate)

 the heavy ions are deflected less than

the lighter ions.
 ions with bigger charged, highly
affected by the field and accelerated
more.
• Step 4: Detection – only ions of a certain mass actually hits the detection plate.
When an ion hits the metal box, its charge is
neutralized 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.

• 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.
• To make sure that all of the ions are detected, you have to vary the strength of
accelerating field.

 When the magnetic field is weak, the lighter ions will hit the detector plate

and when we increase the field, we will focus species having bigger m/z ratio

i.e. heavier ions.

Principle : when the detector is struck by an ion it ejects secondary
electrons, basically amplified the ion current when it is passed on to a
computer for processing and a mass spectrum is produced.
The Nature of Mass Spectra

Mass spectra- Shows the relative current produced by ions of varying mass/charge
ratio.
• A mass spectrum will usually be presented as a vertical bar graph, in which each
bar represents an ion having a specific mass-to-charge ratio (m/z) and the length
of the bar indicates the relative abundance of the ion.

The vertical 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.

• The highest-mass ion in a spectrum is normally considered to be the molecular

ion, and lower-mass ions are fragments from the molecular ion, assuming the
sample is a single pure compound.
Important Terms

• Molecular Ion (M+): A molecule ionized by electron loss but it is not fragmented. Have
high relative abundance. It's an intact ion.

• M represents the mass of the compounds composed of all of the lowest mass isotopes.

• M+1: Isotopes with mass one greater than molecular ion M. Used for calculating the
number of carbons. (M+1/1.1%) ~ Number of carbons

• M+2: Isotopes with mass two greater than molecular ion. Used for calculating presence
of sulfur (~4% intensity), chlorine (~33% intensity) and bromine (~100%).

• Base Peak: The tallest peak in a mass spectrum.

• m/z (mass to charge ratio): if charge of the ion from mass spectrometry is +1(which we
will assume it is), then m/z ratio = atomic mass.
• Intensity(relative abundance)
 not a percent, but used to determine percent

 To calculate percent of abundance, add intensities together, then do part/whole

The mass spectrum of elements
The mass spectrum for boron
Suppose you had 123 typical
atoms of boron. 23 of these would
be 10B and 100 would be 11B.

Relative atomic mass = Relative abundance + Relative abundance the sum of relative
(weight average) of 1*atomic mass of 1 of 2*atomic mass of 2 abundance of 1&2
boron-10 23
E.g. to calculate the relative atomic mass of B:
Ar (B)=(23 x 10) + (100 x 11)/123 = 10.81

From the data we can calculate the % of abundance of each Boron atom i.e.
If you let X = % of boron 10, then 100-X is equal to % of boron-11
Therefore Ar(B) = (X * 10) + [(100-X) * 11)] / 100 =10.81 so, 10X -11X +1100 =100 * 10.81
therefore X = 19 and 100-x = 81
so naturally occurring boron consists of 19% 10B and 81% 11B
boron-11 100
The mass spectrum for zirconium
Isotopes of Zr % abun.
zirconium-90 51.5
zirconium-91 11.2
zirconium-92 17.1
zirconium-94 17.4

zirconium-96 2.8
Suppose you had 100 typical atoms of zirconium, 51.5 of these would be 90Zr, 11.2 would be
91
Zr and so on.

The total mass of these 100 typical atoms would be

(51.5 x 90) + (11.2 x 91) + (17.1 x 92) + (17.4 x 94) + (2.8 x 96) = 9131.8
The average mass of these 100 atoms would be 9131.8 / 100 = 91.3. A r(Zr) = 91.3
Q. Interpret the mass spectrum of the following MS spectra
A. Chlorine B. 2-chloropropane
35
Cl =75 % and 37
Cl = 25%

C. 1,2-dichloroethane D. Bromine
MS spectrum of bromoethane 79
Br =50.5 % and 81
Br = 49.5%

The molecular ion (M+ peak) for

bromoethane is at M/Z=108
 The base peak (relative
abundance=100) occurs at m/z=29
 large m+2 peak occurs at m/z = 110

Q. Guess the molecular formula of spectrum given

belowA B
Fragmentation Patterns
• In simple organic compounds, the most stable molecular ions are those from
aromatic rings, other conjugated π-electron systems and cycloalkanes. Alcohols,
ethers and highly branched alkanes generally show the greatest tendency toward
fragmentation.
• The presence of a functional group, particularly one having a heteroatom Y with
non-bonding valence electrons (Y = N, O, S, X etc.), can dramatically alter the
fragmentation pattern of a compound.

NB. For alkanes, tend to fragment

forming the most stable carbocations in
the following order:
30>20>10
Mass spectrum of hexane(CH3CH2CH2CH2CH2CH3)

 The molecular ion for hexane (with molecular formula C6H14) is at M/Z=86
 The base peak (relative abundance=100) occurs at m/z=57
 A small m+1 peak occurs at m/z = 87
• Mass spectrum of Dodecane (CH3(CH2)10CH3)

 The molecular ion for dodecane (with molecular formula C 12H26) is at M/Z=170

 The base peak (relative abundance=100) occurs at m/z=57 i.e. (CH 3)3C+
 No small m+1 peak occurs at the spectrum.
 There is fragmentation homologous series of hexyl (m/z = 85), pentyl (m/z - 71), butyl
(m/z = 57), propyl (m/z = 43) and ethyl (m/z = 29) cations.
 there is a decrease in molecular mass of each fragment because of consecutive loss of H.
• Alkenes:
– Fragmentation typically forms resonance stabilized allylic
carbocations
• Aromatics:
– Fragment at the benzylic carbon, forming a resonance stabilized
benzylic carbocation (which rearranges to the tropylium ion)

H H H
H C Br H C H C

or

M+
Q1. The diagram shown here provides evidence for the existence of the TWO isotopes.

A. Boron-11 and fluorine-20 C. Boron-10 and boron-11

B. Boron-9 and boron-12 D. Fluorine-19 and fluorine-20

Q2. Peak 1 identifies

A. boron-11 and fluorine-20 C. [ 11B19F3]+

B. boron-9 and boron-12 D. fluorine-19 and fluorine-20

Q3. The difference between peaks 2 and 3 is

A. The fragment at peak 3 contains boron-11 and the fragment at peak 2 contains
fluorine-18

B. The fragment at peak 3 contains an atom of hydrogen not contained in the fragment at
peak 2

C. The fragment at peak 3 contains three atoms of fluorine and the fragment at peak 2
contains only two atoms of fluorine

D. The fragment at peak 2 contains boron-11 and the fragment at peak 3 contains boron-
10

Q4. Which one of the following statements about the mass spectrum of CH3Br is correct?

A. the last two peaks are of equal size and occur at m/z values of 94 and 96

B. the last two peaks have abundances in the ratio 3:1 and occur at m/z values of 94
and 96

C. there is just one peak for the molecular ion with an m/z value of 95
The figure given below is the mass spectrum of Acetylene

Q5. The peak labeled "2" identifies primarily

A. [2H12C2]+ C. [1H212C2]+
B. [3H12C2]+ D. [12C14C]+
Q6. The masses of peaks 2, 3, and 4 are successively smaller in mass by one. This can be
explained by
A. the largest peak containing hydrogen-3, the next largest containing hydrogen-2, and
the smallest containing hydrogen-1
B. successively fewer atoms of hydrogen-1
C. contamination of the sample tested
D. the isotopes of carbon (carbon-12, carbon-13 and carbon-14)
Q7 . Which of the following structures is consistent with the mass
spectrum shown below?

A B C D E
Q8. An unknown compound gives rise to the following infrared and
mass spectra. Propose a structure for the compound. Explain by
citing features from both Infrared (A) and Mass (B) spectra.
A. Infrared spectrum:
B. Mass spectrum:
Q9. The two mass spectra below correspond to two isomers of C5H10O:
3-methyl-2-butanone and 3- pentanone. Draw the two structures.
Match the spectrum with the compound and draw the fragment ion
that corresponds to the base peak.
A. B.