UV visible spectroscopy

1. UV Visible Spectroscopy
Part-1
Dr. Priyanka S. Chaudhari

2. 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

4. 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).

5. 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.

8. • 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.

9. 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.

11. 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

13. Fig: Electronic transitions & UV-Visible spectra in molecule

16. 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*

30. 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

31. • 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

32. 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:

35. • When a beam of light is allowed to pass
through a transparent medium, the rate of
decrease of intensity of radiation with the
thickness of medium is directly proportional
to the intensity of light.
• i.e absorbance is proportional to the
thickness (pathlength) of the solution.

41. Remember that the absorbance of a solution will
vary as the concentration or the size of the
container varies.
Molar absorptivity compensates for this by
dividing by both the concentration and the length
of the solution that the light passes through.
Essentially, it works out a value for what the
absorbance would be under a standard set of
conditions - the light travelling 1 cm through a
solution of 1 mol dm-3.

42. • 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

43. 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

44. 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

47. The importance of concentration
The proportion of the light absorbed will depend on how
many molecules it interacts with.
Suppose you have got a strongly coloured organic dye. If it is
in a reasonably concentrated solution, it will have a very
high absorbance because there are lots of molecules to
interact with the light.
However, in dilute solution, it may be very difficult to see
that it is coloured at all. The absorbance is going to be very
low.

48. 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

49. • 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.

50. 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.

51. 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.

53. 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= A1%
1cmbc

54. •Deviations or
limitations to
Beer’s Law

55. 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

56. 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

57. 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

58.  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

59. • 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

60. • 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

61. 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.

62. • 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.

63. Instrumental deviation:

64. 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.

66. 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.

68. 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.

69. • 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.