UV-VIS SPECTROSCOPY

● Light is an electromagnetic wave form disturbance or photon of energy propagated at 3.0 × 108 m/sec. They
are electromagnetic radiation because they are made of an electrical and a magnetic wave that are in phase and
perpendicular to each other and to the direction of propagation.
● The distance along the direction of propagation for one complete cycle is known as the wavelength, λ.
● The number of waves passing through a fixed point on the time axis per second is known as the
frequency (υ) of the radiation. Usually expressed in Hertz. It has an inverse relationship with wavelength.
υ = c / λ, where c = velocity of light.
● Energy can travel through a vacuum or matter as EMR.
● Electromagnetic radiation is a transverse wave with magnetic and electric components that oscillate
perpendicular to each other. The electromagnetic spectrum is the range of all possible wavelengths and
frequencies of electromagnetic radiation including visible light.
● According to the wave particle duality concept, although electromagnetic radiation is often considered
to be a wave, it also behaves like a particle.

Spectrum - A spectrum is a range of frequencies or wavelengths.

Continuous spectrum
● We observe that when a ray of white light falls on a prism it experiences refraction twice.
● Once when it travels from the rarer medium (air) to a denser medium (glass) and again from the
denser medium (glass) to a rarer medium (air).
● Finally, we observe a band of colors, called spectrum, formed out of a ray of white light. If we
observe this spectrum more closely, the color having a smaller wavelength deviates the most and
vice versa.
● Thus, a spectrum of colors ranging from red to violet is observed where red having the longest
wavelength suffers the least deviation.
● This kind of spectrum is called a continuous spectrum as violet merges into blue, blue into
green and so on.

Emission Spectrum
● Whenever electromagnetic radiation interacts with atoms and molecules of matter, the electrons in these
atoms may absorb energy and jump to a higher energy state, losing their stability.
● In order to regain their stability, they need to move from the higher energy state to the previous lower
energy state.
● To accomplish this job, these atoms and molecules emit radiation in various regions of the
electromagnetic spectrum.
● This spectrum of radiation emitted by electrons in the excited atoms or molecules is known as an
emission spectrum.

Absorption Spectrum
● An absorption spectrum is like a photographic negative of an emission spectrum.
● For observing the absorption spectrum, electromagnetic radiations are bombarded on a sample that
absorbs radiation of certain wavelengths.
● The wavelength of radiation absorbed by the matter contributes to the missing wavelength which leaves
dark spaces in the bright continuous spectrum.
● Each element has its unique line emission or absorption spectrum. The study of the emission
spectrum or absorption spectrum is better known as spectroscopy.
● A beam of radiation from an electric bulb consists of several wavelengths and is known as
polychromatic light, while a beam in which all the rays have the same wavelength is known as
monochromatic light.

LAWS OF ABSORPTION:
Quantitative spectrophotometry is based on the two principal laws of photometry namely Lambert's law
and Beer's law.
The absorption of light by absorbing materials is governed by 2 laws.
● Bouger – Lambert’s Law – It states that the amount of monochromatic light absorbed is
proportional to the thickness of the absorbing material and independent of the intensity of the
incident light.

I = e-kl
Io
Where I = intensity of transmitted light
Io = intensity of incident light
l = pathlength, thickness of the absorbing media in cm.
k = linear absorption coefficient of the absorbing media

using logarithm, ln I/Io = e-kl Changing to common logarithm, 2.303 log10 Io/I = kl

● Beer’s Law – It states that the amount of monochromatic light absorbed by a material is
proportional to the number of absorbing molecules, that is, the concentration of the absorbing
media.
I/Io = e-k’C
Or, 2.303 log10 Io/ I= k’C

Where k’ = absorptivity constant

C = concentration of absorbing media

● Beer – Lambert’s law – The amount of monochromatic light absorbed by a media is proportional to the
concentration of the absorbing substance and to the thickness of the absorbing material. The quantity Io/I
is called the optical density or absorbance and the reverse is known as transmittance (the amount of light
that escapes absorption)
log10 Io/I = ε l C

Where ε = absorptivity or absorption coefficient

If ‘C’ is expressed in grams moles per litre, and ‘l’ in cm, the coefficient ‘ε’is known as extinction
coefficient or molar absorptivity.
Absorbance shares a linear relationship with sample concentration while transmittance has a non-linear
relationship. It is therefore easier to use absorbance as an index of sample concentration.
‘

The relationship between absorbance and transmittance is given as

A = log Io – log I
But Io is set to 100% and log 100 is 2, so
A = 2 – log I

If the optical density of a standard solution is known then an unknown sample concentration can be
calculated from its optical density. The formulae is Concentration of unknown = (OD of the unknown ×
concentration of standard) /OD of standard

Factors that cause deviation from Beer Lambert's Law:

1. The common factor causing deviation from the linearity is the use of a band of wavelength to measure
the absorbance in most colorimeters. The laws of absorption apply only to monochromatic radiation and
not to polychromatic radiation.
2. High sample concentration – At high sample concentration there is some degree of interaction between
the absorbing molecules. This results in a non-linear relationship between OD and concentration. Hence
Beer Lambert's law is primarily used in solutions that are below 10-2 M.
3. Indeterminate instrumental variation causes apparent deviation from the Beer Lambert's Law. This
could be due to stay radiation reaching the detector or sensitivity changes in the detector that are as a
result of voltage variation.
4. Certain systems form more than one complex in solution. All these complexes absorb at different
wavelengths. The OD thus varies for the same concentration depending upon the type of complex in
relative abundance.
BLANK / REFERENCE SOLUTION – To measure the OD of any substance one dissolves it in a solvent.
If the substance does not absorb in the visible region, it is made to absorb in the visible region by adding
some reagents so that it is coloured. The solvents and other agents will also absorb in the region in which
the absorbance due to the substance of interest is being measured. The resulting OD will be a sum of the
individual absorbance due to the substance, the solvent and the reagents. In order to cancel out the OD
contributed by the solvent and the reagents, we prepare the blank, which consists of all reagents used in
the solvent but not the substance of interest. When this solution is read spectrophotometrically a particular
reading is obtained on the OD scale. This reading is subtracted from the OD of the substance to get the
absorbance of the substance of interest.
UV-VIS SPECTROSCOPY & COLORIMETRY :

It is based on the selective absorption of electromagnetic radiation in the wavelength range of 180-800
nm. Electronic transitions in molecules are classified according to the participating molecular orbitals.
There are four possible transitions: n → π*, π → π*, n → σ*, σ → σ*. Transitions among these orbitals
are by the radiation having the same energy as that of the energy difference between the specific orbitals.
The UV-Vis radiation has sufficient energy to cause only two transitions between n → π* and π → π*.
The other transitions, n → σ* and σ → σ* requires higher energies. The molecular structures such as
bonds and functional groups responsible for interaction with electromagnetic radiation are called
chromophores
Transitions to a π* orbital requires the presence of an unsaturated functional group. The outer
nonbonding n electrons form hydrogen bonds with water and alcohols, whereas inner π electrons are
unaffected by solvents. Organic molecules with conjugated double bonds, carbonyl groups, carboxyl
groups, and nitro groups have good absorbance in the UV-Vis region. The absorption maxima is strongly
affected by substituent groups.
In proteins, there are three types of chromophores: peptide bond, certain amino acid side chains
and certain prosthetic groups/ coenzymes. Electronic transition of the peptide bond occur in the far UV
with an intense peak at 190 nm and weak one at 210-220 nm due to π → π* and n → π* transitions. The
aromatic amino acids, phenylalanine, tyrosine and tryptophan have their absorption maxima at 257, 274
and 280 nm, respectively. The proteins that contain prosthetic groups such as haem, falvin, carotenoid
have strong absorption bands in the UV-Vis region. The absorption of UV light by nucleic acids is due to
n → π* and π → π* transitions of the purine and pyrimidine bases that occur between 260 nm and 275
nm.

Instrumentation for UV - Visible spectrophotometer and colorimeter:

● The instruments that are used to study the absorbance or emission of electromagnetic radiation as a
function of wavelength are called spectrometers or spectrophotometers.
● The essential component of spectrophotometer include – (i) a stable and cheap radiant source, (ii) a
monochromator to break the polychromatic radiation into component wavelengths, (iii) transparent vessel
( cuvette) to hold the sample and (iv) a photosensitive detector and associated readout system.
i) Radiant energy sources – Material that can be excited to a high-energy status by a high voltage
electric discharge or by electrical heating serve as a good source of radiant energy. As the electrons of
these materials return to their ground state, they emit radiation of characteristic energies corresponding to
ΔE, the energy difference between the excited and the ground energy level. Sources of ultraviolet
radiation – the most common source are hydrogen lamp and deuterium lamp.
Both the systems consist of a pair of electrodes enclosed in a glass tube provided with a quartz window.
The glass tube is filled with hydrogen or deuterium gas at low pressure. They emit radiation in the range
of 180 – 350 nm. Xenon lamps can also be used.
Source of visible radiation – tungsten filament lamp is commonly used. It is inexpensive and emits
continuous radiation in the region between 350 – 2500nm. Carbon arc may also be used.

ii) Wavelength selectors are of two types – filters and monochromators.

Filters – they operate by absorbing the light in all other regions except for one, which they reflect. Gelatin
filters are made of a layer of gelatin coloured with organic dye and sealed between glass plates. Tinted
glass filers may also be used. One disadvantage of glass filters is their low transmittance.
Monochromator – It resolves the polychromatic radiation into its individual wavelength and isolates these
wavelengths into very narrow bands. The essential components of a monochromator are (i) an entrance
slit that admits the polychromatic radiation from the source (ii) a collimator device such as a lens or
mirror that collects all the polychromatic light on to the dispersion device., (iii) a wavelength resolving
device like a prism or a diffraction grating that breaks the radiation into components wavelength, focusing
lens or a mirror and (iv) an exit slit that allows the monochromatic beam to escape. The entire set up is
mounted in a light tight box so that the assembly will not absorb in the range of wavelength, which are to
be studied. The 2 most widely used monochromators are prism and diffraction grating.
(1) Prism – A prism disperses polychromatic light from the source into its constituent wavelength by
virtue of its ability to refract different wavelengths at different extent; the shorter wavelengths are
diffracted most. The disadvantage of the prism is that the shorter wavelength are dispersed more and the
longer ones less. Hence the red end of the spectrum is not full resolved. Simple glass prisms are used for
visible range. For UV region, silica, fused silica or quartz prisms are used.
(2) Gratings – Grating are used in the monochromators of spectrophotometers operating in ultraviolet and
visible regions. The grating possesses a highly reflective surface etched with a large number of parallel
grooves, which are equally spaced. These grooves are called lines. Principle behind dispersion of
radiation by grafting of radiation by a grating is that it resolves light into its component wavelength by
virtue of constructive reinforcement and destructive interference of reflected radiation. The advantage of
diffraction gratings is that they have a better resolving power than prisms.
iii) Sample containers – Samples are usually held in containers called cuvettes. Cuvettes meant for the
visible region are made up of either ordinary glass or quartz. Since glass absorbs in the UV range, hence
quartz or fused silica cells are used. The standard path length of the cuvette is 1 cm.

iv) Detection devices – the principle of all the detectors used in spectrophotometry is that the incident
light (photons) liberates electrons from the metal or other surface. These electrons would be collected by a
source and measure the amount of current obtained. The current is then proportional to the amount of
light intensity and therefore the measure of it.
The requirement of a detector are – (i) high sensitivity to allow the detection of low levels of radiant
energy, (ii) short response time, (iii) long term stability and (iv) electronic signal that is easily amplified
for typical readout apparatus.
There are 3 types of detectors- photocells, phototubes and photomultiplier tubes
(a) Photovoltaic or photocells or barrier layer detectors: It employs semiconductor materials.
Semiconductors are crystalline and the bonding electrons between the crystals of some semiconductors
can be knocked out of their positions by incident radiation. Commonly used photocell is made of
selenium. A typical photocell consists of a thin coating of selenium over a thin transparent silver film on a
steel base. This arrangement ensures that electrons pass from selenium to silver but not in the reverse
direction. Due to the inability of the electrons to move away from the silver film, the silver film acts as a
collecting electrode. The steel plate functions as the other electrode. The current flowing between the two
electrodes is then measured by a microammeter. Photocells have a long life and are inexpensive and
reliable. They are widely used in colorimeters.

(b) Phototube or photoemission tubes: The components of the phototube are (i) an evacuated glass
envelope with quartz window, (ii) a semi-cylindrical cathode whose inner surface is coated with alkali or
alkaline earth metal oxides and a centrally located metal wire anode. A potential difference of 90 volts is
applied across the electrodes. The quartz window allows the passage of radiation which strikes the
photoemissive surface of the cathode surface. The energy of the photon is transferred to the loosely bound
electrons that gets excited and finally leave the surface and travel towards the anode causing current to
flow in the circuit. If the electron collection is 100 % efficient, the phototube current should be
proportional to the light intensity.
(c) Photomultipliers: these detectors are designed to amplify the initial photoelectric effect and are
suitable for use at very low light intensities. A photomultiplier consists of (i) an evacuated glass tube into
which are sealed the cathode and the anode. (ii) additional electrodes called dynodes. A allowed to pass
between the cathode and anode as the radiation strikes the photocathode, electrons are liberated and the
applied potential difference accelerates the electrons towards the first dynode. Each successive dynode is
at a higher potential and thus act as an amplification stage for the original photon. The applied voltage
causes sufficient electrons to accelerate to knock out other electrons from each dynode surface, causing an
amplification cascade.
⮚ Comparison of UV and visible spectrophotometer:
DOUBLE BEAM SPECTROPHOTOMETER:
It uses a beam splitter that is placed prior to the sample containers. One of the split beams passes through
the blank or reference solution while the other passes through the sample cell. The two transmitted beams
are then compared either continuously or alternately several times in a second. The double beam device
therefore compensates for the voltage fluctuation in the source intensity, the detector signal and amplifier
gain by observing the difference in signal between reference and sample at the same time. The beam
splitting usually occurs after the monochromator. A rotating sector mirror is commonly used for splitting
or chopping the beam. The chopped beams reach sample and reference and then to the detector at
intervals which depend upon the rotational frequency of the chopper. The device then records the ratio of
the reference and the same signals.
Applications of UV - Visible Spectrophotometery:
1. Qualitative analysis - UV - Visible spectra may be used to identify classes of compounds in both the
pure state and in biological preparations. This is done by plotting absorption spectrum curves. Since these
curves are specific for a class of compounds, knowledge of the absorption spectrum can help in
identification of a substance. Example water absorbs at 205 nm, proteins absorb maximally at 280 nm
while nucleic acids absorb at 254 or 260 nm.
2. Quantitative analysis - UV - Visible Spectrophotometery is used for the determination of the unknown
concentration of a given species. The first step in this is to find the absorption band. The unknown
concentration of the solution is determined at this absorption maximum using a set of standards and
plotting the standard graph.
3. Enzyme assay – whenever the product or substrate of an enzyme reaction has a distinct absorption
spectrum; the amount of enzyme present may be determined or its kinetics studied by measuring the rate
of appearance or disappearance of the substance.
4. Molecular weight determination: the molecular weight (M) of a compound can then be readily
calculated on the basis of its absorption data.
M=εcl/A
Where, ε = extinction coefficient
l = pathlength
c = concentration of the compound
A= Absorbance
5. Study of Cis-trans isomerism: Since geometrical isomers differ in spatial arrangement of groups about
a plane, the absorption spectra of the isomers also differ. The trans-isomer is usually more elongated than
its cis counterpart. It is usually therefore, for the trans – isomer to have a higher wavelength of the
maximum absorption.
6. Control of purity: If the absorption maximum is not characteristic of a given compound, it is an
indication of the presence of impurities. Thus carbon disulphide impurity in carbon tetrachloride can be
detected easily by measuring absorbance at 318 nm where carbon disulphide absorbs. Similarly benzene
impurity in commercial absolute alcohol can be measured at 280 nm where alcohol does not absorb.