UV-Visible Prepared by: -

Spectroscopy Dr. Rajendra Kotadiya

The concept of EMR
 • Electromagnetic waves or EM waves are waves that are created as a
result of vibrations between an electric field and a magnetic field. In
other words, EM waves are composed of oscillating magnetic and
Electromagnetic electric fields.
radiation/wave • It refers to the waves of the electromagnetic field, propagating through
space, carrying electromagnetic radiant energy. It includes radio waves,
microwaves, infrared, light, ultraviolet, X-rays, and gamma rays.
Photon
• It is the quantum of the electromagnetic field including
electromagnetic radiation such as light and radio waves, and the force
carrier for the electromagnetic force.
• Photons are massless, so they always move at the speed of light in
vacuum, 299792458 m/s.
• Electromagnetic radiation can be described in terms of a stream of
mass-less particles, called photons, each traveling in a wave-like
pattern at the speed of light.
• Each photon contains a certain amount of energy.
• The different types of radiation are defined by the amount of energy
found in the photons
Properties of EMR

• 1. Wave length (λ) – distance

b/n two successive peaks
(crests/troughs) of waves or
length of one wave
• Unit – cm or nm or angstrom (Å)
or micron
• 1 Å = 10-8cm = 10-10m = 10-4µm
• 1nm=1mµ=10-7cm=10-9m
• 1µm=10-4cm=10-6m=104 Å
• 2. Frequency (ν = nu) = Number of waves produced per seconds
• Unit is cycle per second (Measured in Hz)
• 1 cycle per second is sometimes called Hertz
• Mega cycle per second is common unit (MHz)
• 1MHz = 106 cycles per second
1 1
• Frequency α wave length = ν α λ
• ν =c/λ
𝑐𝑚
𝑠𝑝𝑒𝑒𝑑 𝑜𝑓 𝑙𝑖𝑔ℎ𝑡 𝑐 𝑖𝑛 ( )
• ν= 𝑆𝑒𝑐
𝑤𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ 𝑖𝑛 𝑐𝑚
• Frequency of radiation α its energy
• Velocity = the distance travelled by the wave in one second is called
velocity of wave
• Amplitude = it is the maximum distance a wave extends beyond its
middle position.
• Energy (E) = hc/λ (Electromagnetic radiation possesses a certain amount of energy. The
energy of ultimately indivisible unit of radiation, called the photon)
• Wave number = number of waves passed through a space of 1cm
• Reciprocal of wave length
• Denoted by nu bar
• Unit = cm-1
 Wavelengths in the ultraviolet and visible regions are on
Based on the the order of nanometers.
wavelength, EMR In the infrared region, they are micrometers, but the
reciprocal of wavelength is often used (wavenumbers, in
classified as cm−1).

EMR Wave lengths

Gamma rays LT 0.001nm
X rays 0.01nm-10nm
UV-VIS radiations
UV 200-400nm
Visible 400-800nm
IR radiations
Near IR 0.8-2.5µm
Mid IR 2.5-15µm
Far IR 15-200µm
Microwaves 0.01-1m
Radio waves 1-107m
Numerical
• Calculate the wave number for 2.5µ wavelength.

• Solution
• Wave number = 1/wavelength

• First convert 2.5µ to cm = 2.5x10-4cm

• Wave number = 1/ 2.5x10-4cm = 10000/2.5 cm-1=4000cm-1

Numerical
• Calculate the frequency for 3800Å wavelength.
• Solution
• Frequency = Cycles/sec
𝑐𝑚
𝑠𝑝𝑒𝑒𝑑 𝑜𝑓 𝑙𝑖𝑔ℎ𝑡 𝑐 𝑖𝑛 ( )
• ν= 𝑆𝑒𝑐
𝑤𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ 𝑖𝑛 𝑐𝑚

• First convert 3800Å to cm (1 Å = 10 -8cm ) = 3800 x 10-8cm

𝑐𝑚
• Speed of light 𝑖𝑛 ( )=3x1010cm/sec
𝑆𝑒𝑐
𝑐𝑚
3x1010 (𝑆𝑒𝑐)
• Now, ν =
3800 x 10−8cm
• V = 7.8x1014 sec-1 = 7.8x1014 Hz
Numerical
• Calculate the energy for 3800Å wavelength.
• Solution
• Energy (E) = hc/λ
• h- plank’s constant, c- speed of light and λ – wavelength
• h-6.626x10-27erg/sec
• C- 3x1010cm/sec
• λ – 3800Å [convert 3800Å to cm (1 Å = 10 -8cm ) = 3800 x 10-8cm]
𝑐𝑚
6.626x10−27erg/sec x 3x1010 (𝑆𝑒𝑐)
• Now, E =
3800 x 10−8cm
• E = x erg
Matter
• Matter is everything around you.
• Atoms smallest unit of matter that retains all the chemical properties
of an element.
• Matter is defined as anything that has mass and takes up space (it has
volume)
• It is well known that all matter is comprised of atoms. ...
Atom
• An atom is the smallest unit of
ordinary matter that forms a
chemical element.
• Every solid, liquid, gas, and plasma
are composed of neutral or ionized
atoms. Atoms are extremely small,
typically around 100 picometers
across.
• The atomic nucleus is the small,
dense region consisting
of protons and neutrons at the
center of an atom, discovered in
1911 by Ernest Rutherford based on
the 1909 Geiger–Marsden gold foil
experiment.
 When electromagnetic waves pass through a matter, they are primarily
moving through free space, but may have a chance encounter with the
nucleus or an electron of an atom.
Interaction b/n EMR The science of interaction of EMR with matter is SPECTROSCOPY.
(photon) and matter When a photon does encounter an atomic particle, it transfers energy
(atom) to the particle. The energy may be reemitted back the way it came
(reflected), scattered in a different direction or transmitted forward into
the material.
 Spectroscopy
• study of the absorption and
emission of light and other
radiation by matter, is
spectroscopy.
• More recently, the definition has
been expanded to include the study
of the interactions between
particles such as electrons, protons,
and ions, as well as their
interaction with other particles as a
function of their collision energy
 Ultraviolet-visible spectroscopy or
ultraviolet-visible spectrophotometry
(UV-Visible or UV/Vis) refers to
absorption spectroscopy in the
ultraviolet-visible spectral region.

UV-visible
spectroscopy
This means it uses light in the visible and
adjacent (near-UV and near-infrared
(NIR)) ranges.
Theory/Principle of UV-visible spectroscopy

• Wavelength range for UV is

between 200nm to 400nm
whereas for visible it is 400nm
to 800nm.

• 200nm is highest energy

level whereas 800nm is lowest
energy level in UV-visible
spectroscopy.
Molecular electronic transition
Molecular electronic transitions take place when electrons in a molecule are excited from
one energy level to a higher energy level.

The energy change associated with this transition provides information on the structure
of a molecule and determines many molecular properties such as colour.

This type of transition in organic compounds and some other compounds can be
determined by UV-visible spectroscopy provided that transitions in the ultraviolet (UV)
or visible range of the electromagnetic spectrum exist for this compound.
• An atom is made up of (protons + neutrons) nucleus and electron (figure).
• Electrons are found outside nucleus. As electrons reside apart from nucleus, they have
high potential energy.
• Protons carry positive charge while electrons carry opposite charge i.e negative.
Because opposite electric charges attract each other, negative electrons are attracted
to the positive nucleus.
• This force of attraction keeps electrons constantly moving through the empty space
around the nucleus concentrically.
• They have different energy levels (distance between these levels is specific for
individual atoms)
• In an atom, electrons either available as
• bonding electrons (involved in bonding with other atom) or
• non bonding electrons (does not involved in bonding, n electron) or
• valance electrons which do not participate in chemical bonding in molecule are called as non bonding electrons
or n- electrons. These are located principally in atomic orbital of N, O, S and halogens(X) as a lone pair of
electrons. They can be excited by UV radiation.
• antibonding electrons (An antibonding orbital is a molecular orbital containing an electron outside
the region between the two nuclei. As two atoms approach each other, their electron
orbitals begin to overlap. This overlap forms a molecular bond between the two atoms with its
own molecular orbital shape.)
• Pi electron (π electron): An electron which resides in
the pi bond(s) of a double bond or a triple bond, or in a
conjugated p orbital. They are generally mobile
electrons. Since ∏- bonds are weak bonds, the energy
produced by UV radiation can excite ∏- electrons to
higher energy levels.
• Sigma and pi bonds are chemical covalent bonds. Sigma
and pi bonds are formed by the overlap of atomic
orbitals.
• Sigma bonds are formed by end-to-end overlapping
and Pi bonds are formed when the lobe of one atomic
orbital overlaps another. They are found in saturated
systems like alkane. They require large amount of
energy for their excitation and hence do not show
absorption in UV region. Their absorption band is
appeared in vacuum UV region. Hence, compounds
containing σ- bonds do not absorb in near UV region.
For example saturated hydrocarbons are transparent in
near UV region and thus they can be used as solvents.
• Pi bonds are usually weaker than sigma bonds
• In general, single bonds between atoms are
always sigma bonds. Double bonds are comprised of
one sigma and one pi bond. Triple bonds are comprised
of one sigma bond and two pi bonds.
 • Remember that the diagram isn't intended to be to scale - it shows only
Levels of the relative placing of the different orbitals.
electronic • When light passes through the compound, energy from the light is used to
promote an electron from a bonding or non-bonding orbital into one of
the empty anti-bonding orbitals. The possible electron jumps that light
transitions might cause are:
• The energy requirement order for
excitation for different transitions is
as follows. n→∏* (requires lowest
energy) < ∏→∏* < n→σ* < σ→σ*
(requires highest amount of energy).
• In each possible case, an electron is
excited from a full orbital into an
empty anti-bonding orbital. Each
jump takes energy from the light,
and a big jump obviously needs more
energy than a small one. Each
wavelength of light has a particular
energy associated with it. If that
particular amount of energy is just
right for making one of these energy
jumps, then that wavelength will be
absorbed - its energy will have been
used in promoting an electron.
• We need to work out what the
relationship is between the energy
gap and the wavelength absorbed.
• 1. n→∏* transition
• n→π* transition requires lowest energy due to longer wavelength.
• It is also known as R- band.
• They are further characterized by hypsochromic shift or blue shift observed with an
increase in solvent polarity.
• Observed with Carbonyl compounds (C=O) with hetero atoms and unsaturation
• 2. ∏→∏* transition
• It is due to the promotion of an electron from a bonding π orbital to an anti-
bonding ∏* orbital.
• Energy requirement is between n→ ∏* and n→σ*. Which will be MT 200nm.
• When any double/triple bond compound (hydrocarbon or aromatic compounds) is
present then such transition occurs.
• But the extended conjugation and alkyl substituents shifts the λmax towards longer
wavelength (Bathochromic shift).
• It is also called K band.
• 3. n→σ* transition
• Saturated compounds containing atoms with lone pairs (non-bonding electrons) are
capable of n→σ* transitions.
• Examples are alcohols (methanol at 174nm), aldehydes, ketones, amines, water
(167nm), methyl chloride at 169nm etc. These are used as solvents in UV visible
spectroscopy because they are not getting absorb in UV-vis region.
• These transitions usually need less energy than σ →σ* transitions (the energy
required for n→σ* transitions is approximately 175nm)
• They can be initiated by light whose wavelength is in the range 150 - 250 nm.
• Corresponding absorption bands appear at longer wavelengths in near UV region.
• The number of organic functional groups with n→σ* peaks in the UV region is small.
• 4. σ→σ* transition
• These transitions can occur in such compounds in which all the electrons are
involved in single bonds and there are no lone pair of electrons.
• Energy required for σ→σ* transition is very large so the absorption band occurs
in the far UV region. So this transition can’t normally be observed.
• Every energy level, there will be LUMO and HOMO
• Two terms: - LUMO – lowest unoccupied molecular orbital, HOMO – highest
occupied molecular orbital (from HOMO, 1 electron gets excited after receiving
energy from UV or Visible radiation and reached LUMO) i.e. bonding orbital to
antibonding orbital
• For example, all saturated hydrocarbons comes under this category, like
methane (which has only C-H bonds, and can only undergo σ→σ* transitions)
shows an absorbance maximum at 125 nm.
• Absorption maxima due to σ→σ* transitions are not seen in typical UV-Vis.
spectra (200 - 800 nm)
• So whenever we start spectra, it should start from 800nm to 200nm so
that electron will start to excite from optimum level.
• If it start from 200nm which is the highest energy level in UV-visible range,
then almost all molecules get excited at same time and signal will come at
200nm only.
• When a beam of light (EMR) interact with matter (molecule), there are
some mechanisms occur these are known as an molecular electronic
transition.
• Matter/Atom/Electron which gets interacted with EMR, will then excited
from ground state to one level higher with absorption of radiation. This
electron will try to return to its normal state. During which it emits a
photon which has specific characteristics.
UV-visible
spectrum
 • The term chromophore was previously used to denote a
functional group of some other structural feature of which
gives a color to compound.
• For example- Nitro group is a chromophore because its
presence in a compound gives yellow color to the
compound.
• But these days the term chromophore is used in a much
broader sense which may be defined as “any group which
exhibit absorption of electromagnetic radiation in a visible
Chromophores: or ultra-visible region “It may or may not impart any color
to the compound.
• Some of the important chromophores are: ethylene,
acetylene, carbonyls, acids, esters and nitrile groups etc.
• A carbonyl group is an important chromophore, although
the absorption of light by an isolated group does not give
rise to any colour in the ultra-violet spectroscopy.
• Types of chromophores:
• Two types of chromophores are known.
• Chromophores in which the groups have π electrons undergo π-π*
transitions. For examples:-ethylenes, acetylenes etc. (C=C, CΞC)
• Chromophores having both π- electrons and n (non-bonding) electrons
undergo two types of transitions. i.e., π-π* and n-π*, for examples: -
carbonyls, nitriles, azo compounds and nitro compounds etc.
 There is no set rule for the identification of a chromophore.
The change in position as well as the intensity of the
absorption depends upon a large number of factors.
Following points may be useful.
1. Spectrum having a band near 300 mµ may possess two
or three conjugated units.
2. Absorption bands near 270-350 mµ with very low
Identification of intensity ɛmax 10-100 are because of n-π* transitions of
carbonyl group.
chromophores: 3. Simple conjugated chromophores likes dienes or,α β –
unsaturated ketones have εmax values, i.e., from 10,000
to 20,000.
4. The absorption with ɛmax value between 1, 000-10,000
reveals the presence of an aromatic system. If aromatic
nucleus is substituted with groups which can extends
the chromophore, the absorption take place at still
higher value of extinction coefficients.
 • It is a group which itself does not act as a chromophore but when
attached to a chromophore, it shifts the adsorption towards longer
wavelength along with an increase in the intensity of absorption.
• Some commonly known auxochromic groups are: -OH, -NH2, -OR, -
NHR, and –NR2.
• For example:- When the auxochrome –NH2 group is attached to
benzene ring.
• Its absorption change from λ max 225 (ɛmax 203) to λmax 280
(εmax1430).
• All auxochromes have one or more non-bonding pairs of electrons.
AUXOCHROMES: • If an auxochromes is attached to a chromophore, it helps is extending
the conjugation by sharing of non-bonding pair of electrons as shown
below.

• CH2 = CH – NR2---------------->CH2-CH-NH2

• The extended conjugation has been responsible for bathochromic

effect of auxochromes.
Explain electronic transitions 5 marks
• What is spectroscopy?
• Matter – Atom – Electron – its interaction with EMR
• Types of electrons
• Transitions types – diagram – with explanation
• HOMO and LOMO
• Main points – energy level, examples of materials observed such
transition
 • Absorbing compound
• Chromophores
Factors • Auxochromes
affecting • They show addition effect for wavelength maxima
when they are involved
absorption in • Solvent effect
• Benzene (255nm), CCl4 (265nm) and CHCl3 (240nm)
UV visible • They show absorption in UV range. Hence they should
spectroscopy be avoided
• Temperature
• Low temperature is suitable for the UV visible
spectroscopy because in low temperature any
compound will give higher absorption
• Inorganic moieties
• They increase the absorption in UV-vis spectroscopy
• Example CrO42-, MnO4-, Ag, Au etc
Molar extinction coefficient (ε) is a measure
of how strongly a chemical species or
substance absorbs light at a particular
wavelength
It is intrinsic property of chemical species i.e
dependent upon their chemical composition
and structure.
Four types of shifts occurs;
1. Bathochromic shift (Red shift)
2. Hypochromic shift (Blue shift)
3. Hypochromic shift (decrease in ε)
4. Hyperchromic shift (increase in ε)
 • If the absorption wavelength is increased towards the higher λ or
towards the red shift then it is known as bathochromic shift or
red shift.
• There are four reasons to it
• Addition of chromophores
• Addition of auxochromes
• Solvent effect
• Red shift is caused when excited state is more polar as
Bathochromic compared to ground state so the polar solvents stabilize
excited state more than ground state. So overall there is
decrease in the energy gap between excited and ground
shift state resulting in red shift. When ground state is more
polar than excited state, the polar solvents
stabilize ground state more than excited state. So
overall there is increase in the energy gap between
excited and ground state resulting in blue shift.
• Or in carbonyl compounds (polar compound), if we are
using solvent with decreasing polarity, then it will be
responsible for red shift where n-π* transition (lowest
energy) takes place.
• Increasing conjugation
• Increase in double/triple bond
 • If the absorption wavelength is decreased towards the
lower λ or towards the blue color then it is known as
hypsochromic shift or blue shift.
• Shift towards shorter wavelengths.
• Reasons
• Removal of chromophores
Hypsochromic • Removal of auxochromes
shift • Solvent effect
• Aniline gives absorption at 280nm but when it is
dissolved in acid then it gives 203nm as it leads to
gives loss of electron pair (it losses conjugation in
acidic medium)
• Removal of conjugation
 Shifting of ε (molar absorptivity) or
absorption intensity towards higher values.

Introduction of auxochromes generally

Hyperchromic increase intensity of absorption.
shift
Example:

• For Pyridine, εmax= 2750 and substitution with methyl

group (2-methyl-pyridine) it is 3560.
 Shifting of ε (molar absorptivity) or
absorption intensity towards lower
values.
Hypohromic
shift
Example:
For biphenyl, εmax= 19000 and Substitution of methyl group
substitution with methyl group leads to distortion of geometry
(2-methyl-phenyl) it is 10250. of molecule
Instrumentation
Radiation source
 Ideal radiation source requirements
1. It should be stable and should not allow fluctuations.
2. It should emit light of continuous spectrum of high and uniform intensity over the
entire wavelength region in which it’s used.
3. It should provide incident light of sufficient intensity for the transmitted energy to be
detected at the end of optic path.
4. It should not show fatigue on continued use.

• Various UV radiation sources are as follows

a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp
• Various Visible radiation sources are as follows
a. Tungsten lamp
b. Mercury vapour lamp
c. Carbon-one lamp
 • The electrical excitation of deuterium or hydrogen at low
pressure produces a continuous UV spectrum.
• The mechanism for this involves formation of an excited
molecular species, which breaks up to give two atomic
Radiation species and an ultraviolet photon.
source • Both Deuterium and Hydrogen lamps emit radiation in the
range 160 - 375 nm.
• Quartz windows must be used in these lamps, and quartz
cuvettes must be used, because glass absorbs radiation of
wavelengths less than 350 nm.
• Generally two types of lamp can be used
• H2-D2 lamp
• Hydrogen – Deuterium lamp
• Covers 200-375nm wavelength
• Beyond 375nm range, intensity of the radiation will decreases and
• hence it can not be used for due to this reason
• Tungsten filament lamp
• Covers 375nm to 800nm wavelength for UV-visible spectroscopy
• But it has capacity to cover 350nm to 750nm wavelength

• A deuterium lamp has a tungsten filament and a Ni plate anode filled with deuterium
gas under low pressure. When subjected to a high voltage it produces a continuous
spectrum in the UV region due to formation of excited molecular species. It emits
radiation in the range of 160 – 375 nm. The lamp envelope is made of optical grade
silica or quartz and exhibits excellent ultraviolet transmissions.
• An arc is struck between the anode and cathode. The cathode is first heated and
begins to emit electrons. A high voltage is then applied across the electrodes and the
arc is established. This produces enough heat to excite molecular deuterium and main
continuous emission. The heating current can then be switched off. The striking
process takes a few seconds and emission is carried on automatically as a stable and
uniform output.
Monochromator
 Monochromator or filter or wavelength selector
• The most important design feature of the instrument is the monochromator
and its purpose is to resolve polychromatic radiation into individual
wavelengths.
• All monochromators contain the following component parts;
• An entrance slit (accommodate the source radiation)
• A collimating lens (to pass the light through the system)
• A dispersing device (to spread the radiation into its constituent wavelengths, a prism or a
grating which converts polychromatic light into VIBGYOR, it has 1200 to 1400 grooves
[very small prism] per mm length)
• A focusing/reflecting lens
• An exit slit (to select the desired wavelength)
• Polychromatic radiation (radiation of more than one wavelength) enters the
monochromator through the entrance slit.
• The beam is collimated, and then strikes the dispersing element at an angle.
• The beam is split into its component wavelengths by the grating or prism.
• By moving the dispersing element or the exit slit, radiation of only a particular
wavelength leaves the monochromator through the exit slit.
How it works?
Sample container
 • A variety of sample cells available for UV region.
• The choice of sample cell is based on
• the path length, shape, size
sample • the transmission characteristics at the desired
wavelength
containers or • the relative expense
sample cells or • The cell holding the sample should be transparent to the
wavelength region to be recorded.
cuvettes • Quartz or fused silica cuvettes are required for
spectroscopy in the UV region. (because they are
transparent to UV and visible light)
• Glass cuvettes can be used for visible range wavelength.
• The thickness of the cell is generally 1 cm.
• Cells may be rectangular in shape or cylindrical with flat
ends.
Detector
 The purpose of a detector is converting radiant
energy received into an electrical signal which can
be multiplied and measured as a flowing current.

Detectors
In order to detect radiation, three types of
photosensitive devices are used
Photovoltaic
Photomultiplier Charged Phototubes or
Photodiode cells or barrier-
tubes (PMT) coupled photo-emissive
array detector layer cell
detector detector tubes detector
detector
 Photomultiplier tube Dynode electrode where multiplication of electrons takes
place

▪ The photomultiplier is an evacuated glass or

quartz tube with a photosensitive faceplate called
the photocathode in which the absorption of a
photon from the radiation of light results in the
emission of an electron.
▪ The electron is released and focused to the first of
nine electrodes known as a channel or dynode
electrodes.
▪ Additional electrons are generated and multiplied
by each dynode until they reach the end of the
dynode chain.
▪ The electrons are eventually collected at an anode
which is the collection electrode, and the
electrical signal is amplified and measured at
ground potential.
▪ Which leads to generation of electrons and this
will be multiplied time to time (each original
photon has produced 106 - 107 electrons) and
Primary electron finally reaches anode and then to amplifier.
• Photomultipliers are very sensitive to UV and visible radiation.
• They have fast response times.
• Intense light damages photomultipliers; they are limited to measuring
low power radiation.
• Photodiode array detector
• Diodes will convert radiation to electric signals
• It covers entire range of UV-vis (200-800nm)
• Mainly used in HPLC
• Charged coupled detector
• Here silicon surface is used to catch the photons from the radiation which convert
radiation to digital signals
• Photocell
• This is simple and cheap. It is constructed on a piece of selenium metal coated with
light sensitive material. When irradiated, sufficient energy is generated to release
electrons, and a current flow is established. No external power supply is required.
Wavelength range is about 400 – 750 nm and is ideal for use in colorimeters.
• Photovoltaic cell
 Traditional Chemistry

Life Science

Microbiology

Food & Agriculture

Material Science
Applications Optical Components

Pharmaceutical Research

Petrochemistry

Cosmetic Industry

Quality Control
Applications of UV-visible spectroscopy
• 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. 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
• 4. Qualitative analysis
• UV absorption 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.
• 5. Dissociation constants of acids and bases.
PH = PKa + log [A-] / [HA]
From the above equation, the PKa value can be calculated if the ratio
of [A-] / [HA] is known at a particular PH. and the ratio of [A-] / [HA]
can be determined spectrophotometrically from the graph plotted
between absorbance and wavelength at different PH values.
• 6. Chemical kinetics
• Kinetics of reaction can also be studied using UV spectroscopy. The
UV radiation is passed through the reaction cell and the absorbance
changes can be observed.
• 7. 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 analysed by 0.5% H2SO4 in methanol at the
wavelength 284 nm.
• Molecular weights of compounds can be measured
• Spectrophotometrically by preparing the suitable derivatives of these
compounds.
For example, if we want to determine the molecular weight of amine
then it is converted in to amine picrate. Then known concentration of
amine picrate is dissolved in a litre of solution and its optical density
is measured at λmax (380 nm). After this the concentration of the
solution in gm moles per litre can be calculated by using the following
formula.
"c" can be calculated using above equation, the weight "w" of amine
picrate is known. From "c" and "w", molecular weight of amine picrate
can be calculated. And the molecular weight of picrate can be
calculated using the molecular weight of amine picrate.
• 9. As HPLC detector
• A UV/Vis spectrophotometer may be used as a detector for HPLC. The
presence of an analyte gives a response which can be assumed to be
proportional to the concentration. For more accurate results, the
instrument's response to the analyte in the unknown should be
compared with the response to a standard; as in the case of
calibration curve.
 Single component analysis

Applications Multi component analysis

Spectrophotometric
titrations
Methods for
multicompon
ent analysis
 • If a sample contains two absorbing drugs (X and Y) each of which abs
orbs at the λmax different from the other, it may be possible to deter
mine both drugs by the technique of simultaneous equations
(Vierodt's method).
• The information required is
• (a) The absorptivities of X at and λ1 and λ2 are ax1 and ax2 respectively
• (b) The absorptivities of Y at and λ1 and λ2 are ay1 and ay2 respectively.
•
Simultaneous (c) The absorbances of the diluted sample at λ1 and λ2 are
A1 and A2 respectively.

estimation • Conditions to fulfil this criteria:

• λmax of two-component should be reasonably dissimilar.
• Two-component should not interact chemically, thereby negating the
initial assumption that the absorbance.
Let Cx & Cy be the concentrations of X and Y respectively in the dil
uted sample.
Two equations are constructed based upon the fact that at λ1 and λ2,
the absorbance of the mixture is the sum of the individual absorbance of
X and Y.
• Consider
• The absorptivities of X at λ1 and λ2, ax1 and ax2, respectively
• The absorptivities of Y at λ1 and λ2, ay1 and ay2, respectively
• The absorbance of the dilute sample at λ 1 and λ2, A1 and A2, respectively
• X, have concentration c x and Y have concentration cy in dilute sample
• According to the fact, the concentration of mixture is the sum of the
individual concentrations of X and Y.
• So, at λ1 , A1 = ax1 b cx + ay1 b cy (1) and At λ2 , A2 = ax2 b cx + ay2 b cy (2)
• If cell is 1 cm, b = 1 equation 2 become, cy = (A-ax2cx)/ay2
• Substituting value of cy in equation (1) and rearranging it, we will get
• cx = (A2ay1-A1ay2)/(ax2ay1-ax1ay2)
• cy = (A1ax2-A2ax1)/(ax2ay1-ax1ay2)
 Spectrophotometric titrations
• If at least one species in a complexation titration absorbs
electromagnetic radiation, we can identify the end point by monitoring
the titrand’s absorbance at a carefully selected wavelength.
• For example, we can identify the end point for a titration of Cu2+ with
EDTA, in the presence of NH3 by monitoring the titrand’s absorbance at a
wavelength of 745 nm, where the Cu(NH3)42+ complex absorbs strongly.
• At the beginning of the titration the absorbance is at a maximum.
• As we add EDTA the concentration of Cu(NH3)42+, and thus the
absorbance, decreases as EDTA displaces NH3.
• After the equivalence point the absorbance remains essentially
unchanged.
• The resulting spectrophotometric titration is shown below in panel
(a).
• Note that the titration curve’s y-axis is not the actual absorbance, A,
but a corrected absorbance, Acorr
• Acorr = A × (VEDTA + VCu)/VCu
• where VEDTA and VCuu are, respectively, the volumes of EDTA and Cu.
• Correcting the absorbance for the titrand’s dilution ensures that the
spectrophotometric titration curve consists of linear segments that
we can extrapolate to find the end point.
• Other common spectrophotometric titration curves are shown in
panels b-f.