UV-Visible absorption spectroscopy

Contents:
Introduction to Spectroscopy
Basic Principle of UV-Vis spectroscopy
Origin & theory of UV spectra
Type of Electronic transitions
Fundamental Laws of absorption
Instrumentation
Applications
Basic principles:
• Ultraviolet (UV) and visible radiation comprise only a
small part of the electromagnetic spectrum
• The energy associated with electromagnetic
radiation is defined by the following equation:
E = hν
where E = energy (in joules),
h = Planck’s constant (6.62 × 10-34 Js), and
ν = frequency (in seconds).
Electromagnetic radiation can be considered a combination
of alternating electric and magnetic fields that travel
through space with a wave motion. Because radiation acts
as a wave, it can be classified in terms of either wavelength
or frequency, which are related by the following equation:
C= ν × λ
where n is frequency (in seconds), c is the speed of light
(3 × 108 ms-1), and l is wavelength (in meters).

In UV-visible spectroscopy, wavelength usually is

expressed in nanometers (1 nm = 10-9 m).
radiation with shorter wavelength = higher energy.
In UV-visible spectroscopy,
The low-wavelength UV light = the highest energy.
• Ultraviolet light: 10 to 400 nm
Near UV region: 200-400 nm
Far UV region : below 200 nm

• Visible light: 400 to 800 nm

Ultraviolet/visible spectroscopy involves the

absorption of ultraviolet light by a molecule causing
the promotion of an electron from a ground electronic
state to an excited electronic state.
Electronic excitation spectroscopy:

The spectroscopy which utilizes the ultraviolet

(UV) and visible (VIS) range of electromagnetic
radiation, is frequently referred to as Electronic
Spectroscopy.

Photon absorption promotes an electron from

its ground state to an excited state.
 Origin of UV-visible spectra
• When radiation interacts with matter, a number of
processes can Occur.
• absorption of radiation by matter causes the energy
content of the molecules or atoms to increase.
• The total potential energy of a molecule generally is
represented as the sum of its electronic, vibrational, and
rotational energies
Etotal = Eelectronic + Evibrational + Erotational

Eelectronic > Evibrational > Erotational

Fig: Electronic transitions & UV-Visible spectra in molecule
 Origin of absorption
• Valence electrons can generally be found in one of three types
of electron orbital:
1. single, or σ, bonding orbitals;
2. double or triple bonds (π bonding orbitals);
3. 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*
Types of electronic transition
 Beer-Lambert Law
• When a beam of light is passed through a transparent
cell containing a solution of an absorbing substance,
reduction of the intensity of the light may occur.

• This due to:

1. reflection at the inner & outer surfaces of the cell
2. scatter by particles in the solution
3. absorption of light by molecules in the solution
• The reflections at the cell surfaces can be
compensated by -a reference cell containing
the solvent only.

• Scatter may be eliminated by filtration of the

solution, the intensity of light absorbed is then
given by:

Iabsorbed = I0 – IT
When light passes through or is reflected from a sample,
the amount of light absorbed is the difference between the
incident radiation (Io) and the transmitted radiation (I).

The amount of light absorbed is expressed as either

transmittance or absorbance.

Transmittance usually is given in terms of a fraction of 1 or

as a percentage and is defined as follows:
• Lambert’s law shows that there is logarithmic
relationship between the transmittance & length
of the path.
• Beer’s observed a similar relationship between
transmittance & the concentration of the
solution.
• Beer’s law is defined as follows:
The intensity of a beam of monochromatic
radiation decreases exponentially with the
increase in concentration of the absorbing
substance arithmetically.
• More simply it is stated as absorption is
proportional to concentration
Beer’s law tells us that absorption is proportional
to the number of absorbing molecules – i e to the
concentration of absorbing molecules (this is only
true for dilute solutions) –
Lambert’s law tells us that the fraction of
radiation absorbed is independent of the intensity
of the radiation.
Combining these two laws, we can derive the
Beer-Lambert Law:

A = log10(Io/I) = log10(100/T) = kcL

 A = log10 (Io/I)
= log10 (100/T)
= kcL
where Io = the intensity of the incident radiation
I = the intensity of the transmitted radiation
ε = a constant for each absorbing material, known
as the molar absorption coefficient (called the molar
extinction coefficient in older texts) and having the units
mol-1 dm3 cm-1, but by convention the units
are not quoted
ι = the path length of the absorbing solution in cm
c = the concentration of the absorbing species in
mol dm-3
The importance of concentration

The proportion of the light absorbed will depend on how

many molecules it interacts with.

Strongly colored dye -

higher absorbance as no of molecules interacting with
radiation is high

Dilute solution-
Low absorbance
Lambert's Law states that each layer of equal thickness of
an absorbing medium absorbs an equal fraction of the
radiant energy that traverses it.

The fraction of radiant energy transmitted by a given

thickness of the absorbing medium is independent of the
intensity of the incident radiation, but the radiation does
not alter the physical or chemical state of the medium.

If the intensity of the incident radiation is Io and that of the

transmitted light is l, then the fraction transmitted is:
l/lo = T
The percentage transmission is
%T = l/lo x 100
• The Beer-Lambert Law states that the concentration of a substance in
solution is directly proportional to the 'absorbance ', A, of
the solution.
• Absorbance A = constant x concentration x cell length
• The law is only true for monochromatic light, that is light of a single
wavelength or narrow band of wavelengths, and provided that the physical or
chemical state of the substance does not change with concentration.
• When monochromatic radiation passes through a homogeneous solution in a
cell, the intensity of the emitted radiation depends upon the thickness (l) and
the concentration (C) of the solution.
• Io is the intensity of the incident radiation and I is the intensity of the
transmitted radiation. The ratio I/Io is called transmittance.
• This is sometimes expressed as a percentage and referred to as
%transmittance.
Mathematically, absorbance is related to percentage
transmittance T by the expression:

A = log10(Io/I) = log10(100/T) = kcL

where L is the length of the radiation path through the
sample, c is the concentration of absorbing molecules in
that path, and k is the extinction coefficient - a constant
dependent only on the nature of the molecule and the
wavelength of the radiation.
If, in the expression A = kcl, c is expressed in molar-1 and l
in m, then k is replaced by the symbol τ and is called the
molar absorption coefficient.

The units of τ are mol-1m2. τ was formerly called the molar

extinction coefficient and concentrations were
often expressed as mol l-1, mol dm-3 or M and the cell
length in cm to give units mol-1lcm-1, mol-1dm3cm-1 and
M-1cm-1
respectively. C Sometimes is expressed in g dm-3(gl-1) and l
in cm. In this case, k is replaced by A (sometimes E). A is
known as the specific absorption coefficient.
Specific absorbance:
• Absorbance of a specified concentration in a
cell of specified pathlength
• i.e A(1%, 1cm), which is the absorbance of a
1g/100ml (1%w/v) solution in a 1cm cell.

A= A 1%
1cm bc
• Deviations or
limitations to
Beer’s Law
Deviations from Beer’s Law:

• In Beer’s law it states that if we plot

absorbance A against concentration C a
straight line passing through the origin is
obtained, but usually deviation from linear
relationship between concentration &
absorbance & an apparent failure of beer law
 There are two types of deviation
• Positive deviation
When a small change in concentration produces
a greater change in absorbance

• Negative deviation
When a larger change in concentration produces
a greater change in absorbance
• Real deviation to Beer’s Law:

• Apparent deviation:
 Instrumental deviation: stray radiation, improper slit
width, fluctuation in single beam& when
monochromatic light is not used can influence the
deviation
 Chemical deviation: factors like association, dissociation,
ionization, faulty development of color, refractive index
at high concentration
LIMITATIONS OF THE BEER-LAMBERT LAW
The linearity of the Beer-Lambert law is limited by chemical and
instrumental factors.
Causes of nonlinearity include :
·Deviations in absorptivity coefficients at high concentrations
(>0.01M) due to electrostatic interactions between molecules in
close proximity
· scattering of light due to particulates in the sample
· fluoresecence or phosphorescence of the sample
· changes in refractive index at high analyte concentration
· shifts in chemical equilibria as a function of concentration
· non-monochromatic radiation, deviations can be minimized by
using a relatively lat part of the absorption spectrum such as the
maximum of an absorption band
· stray light
 Deviation may occur due to presence of impurities that
fluoresce or absorb at the absorption wavelength. This
interference introduces an error in the measurement of
absorption of radiation penetrating the sample.
 Deviation may occur if monochromatic light is not used.
 If width of slit is not proper & therefore it allows
undesirable radiation to fall on the detector. These
undesirable radiation might be absorbed by impurities
present in the sample.
 If the solution species undergoes polymerisation
• Example:
• benzyl alcohol in CCl4 in high concentration
exist in polymeric form, dissociation of this
polymer increases with dilution, due to this
change, absorbance will change.
• Beers law can not be applied to the suspension
but can be estimated colorimetrically after
preparing a reference curve with known
concentration
 Real deviation:
• Beer’s Law is successful in describing the absorption
behaviour of media containing low analyte concentration, in
this sense it is limiting Law.
• At high concentration (usually > 0.01 M) the average distance
between the molecules responsible for absorption is
diminished, because each molecule affects the charge
distribution of its neighbours. This interaction alter the ability
of molecule to absorb a given wavelength of radiation.
• The extent of interaction depends upon concentration,
• So deviations from the linear relationship between
absorbance & concentration.
• A similar effect is occur in media containing
low absorber concentrations but high
concentration of other species (electrolyte)

• Deviation arise because molar absorptivity

depends upon Refractive index of the
medium.
Instrumental deviation:
Deviations due to polychromatic radiation :

Strict adherence to Beer's law is observed only with truly

monochromatic radiation.
Beers law required monochromatic radiation because absorptivity
is a constant at a single wavelength & varies with a change in
wavelength.
The possibility of error due to practical impossibility of obtaining
monochromatic radiation. So it is minimized by the selection of a
spectral region where a change iin a absorptivity with a change in
wavelength is very small.
Monochromators are used to isolate portions of the output from
continuum light sources, hence a truly monochromatic radiation
never exists and can only be approximated, i.e. by using a very
narrow exit slit on the monochromator.
Deviations in presence of stray radiation :

The radiation exiting from a monochromator is

contaminated with small amounts of scattered or stray
radiation, which reaches the exit slit as a result of
scattering & reflections from various internal surfaces.

Stray radiation differs greatly in wavelength from that of

principal radiation.

At high concentration & at longer path length, stray

radiation cause significant deviations from linear
relationship between absorbance & pathlength.
 Chemical deviation:
• it arises when analyte dissociates, associate
or reacts with a solvent to produce a product
having a different absorption spectrum from
the analyte.
• A chromophore (literally color-bearing) group is a functional group,
not conjugated with another group, which exhibits a
• characteristic absorption spectrum in the ultraviolet or visible
region. Some of the more important chromophoric groups are:
• If any of the simple chromophores is conjugated with another (of
the same type or different type) a multiple chromophore is
• formed having a new absorption band which is more intense and at
a longer wavelength that the strong bands of the simple
• chromophores.
• This displacement of an absorption maximum towards a longer
wavelength (i.e. from blue to red) is termed a bathochromic shift.
• The displacement of an absorption maximum from the red to
ultraviolet is termed a hypsochromic shift.