UV-VISIBLE SPECTROSCOPY

Mr. P.Prachet,
Assistant Professor,
Department of Pharmaceutical Analysis,
Chalapathi Institute of Pharmaceutical Sciences 1
Introduction
• The UV-visible spectroscopy is one of the oldest instrumental techniques of
analysis and is the basis for a number of ideal methods for the determination
of micro and semi-micro quantities of analytes in a sample.
• UV-visible spectrum results from the interaction of electromagnetic radiation
in the UV-visible region with molecules, ions or complexes.
• These determinations finds applications in research, industry, clinical
laboratories and in the chemical analysis of environmental samples.
• Ultra violet spectroscopy is concerned with the study of absorption of UV
radiation which ranges from 200nm to 400nm.
• Compounds which are coloured, absorb radiation from 400nm to 800nm.
• Compounds which are colourless absorb radiation the UV region.
• In both UV as well as visible spectrosopy, only the valence electrons absorb
the energy, thereby the molecule undergoes transition from ground state to
excited state.
Origin of spectrum
• The absorption of radiation in the UV-visible region of the spectrum is
dependent on the electronic structure of the absorbing species like atoms,
molecules, ions or complexes.
• A given electronic energy level has a number of vibrational energy levels in
it and each of the vibrational energy level has a number of rotational energy
levels in it.
• When a photon of a given wavelength interacts with the molecule it may
cause a transition.

Theory
• Many molecules absorb ultraviolet or visible light.
• Any molecules has either n, pi, sigma or a combination of these electrons n
these bonding and non bonding electrons absorb the characteristic radiation
and undergoes transition from ground state to excited state.
Electronic transitions
• The transitions that results in the absorption of electromagnetic radiation in
this region of the spectrum are transitions between electronic energy energy
levels
• As a molecule absorbs energy, an electron is promoted from an occupied
orbital to an unoccupied orbital of greater potential energy i.e. transition is
from The highest occupied molecular orbital (HOMO) to the lowest
occupied molecular orbital (LUMO)
• For most molecules, the LUMO are the sigma orbitals, which corresponds to
sigma bonds. The pi orbitals lie at somewhat higher energy levels, and
orbitals that hold unshared pairs, the nonbonding (n) orbitals lie at even
higher energies. The unoccupied or antibonding orbitals are the orbitals of
higest energy .
1. n Π* Transitions
• Its require lowest energy.
• The peak is called R band, where n electron present.
• Double bonds and triple bonds are present .
E.g. Aldehydes or ketones.
2. Π Π* Transitions
• This type of transition gives rise to B, E and K bands .
• B bands : Aromatic & hetero aromatic systems.
• E bands :Aromatic systems.
• K bands :Conjugated systems.
• Its require energy between n σ and n Π*
• Wavelength is in the range 174 - 178 nm.
E.g. Beta carotene, lycophene etc.

3. n σ* Transitions
• These transitions occur in saturated compounds containing atoms with lone
pairs (non-bonding electrons).
• These transitions usually requires less energy than σ - σ * transitions.
• They can be initiated by light whose wavelength is in the range 180 - 250
nm.
4. σ σ*Transitions
• Its require highest energy.
• These transitions occur in saturated compounds (specially hydrocarbons).
• Wavelength is in the range 125 - 135 nm.
E.g. Methane, ethane, propane etc.

Chromophores
It is defined as any isolated covalently bonded group that shows a
characteristic absorption in the ultraviolet or visible region. Some of the
important chromophores are ethylenic, acetylenic, carbonyls, acids, esters, and
nitrile group etc. –CH2–, – CΞC –, – COOH–, – CΞN etc.

Types of Chromophores
There are two types of chromophores such as
a. Chromophores in which the group contains π electrons and they undergo
π → π * transitions. Such chromophores are ethylene, acetylenes etc.
b. Chromophores which contains both π electrons and n (non-bonding)
electrons. Such chromophores undergo two types of transitions i.e., π → π* and
n → π*. Examples of this type are carbonyls, nitriles, azo compounds, nitro
compounds etc.

Azo compound Nitro methane

Identification of a chromophore depends on a number of factors as follows:

1. Spectrum consisting of a band near 300 mµ may contain two or three
conjugated units.
2. Absorption bands near 270-350 mµ with very low intensity εmax 10-100 are
due to n → π*transitions of the carbonyl group.
3. Simple conjugated chromophores such as dienes or α, β- unsaturated ketones
have high εmax values, i.e., 10,000 to 20,000.
4. The absorption with εmax values between 1000-10,000 shows an aromatic
system.
Auxochromes
• It is defined as any group which does not itself act as a chromophores but
whose presence brings about a shift of the absorption band towards the red end
of the spectrum (longer wavelength).
• An auxochrome group is called color enhancing group. Auxochromic groups do
not characteristic absorption above 200 mµ. Common auxochromic groups are
– OH, -OR, -NH2, -NHR, -NR2, -SH etc.
• The effect of the auxochrome is due to its ability to extend the conjugation of a
chromophore by sharing of its non-bonding electrons.

Absorption and intensity shifts

1. Bathochromic shift/effect (Red shift)
2. Hypsochromic shift/effect (Blue shift)
3. Hyperchromic effect
4. Hypochromic effect
1. Bathochromic shift/effect (Red shift)
It is an effect due to which the absorption maximum is shifted towards
longer wavelength for the presence of an auxochrome or by the change of
polarity of solvent. The n → π* transition for carbonyl compounds
experiences red shift when the polarity of solvent decreased.
2. Hypsochromic shift/effect (Blue shift)
• It is an effect due to which the absorption maximum is shifted towards shorter
wavelength for the removal of conjugation (auxochrome) or by the change of
polarity of solvent.
• In aniline, absorption maximum occurs at 280 mµ as the pair of electrons on
nitrogen atom is in conjugation with π bond system of the benzene ring but in
acidic solutions, a blue shift occurs at shorter wavelength (~203 mµ). In aniline
ion formed in acidic solution, the electron pair is no longer present and hence
conjugation is removed.

Aniline Aniline ion

3. Hyperchromic shift/effect
It is an effect due to which the intensity of absorption maximum (εmax)
increases. For example, the B-band for pyridine at 257 mµ εmax 2750 is shifted
to 262 mµ εmax 3560 for 2methyl pyridine. Introduction of an auxochrome
usually increases intensity of absorption.
 Pyridine 2-methyl pyridine

4. Hypochromic effect
It is defined as an effect due to which the intensity of absorption maximum
decreases, i.e., extinction coefficient, εmax . For example, biphenyl absorbs
at 250 mµ εmax 19000 whereas 2-methyl biphenyl absorbs at 237 mµ, εmax
10250 (εmax decreases). Introduction of methyl group distorts the geometry
of the molecules thus, cause hypochromic effect.

Choice of solvents
• It should not itself absorb radiations in the region under investigations.
• It should be less polar so that it has minimum interaction with the solute
molecule.
• Most commonly: 95%ethanol.
• Cheap, good dissolving power, does not absorb radiation above 210nm.
Typical examples

Solvent effect
• The position and intensity of an absorption band may shift when the spectrum
is recorded in different solvents.
• A dilute sample solution is prefered for analysis.
• Most commonly used solvent is 95% ethanol. It is best solvent as it is cheap,
transparent down to 210nm.
• Position as well as intensity of absorption maxima get shifted for a particular
chromophore by changing the polarity of solvent.
• By increasing polarity of solvent → dienes, conjugated hydrocarbons→ no
shift.
• α, β unsaturated carbonyl compound show 2 different shifts.

Effect of solvent polarity on the various types of bands

K-band
• Due to conjugated enes & enones are affected differently by changing the
polarity of the solvent.
• K bands due to conjugated dienes are not affected by changing the polarity
of the solvent, while these bands due to enones shows a red shift by
increasing the polarity of solvent.
R band
• The absorption shifts to shorter wavelength (blue shift) with increasing
polarity of solvent.

B band
• The position as well as the intensity of the band is not shifted by increasing
the polarity of the solvent.
• But the heterocyclic aromatic compound, a marked hyperchromic shift is
observed by increasing the polarity of the solvent.

Lambert’s Law
When a monochromatic light passes through an absorbing medium at right
angles to the plane of surface of medium or solution, the rate of decrease in
intensity with thickness of medium (b) is proportional to the intensity of
incident light.
In other words the intensity of transmitted light decreases exponentially as the
thickness of medium increases arithmetically.
Beer’s Law
Bernard and Beer independently stated that ‘The intensity of incident light
decreases exponentially as the concentration of absorbing medium increases
arithmetically. This is similar to Lambert’s law and thus,
Where ε is the Molar extinction coefficient, a constant dependent upon the
wavelength of incident radiation and the nature of absorbing material and the
concentration is expressed in gram mole/litre
Since absorbance A= log Io /It
We can infer that
A= εbc (Equation of beer – Lambert’s law)
Where, A – Absorbance or optical density
ε – Molar extinction coefficient
c – Concentration of the drug (mol/lit)
b – Path length (normally 10mm or 1cm)
Or A= abc
a –absorptivity, if concentration is expressed in grams/litre

Deviations from Beer lambert’s law

A system is said to obey Beer’s law, when a plot a graph of concentration and
absorbance gives a straight line.
• When a non-linear curve is obtained then the system is said to undergo
deviation from beer law.

There are two types deviation

Positive deviation: It results in when small change in concentration produce a
greater change in absorbance.
Negative deviation: It results in when a large change in concentration
produces smaller change in absorbance.
These deviations from the Beer law can be classified into three categories
Real deviations – These are fundamental deviations due to the limitations of
the law itself.
Chemical deviations – These are deviations observed due to specific chemical
species of the sample which is being analyzed.
Instrumental deviations – These are deviations which occur due to how the
absorbance measurements are made.

Real deviation and limitation

• Beer’s law describes the absorption behaviour of dilute solutions only so it is
a limiting law.
• At high concentration (exceeding 0.01M) solute molecules can cause
different charge distribution on their neighboring species in the solution.
• Since at high concentration it result in a shift in the absorption wavelength of
the analyte.
• Some large ions or molecules show deviations even at very low
concentrations.
For e.g. methylene blue absorptivity at 436 nm fails to observe beer’s law even
at concentrations as low as 0.01M.
• High analyte concentrations alter the refractive index (η) of the solution ,it
affect the absorbance obtained.

Chemical deviation and limitation

Occurs due to some chemical phenomenon
• Association, dissociation and interaction with the solvent to give a different
product .
• Example, phenol red undergoes a resonance transformation when moving
from the acidic form (yellow) to the basic form (red).
• Due to this resonance, the electron distribution of the bonds of molecule
changes with the pH of the solvent in which it is dissolved.

Instrumental deviation and limitation

1. Polychromatic radiation
• Beer law is strictly followed when a monochromatic source of radiation
exists. It is common to use a polychromatic source of radiation with
continuous distribution of wavelengths along with a monochromator is used
to create a monochromatic beam from this source.
2. Stray radiation
• Scattered radiation is the radiation from the instrument that is outside the
nominal wavelength band selected.
• This radiation is due to reflection and scattering by the surfaces of lenses,
mirrors, gratings, filters and windows.
• The wavelength of the stray radiation is different from the wavelength band
selected.
• The radiation exiting from a monochromator is often contaminated with
minute quantities of scattered or stray radiation.
• If the analyte absorb at the wavelength of the stray radiation, a deviation
from Beer law.

3. Mismatched cell
• If the cells holding the analyte and the blank solutions are having different
path-lengths, or unequal optical characteristics it leads to deviation from
Beer’s law.

4. Due to improper slit width

• If the width of the slit is not proper, deviations are occurs due to undesirable
radiation to fall on the detector.
• These undesirable radiation are absorbed by the impurities present in the
solution of sample.
• It leads to change in the absorbance of the sample.

Instrumenation
1. Light source
2. Monochromator
3. Detector
4. Read out device
Source of radiant energy
Requirements of an ideal source
• It should be stable and should not allow fluctuations.
• It should emit light of continuous spectrum of
• high and uniform intensity over the entire wavelength region in which it’s used.
• It should provide incident light of sufficient intensity for the transmitted
energy to be detected at the end of optic path.
• It should not show fatigue on continued use.

1. Tungsten halogen lamp

• Its construction is similar to a house hold lamp.
• The bulb contains a filament of Tungsten fixed in evacuated condition and
then filled with inert gas.
• The filament can be heated up to 3000k, beyond this tungsten starts
sublimating.
• It is used when polychromatic light is required. To prevent this along with inert
gas some amount of halogen is introduced (usually Iodine).
• Sublimated form of tungsten reacts with iodine to from tunsgten iodine lamp.
2. Deuterium Lamp:
• It is used in UV region and tungsten lamp is used in Visible region.
• A deuterium arc lamp (or simply deuterium lamp) is a low-pressure gas-
discharge light source often used in spectroscopy when a continuous
spectrum in the ultraviolet region is needed.
• Mechanism: Formation of an excited molecular species, which breaks up to
give2 atomic species and an ultraviolet photon.
• Tungsten Lamp Deuterium Lamp
• Which migrates back to the hot filament where it decomposes and Tungsten
get deposited.

Demerit
• It emits the major portion of its radiant energy in near IR region of the
spectrum.

3. Hydrogen discharge lamp

• In Hydrogen discharge lamp pair of electrodes is enclosed in a glass tube
(provided with silica or quartz window for UV radiation to pass trough)
filled with hydrogen gas.
• When current is passed trough these electrodes maintained at high voltage,
discharge of electrons occurs which excites hydrogen molecules which
in turn cause emission of UV radiations in near UV region.
• They are stable and robust.
4. Xenon discharge lamp
• It possesses two tungsten electrodes separated by some distance.
• These are enclosed in a glass tube (for visible) with quartz or fused silica
and xenon gas is filled under pressure.
• An intense arc is formed between electrodes by applying high voltage. This
is a good source of continuous plus additional intense radiation. Its intensity
is higher than the hydrogen discharge lamp.

Demerits
• The lamp since operates at high voltage becomes very hot during operation
and hence needs thermal insulation.

5. Mercury arc lamp

In mercury arc lamp, mercury vapor is stored under high pressure and
excitation of mercury atoms is done by electric discharge.

Demerit
• Not suitable for continuous spectral studies,(because it doesn’t give
continuous radiations).
Mirrors
• These are used to reflect, focus or collimate light beams in
spectrophotometer.
• To minimize the light loss, mirrors are aluminized on their front surfaces.

Slits
• Slit is an important device in resolving polychromatic radiation into
monochromatic radiation.
• To achieve this, entrance slit and exit slit are used.
• The width of slit plays an important role in resolution of polychromatic
radiation.

Monochromators
• It is a device used to isolate the radiation of the wavelength from
wavelength of the continuous spectra. Following types of monochromatic
devices are used.
1.Filters
2.Prisms
3.Gratings
Filters
• Selection of filters is usually done on a compromise between peak
transmittance and band pass width; the former should be as high as possible
and latter as narrow as possible.
• Absorption filters- work by selective absorption of unwanted radiation and
transmits the radiation which is required.
E.g. Glass and Gelatin filters.

Merits
• Simple in construction
• Cheaper
• Selection of the filter is easy

Demerits
• Less accurate.
• Band pass (bandwidth) is more (±20-30nm) i.e. if we have to measure at
400nm; we get radiation from 370-430nm. Hence less accurate results are
obtained.
Interferance filters
• Works on the interference phenomenon, causes rejection of unwanted
wavelength by selective reflection.
• It is constructed by using two parallel glass plates, which are silvered
internally and separated by thin film of dielectric material of different
(CaF2, SiO, MgF2) refractive index. These filters have a band pass of 10-
15nm with peak transmittance of 40-60%.

Merits
• Provide greater transmittance and narrower band pass (10- 15nm) as
compare to absorption filter.
• Inexpensive .
• Additional filters can be used to cut off undesired wavelength.

Prism
• Prism is made from glass, Quartz or fused silica.
• Quartz or fused silica is the choice of material of UV spectrum.
• When white light is passed through glass prism, dispersion of polychromatic
light in rainbow occurs. Now by rotation of the prism different
wavelengths of the spectrum can be made to pass through in exit slit on
the sample.
• The effective wavelength depends on the dispersive power of prism
material and the optical angle of the prism.

Gratings
• Are most effective one in converting a polychromatic light to
monochromatic light. As a resolution of +/- 0.1nm could be achieved
by using gratings, they are commonly used in spectrophotometers.
 Gratings are of two types
1. Diffraction grating
2. Transmission gratings

Diffraction grating
• More refined dispersion of light is obtained by means of diffraction
gratings.
• These consist of large number of parallel lines ( grooves) about 15000-
30000/ inch is ruled on highly polished surface of aluminum.
• These gratings are replica made from master gratings by coating the
original master grating with a epoxy resin and are removed after setting.
• To make the surface reflective, a deposit of aluminum is made on the
surface. In order to minimize to greater amounts of scattered radiation and
appearance of unwanted radiation of other spectral orders, the gratings are
blazed to concentrate the radiation into a single order.
Transmission grating
• It is similar to diffraction grating but refraction takes place instead of
reflection. Refraction produces reinforcement. this occurs when radiation
transmitted through grating reinforces with the partially refracted radiation.
Advantages
• Grating gives higher and linear dispersions compared to prism monochromator.
• Can be used over wide wavelength ranges.
• Gratings can be constructed with materials like aluminium which is resistant to
atmospheric moisture.
• Provide light of narrow wavelength.
• No loss of energy due to absorption.
Sample Holders
Glass or Quartz was used but mostly cuvette made up of Quartz was used.
Detectors
• Device which converts light energy into electrical signals, that are displayed on
readout devices.
• The transmitted radiation falls on the detector which determines the intensity of
radiation absorbed by sample.
The following types of detectors are employed in instrumentation of absorption
spectrophotometer
1. Barrier layer cell/Photovoltaic cell
2. Phototubes/ Photo emissive tube
3. Photomultiplier tube

Requirements of an ideal detector

• It should give quantitative response.
• It should have high sensitivity and low noise level.
• It should have a short response time.
• It should provide signal or response quantitative to wide spectrum of radiation
received.
1. Barrier layer cell/Photovoltaic cell
• The detector has a thin film metallic layer coated with silver or gold and acts as
an electrode.
• It also has a metal base plate which acts as another electrode.
• These two layers are separated by a semiconductor layer of selenium.
• When light radiation falls on selenium layer, electrons become mobile and are
taken up by transparent metal layer.
• This creates a potential difference between two electrodes & causes the flow
of current.
• When it is connected to galvanometer, a flow of current observed which is
proportional to the intensity and wavelength of light falling on it.
2. Photo tubes/Photo emissive tubes
• Consists of a evacuated glass tube with a photocathode and a collector
anode.
• The surface of photocathode is coated with a layer of elements like cesium,
silver oxide or mixture of them.
• When radiant energy falls on photosensitive cathode, electrons are emitted
which are attracted to anode causing current to flow.
• More sensitive compared to barrier layer cell and therefore widely used.
3. Photo multiplier tubes
• The principle employed in this detector is that, multiplication of photoelectrons
by secondary emission of electrons.
• In a vacuum tube, a primary photo-cathode is fixed which receives radiation
from the sample.
• Some eight to ten dynodes are fixed each with increasing potential of 75-100V
higher than preceding one.
• Near the last dynode is fixed an anode or electron collector electrode.
• Photo-multiplier is extremely sensitive to light and is best suited where weaker
or low radiation is received.
4. Silicon photodiode
• For photodiode array spectrophotometers, a white light passes through
sample.
• The grating polychromator disperses the light into the component
wavelengths,
• All wavelengths are measured simultaneously.
• Resolution depends upon the distance between the diodes and amount of
dispersion.
Instrument design
• Depending upon the monochromators (filters or dispersing device) used to
isolate and transmit a narrow beam of radiant energy from the incident light
determines whether the instrument is classified as Photometer or a
Spectrophotometer.
• Spectrophotometers used here detects the percentage transmittance of light
radiation, when light of certain intensity & frequency range is passed
through the sample.
• Both can be a single beam or double beam optical system.

1. Single beam spectrophotometer

• Light from the source is carried through lens and/or through aperture to pass
through a suitable filter.
• The type of filter to be used is governed by the colour of the solution.
• The sample solution to be analysed is placed in cuvettes.
• After passing through the solution, the light strikes the surface of detector
(barrier-layer cell or phototube) and produces electrical current.
• The output of current is measured by the deflection of needle of light-spot
galvanometer or micro ammeter. This meter is calibrated in terms of
transmittance as well as optical density. The readings of solution of both
standard and unknown are recorded in optical density units after adjusting
instrument to a reagent blank.

Advantages
• Simple in construction.
• Easy to use and economical.

Disadvantages
• Any fluctuation in the intensity of radiation sources affects the absorbance.
• Continuous spectrum is not obtained.

2. Double beam UV-Spectrophotometer

• Double beam instrument is the one in which two beams are formed in the space
by a U shaped mirror called as beam splitter or beam chopper .
• Chopper is a device consisting of a circular disc. One third of the disc is opaque
and one third is transparent, remaining one third is mirrored. It splits the
monochromatic beam of light into two beams of equal intensities.

Advantages
• It facilitates rapid scanning over wide λ region.
• Fluctuations due to radiation source are minimised.
• It doesn’t require adjustment of the transmittance at 0% and 100% at each
wavelength.
• It gives ratio of intensities of sample & reference beams simultaneously.

Disadvantages
• Construction is complicated.
• Instrument is expensive.

Applications
1. Detection of Impurities
UV absorption spectroscopy is one of the best methods for determination of
impurities in organic molecules. Additional peaks can be observed due to
impurities in the sample and it can be compared with that of standard raw
material. By also measuring the absorbance at specific wavelength, the
impurities can be detected.
Benzene appears as a common impurity in cyclohexane. Its presence can be
easily detected by its absorption at 255 nm.
2. Structure elucidation of organic compounds.
UV spectroscopy is useful in the structure elucidation of organic molecules,
the presence or absence of unsaturation, the presence of hetero atoms.
From the location of peaks and combination of peaks, it can be concluded that
whether the compound is saturated or unsaturated, hetero atoms are present or
not etc.
3. Qualitative analysis
UVabsorption spectroscopy can characterize those types of compounds which
absorbs UV radiation. Identification is done by comparing the absorption
spectrum with the spectra of known compounds.
UV absorption spectroscopy is generally used for characterizing aromatic
compounds and aromatic olefins.
4. Quantitative analysis of pharmaceutical substances
Many drugs are either in the form of raw material or in the form of formulation.
They can be assayed by making a suitable solution of the drug in a solvent and
measuring the absorbance at specific wavelength.
Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the wavelength
284 nm.
Quantitative analysis

UV absorption spectroscopy can be used for the quantitative determination

of compounds that absorb UV radiation. This determination is based on

Beer’s law which is as follows.

A = log I0 / It = log 1/ T = – log T = abc = εbc

Where ε is extinction co-efficient, c is concentration, and b is the length of

the cell that is used in UV spectrophotometer.

Other methods for quantitative analysis are as follows.

a. calibration curve method

b. simultaneous multicomponent method

c. difference spectrophotometric method

d. derivative spectrophotometric method

THANK
YOU