Dept.

of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

UV/Visible Spectroscopy
Swapnil J. Dengale

Introduction:

Spectroscopy per se involves the measurement and interpretation of EMR (Electro Magnetic
Radiations) absorbed or emitted when the molecules or atoms or ions of sample moves from one
energy state to another energy state. On passing EMR in the UV and visible region through compound
with multiple bonds, a portion of radiation is normally absorbed by the compound. The amount of
absorption depends on the wavelength of the radiation and the structure of the compound. The
absorption of radiation is due to the subtraction of energy from the radiation beam when electrons in
orbitals of lower energy are excited into orbitals of higher energy. Since this is an electron excitation
phenomenon, UV spectroscopy is sometimes called as electronic spectroscopy.

Definition of spectroscopy:

The interaction of EM radiation and matter are the subject of the science called “spectroscopy”.
Spectroscopic analytical methods are based on measuring the amount of radiation produced or
absorbed by molecular or atomic species of interest.

By looking at the definition of spectroscopy, it is imperative to understand the EMR spectrum and its
properties.

EMR Spectrum:

• EMR or light, is a form of energy. Whose behavior is described by the properties of both waves
and particles

• The optical properties of EMR such as diffraction are explained best by describing light as a
wave.

• Many of the interactions between EMR and matter such as absorption and emission, however,
are better described by treating light as particle or photon

• EMR consists of discrete packets of energy, which we call photons.

• EMR consists of oscillating electric and magnetic fields that propagate through space along a
linear path with constant velocity

• Oscillation in the electric and magnetic fields are ┴r to each other

• In vacuum travels at the speed of light [C= 2.99792x108 m/s]

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

• Moves through medium other than a vacuum with a velocity V, which is less than the speed
of light in vacuum.

Fig. 1 Light as an electromagnetic wave

In order to use light wave for any meaningful purposes, the qualification (characterization) of waves
have to be understood first.

Fig. 2

Wavelength (λ):

The distance between consecutive crests (or troughs) is called as wavelength. It can be measured in
cm (10-2 m) , mm, µm (10-6m), nm (mµ or 10-9mt), A° (10-10).

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Frequency (ν):

The number of waves per second or Number of wavelength unit passing through a given point in unit
time. It can be measured in Hz, KHz, MHz, GHz ... or CPS.

Wavenumber (ῡ):

Number of waves per centimetre. (cm-1)

Relationship between wavelength, frequency and energy:

The energy content of a photon can be given by the equation,

E = h.v eq(1)

Where,

E= Energy of radiation (Joules, ergs)

H = Planks constant

V (nu) = Frequency (Hz)

According to the definition of frequency,

v = C/λ ,

where,

C= speed of light (3.0 x 108 m/s), λ= Wavelength.

So, substituting the value of frequency (v) in equation 1,

E = h. C/λ eq(2)

Hence, there are 2 important takeaways above equations.

Eq (1) proves that energy of electromagnetic radiation (E) is directly proportional to the frequency (v).

Eq (2) proves that energy of electromagnetic radiation (E) is inversely proportional to wavelength (λ).

That means as wavelength of light increases, the energy within it decreases and vice-versa.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Different regions of EMR spectrum:

The following table lists the names of different spectral regions, the range of frequencies and
wavelengths in those regions, and the type of transition that can occur when a photon in these spectral
ranges interacts with matter.

Type of Radiation Frequency Range (Hz) Wavelength Range Type of Transition

20 24
gamma-rays 10 -10 <1 pm nuclear
17 20
X-rays 10 -10 1 nm-1 pm inner electron
15 17
ultraviolet 10 -10 400 nm-1 nm outer electron
14
visible 4-7.5x10 750 nm-400 nm outer electron

14 14 outer electron molecular

near-infrared 1x10 -4x10 2.5 µm-750 nm
vibrations
13 14
infrared 10 -10 25 µm-2.5 µm molecular vibrations

11 13 molecular rotations,
microwaves 3x10 -10 1 mm-25 µm
electron spin flips*
11
radio waves <3x10 >1 mm nuclear spin flips*

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 Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Types of spectroscopy:

Spectroscopy is classified based on the following criteria

a) Depending upon the EMR region employed

b) Depending upon the nature of analyte being analysed
c) Depending upon the interaction of EMR with matter

Depending upon the EMR region employed:

Spectroscopy is classified based on the region of EMR spectrum used for the interactions with the
matter.

Spectroscopy

UV-Spectroscopy IR-Spectroscopy X-ray diffraction NMR Spectroscopy

Uses UV region Uses IR region Uses X-ray region Uses radio frequency
region

Depending upon the interaction of EMR with matter:

1. Absorption spectroscopy: Energy levels have well defined values (i.e they are quantized)
absorption only occurs when the photon’s energy matches the difference in energy, ΔE,
between two energy levels. A plot of absorbance as a function of the photon’s energy is called
an “absorbance spectrum”.

E
n

h

5
E
o

Absorption
 Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

2. Emission spectroscopy: Emission of a photon occurs when an analyte in a higher-energy state

returns to a lower-energy state. The higher-energy state can be achieved in several ways,
including thermal energy, radiant energy from a photon, or by a chemical reaction.

E
n

h

E
o

Emission

Depending upon the nature of analyte being analysed:

1. Atomic spectroscopy: Changes in the energy take place at atomic level. Further subdivided
into Atomic Absorption spectroscopy (AAS) & Atomic Emission Spectroscopy (AES).
2. Molecular spectroscopy: Changes in energy takes place at molecular level. Further subdivided
into Molecular absorption & Molecular emission spectroscopy

Spectroscopy

Molecular Atomic Spectroscopy

Spectroscopy

Atomic Absorption Atomic Emission

Molecular Absorption Molecular Emission Eg. AAS spectroscopy Eg. Flame photometry
Eg. UV spectroscopy Eg. Fluorescence
spectroscopy
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Types of transitions involved in UV/visible spectroscopy:

When electrons absorb radiations (photons) and become excited, they move from bonding orbital
(ground state) to anti-bonding orbital (excited state). Bonding electrons like σ and π move to excited
states like σ* and π* respectively. While non-bonding electrons (n) move to either of the antibonding
orbitals (excite levels) like σ* and π*.

1. σ σ* Transition:

A transition of an electron from a bonding σ orbital to the higher energy antibonding (excited) σ*
orbital is designated σ σ* Transition. Sigma bonds are, in general, very strong, this therefore, is a
high energy process and these transitions require very short wavelength i.e. high energy UV light
approximately around 150 nm. Alkanes eg. Methane, Ethane, Cyclopropane show this type of
transition.

2. n σ* Transition:

This transition involves saturated compounds with one hetero atom (S, O, N) with lone pair of
electrons. The energy required for the transition is less when compared to σ σ* transition. The peaks
due to absorption bands of such transition occur in near UV region i.e. between 170 nm – 190 nm.

Eg. Water absorbs at 167 nm. Methanol at 174 nm & methyl chloride at 169 nm.

These kind of transitions are particularly sensitive to hydrogen bonding and thus exert solvent effect
on UV spectrum.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

3. ππ* transition:

This transition is available in compounds with unsaturated centres eg. Simple alkenes, carbonyl
compounds etc. This transition requires lesser energy than n σ* transition. In simple unconjugated
alkene, an absorption band at 190 nm is due to such transition. The band due to ππ* transition in a
compound with conjugated π system is usually intense (ɛmax > 10000) and is frequently referred to
as K-band (German Konjugierte).

4. n π* transitions:

Unsaturated molecules having atoms like sulphur, oxygen and nitrogen exhibit n π* transition.
These atoms contain lone pair of electrons which are promoted from ground state to antibonding
excited state π*. Aldehydes, Ketones and nitro group compounds show this type of transition. This
transition involves least amount of energy than all other transitions and therefore, this transition gives
rise to an absorption band at longer wavelength.

Eg. In saturated aliphatic ketones, n π* transition around 280 nm is the lowest energy transition.

n π* transition is forbidden by symmetry consideration, thus the intensity of the band due to this
transition is low, although the wavelength is longer.

This kind of transitions arise due to the presence of radical group containing lone pair of electrons
within a structure, that’s why the absorption band due to such transition is called as R-band. (R stands
for Radikal).

Absorption Characteristics of Some Common Chromophores:

Chromophore Example Solvent λmax (nm) ɛmax Type of

transition
Alkene C6H13HC=CH2 n-Heptane 177 13,000 pp*
Alkyne C5H11C≡C n-Heptane 178 10,000 pp*
196 2,000 _
225 160 _
Carbonyl (CH₃)₂C=O n-Hexane 186 1,000 ns*
n-Hexane 280 16 np*
180 Large ns*
293 12 np*
Carboxyl CH₃COOH Ethanol 204 41 np*
Amido O Water 214 60 np*
C H 3C N H 2
Azo H3CN NCH3 Ethanol 339 5 np*
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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Nitro CH3NO2 Isooctane 280 22 np*

Nitroso C4H9NO Ethyl ether 300 100 _
665 20 np*
Nitrate C2H5ONO2 Dioxane 270 12 np*

Laws of photometry:

Beer’s Law:

“The intensity of a beam of parallel monochromatic radiation decreases exponentially with the
number of absorbing molecules. More simply it is stated that the absorbance is proportional to the
concentration”.

OR

“The intensity of a beam of monochromatic light decreases exponentially as the concentration of

the absorbing substances increases arithmetically”

Lambert’s law:

when a beam of monochromatic light is passed through a substance, a absorption of light is directly
proportional to the path length of the sample of substance.

OR

The intensity of a beam of parallel monochromatic radiation decreases exponentially as it passes

through a medium of homogeneous thickness.

OR

More simply it is stated that the absorbance is proportional to the thickness (pathlength) of the
solution

Relationship between Absorbance and Transmittance:

Before learning the derivation of Beer-Lambert’s law, it is imperative to understand the relationship
between Absorbance and Transmittance.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

P0 P
(power in) (power out)

B(path through sample)

The diagram shows a beam of monochromatic radiation of radiant power P0 directed at a sample
solution. Absorption takes place and the beam of radiation leaving the sample has radiant power P.
the amount of radiation absorbed may be measured in a number of ways.

Transmittance (T) = P/P0

% Transmittance = P/P0*100

Absorbance = - log (T) = -log (P/P0) or = log (1/T)

Example: If no light is absorbed then P=Po and A=0. If P0 is 100 and P is 10 then Transmittance is 10%
whilst Absorbance is 1.

Derivation of Beer-Lambert’s law:

According to Lambert’s law, transmittance is exponentially proportional to path length (b).

So,

T = P/P0= 10-kb ----- 1 k is a constant T is transmittance

The fraction of radient energy transmitted put it in a logarthemic form

log T = log P/P0= -Kb ----- 2

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

As per Beer’s law, transmittance is exponentially proportional to concentration.

So,

T = P/P0= 10-k1C ----- 3 k1 is a new constant

put it in a logarthemic form log T = log P/P0= - K1C ----- 4

Combining the equations 2 and 4

We have Beer-Lambert’s law which describes the dependence of T on both the path length and the
concentration

T = P/P0= 10-abC ----5 where ‘a’ is a combined constant of k and K1

called as absorptivity.

put it in a logarthemic form log T = log P/P0= -abc ----- 6

It is more convenient to omit the negative sign on the right hand side of the equation and to define
the new term absorbance

A = -log10 T = log1/T = log10 P0/P =a b C Where A is the absorbance.

Sometimes absorptivity can be denoted by epsilon ε.

A = a.b.C or/ A = ε. b. C

Molar Absorptivity Vs Specific Absorptivity:

Other common names for absorptivity are absorption index and extinction coefficient.

If the absorbance is divided by the product of concentration of the solution (in g/L) and solution path
length (in cm) the dividend is known as absorptivity,

a = A/bc

b= path length and c= concentration of the solution

Specific Absorbance Or Specific Absorptivity E1%1cm OR A1%1cm:

It is the absorbance of a solution of unit concentration and unit path length.

E1%1cm It is the absorption [log10 (Po/P)] of a 1% w/v solution in a cell with 1cm path length.

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Molar Absorptivity, or Molar Extinction Coefficient:

• Molar absorption index or Molar absorption coefficient, ε. It is the product of absrptivity (a)
and molecular weight (M),

ε = a.M

• Molar absorptivity can be obtained by dividing absorbance with the product of concentration
(expressed in g moles per litre) and path length (cm). it has the unit of 1000 cm2 mol-1 but the
units are, by convention, never expressed

Conditions for Beer-Lambert’s Law:

Beer lambert’s law is applicable under the following conditions:

• The light used must be monochromatic

• The concentration must be weak (As dilute as 10mM)

• The solution must not fluoresce and must be homogeneous

• The solute must not undergo photochemical reactions

If the specifies conditions are not met then there are deviations from BL law, meaning the absorbance
is not proportional to the concentration of an analyte.

Deviations from BL law are classified into:

1. True deviations
2. Chemical deviations
3. Instrumental deviations

True deviations:

 Due to the disturbance of charge distribution:

At high concentrations, solute molecules can cause different charge distribution on their neighboring
species in the solution. Since UV-visible absorption is an electronic phenomenon, high concentrations
would possibly result in a shift in the absorption wavelength of the analyte. At times, even electrolyte
concentrations (such as those present in buffers) play an important role in altering the charge
distributions and affecting UV-visible absorbance. 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
Lambert law even at concentrations as low as 10μM.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

 Due to change in the refractive index:

High analyte concentrations can also possibly alter the refractive index (η) of the solution which in
turn could affect the absorbance obtained. If the addition of solute causes a significant change in the
refractive index of the solution.

Chemical deviations:

Chemical deviations occur due to chemical phenomenon involving the analyte molecules due to
association, dissociation and interaction with the solvent to produce a product with different
absorption characteristics. For 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. Since
UV-visible spectroscopy is an electron-related phenomenon, the absorption spectrum of the sample
changes with the change in pH of the solvent.

Instrumental deviations:
 Due to polychromatic radiation (Bandpass effect):

Beer-Lambert law is strictly followed when a monochromatic source of radiation exists. In

practice, however, it is common to use a polychromatic source of radiation with continuous

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distribution of wavelengths along with a filter or a grating unit (monochromators) to create a

monochromatic beam from this source. For example (see figure below), consider a molecule
having molar absorptivities ε’ and ε” at wavelengths λ’ and λ”. The absorbance (Am) for such a
species can be calculated as:


 Equation to calculate absorbance of a sample with polychromatic light source.
When the molar absorptivities are the same at both wavelengths (i.e. ε’ = ε”) , the relationship
between absorbance and concentration follows Beer-Lambert law to obtain a straight line.
However, as the difference between ε’ and ε” increases, the deviations from linearity also
increases.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

 Due to presence of stray radiation:

Stray radiation or scattered radiation is defined as radiation from the instrument that is outside
the nominal wavelength band selected. Usually the wavelength of the stray radiation is very
different from the wavelength band selected. It is known that radiation exiting from a
monochromator is often contaminated with minute quantities of scattered or stray radiation.
Usually, this radiation is due to reflection and scattering by the surfaces of lenses, mirrors,
gratings, filters and windows. If the analyte absorbs at the wavelength of the stray radiation, a
deviation from Beer-Lambert law is observed similar to the deviation due to polychromatic
radiation.

 Due to mismatched (uneven) cells or cuvettes:

If the cells holding the analyte and the blank solutions are having different path-lengths, or unequal
optical characteristics, it is obvious that there would be a deviation observed in Beer-Lambert law.
This is due mainly to the change in the path-length. In today’s instrument this problem is generally not
observed, however if it is present, appropriate linear regression to quantify this deviation must be
made.

CHROMOPHORES AND AUXOCHROMES:

Chromophore:

It is a covalently unsaturated group which is responsible for absorption of UV or visible radiation and
impart colour to the compound. Chromophores can be identified by unsaturated linkages within a
compound such as -C=C-, -N=N-. In unsaturated linkages, the π electrons are loosely bound. These
loosely bound electrons require less energy for electronic transition and the absorption bond occur in
near UV region.

Eg; ethylenic, acetylenie, carbonyl, acids, esters, nitro and nitrile group. C=C, C=O, NO2 etc

• There are two types of chromophore:

I. Independent chromophore: single chromophore is sufficient to import color to the compound e.g.
Azo group

II. Dependent chromophore: Where more than one chromophore is required to produce color. e.g.
acetone having one ketone group is colorless where as diacetyl having two ketone group is yellow.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Auxochrome:

An auxochrome represents a saturated group containing unshared electrons which when attached to
a chromophore changes both the intensity as well as the wavelength of absorption maximum.

Example: -OH, -NH2, Cl etc.

Types of shifts in UV-spectrum:

Bathochromic shift (Red shift):

bathochromic effect is a shift of an absorption maximum towards longer wavelength. OR shift of

lambda max (λmax) to longer side or less energy. It may be produced by a change of medium or by
the presence of an auxochrome or by the change of solvent.

The n-π* transition for carbonyl compounds experiences bathochromic shift when the polarity of
the solvent is decreased.

Hypsochromic shift (Blue shift):

Hypsochromic effect is a shift of an absorption maximum towards shorter wavelength. OR shift of

lambda max (λmax) to shorter side and higher energy. This may be caused by a change of medium,
solvent, substitution or removal of conjugation.

Example: the conjugation of lone pair of electrons on the nitrogen atom of aniline with the π bond
system of the benzene ring is removed on protonation. Aniline absorbs at 280nm, but in acid solution
the main peak is almost identical with that of benzene, being at 203nm due to blue shift. In case of
C6H5N+ H3 ions formed in acidic solution, the electron pair is no longer present and hence conjugation
is removed.

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Hyperchromic shift:

This effect leads to increased absorption intensity i.e., ε max increases. The introduction of
auxochrome usually increases the intensity of absorption.

Example: pyridine absorbs at 257 nm εmax = 2750 is shifted to 262nm εmax = 3560 for 2- methyl
pyridine. The introduction of auxochrome usually increases the intensity of absorption.

Hypochromic shift:

This effect leads to decreased absorption intensity i.e., ε max decreases. The introduction of a group
which distorts the geometry of the molecule causes hypochromic effect.

Example: biphenyl absorbs at 250nm, εmax = 19000 where 2-methyl biphenyl absorbs at 237nm, εmax
= 10250 It is due to distortion caused by methyl group in 2-methyl biphenyl

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Instrumentation and working of UV/Visible spectrophotometer:

Instrumentation

Radiation sources Monochromator Filter Sample cell Detector

UV Radiation Prism Grating

D2 discharge lamp Absorption filter Interference filter

H2 discharge lamp

Visible Radiation Photomultiplier Tube Barrier layer cell Photodiodes

Xenon discharge lamp

Tungsten lamp

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Radiation (Light)sources:

UV region:

a) D2 discharge lamp
b) H2 discharge lamp

Visible region:

a) Xenon discharge lamp

b) Tungsten lamp

Ideal requirements of source to be used in spectroscopy are as following:

 Needs to cover region from 200-800 nm with sufficient power and must be constant over the
time taken to make the measurement.
 The source should provide continuous radiation
 The source must generate beam with sufficient power for ready detection and measurement
 It should be stable. Either a fully charged high capacity battery or an A.C supply with a
transformer or voltage regulator is necessary to provide constant intensity.

Deuterium lamp:

This lamp consists of a silica or quartz envelope containing D2 filled under low pressure. The
mechanism by which continuous radiation is produced by this source involves the formation of an
excited molecule D2* by absorption of electrical energy. This species then dissociates to give 2
deuterium atoms plus an Ultraviolet photon.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

D2 + Ee - D2* - D’ + D’’ + hv (Photon)

Where, Ee is the electrical energy absorbed by the molecule.

When voltage is applied across electrodes, an arc is formed between heated, oxide coated filament
and a metal electrode, which gives away high intensity beam. The intensity of D2 discharge lamp is
about 3-5 times the intensity of H2 discharge lamp. The D2 lamp provides continuous spectrum in the
region of 160 to 375 nm. At longer wavelengths (> 360 nm), the lamp generates emission lines. These
lines are nuisance but they are useful for wavelength calibration.

H2 discharge lamp:

It is widely used UV radiation source, consists of 2 electrodes enclosed in a glass tube with a quartz
window. The glass tube is filled with H2 gas under relatively high pressure. When voltage is applied
across the electrodes, the hydrogen molecules are excited to higher energy level. As like D2 discharge
lamp, while returning to the ground state, the electrons emit radiation in the near UV region (180-350
nm). H2 discharge lamps are stable and robust but their intensity is significantly less than D2 lamp.

Xenon discharge lamp:

Xenon lamp is an electric discharge lamp which utilizes ionized xenon gas to produce an extremely
intense white light light for short duration. It consists of a sealed tube made up of glass or fuse quartz
which is filled with xenon gas at a pressure of 10-30 atmospheres. Additionally it contains a pair of
tungesten metal electrodes which can carry electric current to the gas. The electrodes are connected
to a capacitor which is charged at a high voltage.

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The xenon lamp generates significant amounts of UV radiation and shows spectral lines in the UV
region. Its intensity is higher than the hydrogen discharge lamp. The disadvantage of xenon lamp is
that the UV radiation released by it causes generation of ozone by ionization of the oxygen molecules.

Tungsten lamp:

It is simmilar to an electric bulb. Tungsten is the most widely used and most suitable material for lamp
filaments. It is used when polychromatic light is required. It consists of tungsten filament enclosed in
a vacuum bulb, which is heated electrically at a higher temperature to produce white light. It requires
a potential of 3-220 volts. It emits continuous radiations over wide wavelenght region (350 – 2500
nm). The energy obtained from a tungsten lamp can be used in wavelength regions above 375 nm. It
is available in different shapes and sizes suitable for fixing into different instruments.

Emission profile of a tungsten filament and a deuterium arc lamp

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Wavelength Selectors:

Classified into 2 categories:

1) Dispersive type
2) Non-dispersive type

Dispersive type further divided into:

1) Prism monochromators:
a. Refractive type
b. Reflective type
2) Grating monochromators:
a. Diffraction grating
b. Transmission grating

Non-dispersive type further divided into:

1) Absorption filter
2) Interference filter

Monochromator:

A monochromator is a device which converts a polychromatic beam of light into a monochromatic

beam. Basic components of monochromators are as follows:

a) Entrance slit: It defines the incoming beam of polychromatic light.

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b) Collimators: it collimates (make parallel) the radiations.

c) Dispersing device: it disperses the radiations with respect to component wavelength.
d) Exit slit: It selects the narrow band of dispersed spectrum for observation by detector.

Prism monochromators:

The prisms are usually made-up of glass, quartz or fused silica. They disperse the polychromatic light
falling onto it into its component rainbow colours according to their wavelength. Resolution depends
upon the size and refractive index of the prism. Prisms are usually used in inexpensive instruments.

Advantage: cost effective, wavelengths do not overlap.

Disadvantage: Significant portion of the radiation is absorbed reducing the intensity of radiation. Very
high bandpass (30-150 nm).

Refractive type prism monochromator:

Light from the radiation sources passes through the entrance slit and falls on collimator. The radiations
are collimated and dispersed into component wavelengths by the prism. Entrance slit selects any one
of the seven colours i.e. VIBGYOR. The required wavelength is selected either by rotating the prism or
by keeping the prism stationary and moving the exit slit. Refraction in the prism occurs as per snell’s
law.

Reflective type prism monochromator:

Its working is similar refractive type but a reflective surface is present on one side of the prism. The
dispersed radiation gets reflected & can be collected on the same side of the source of light.

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Grating monochromator:

Gratings are made up of glass, quartz and alkyl halides like KBr. They are highly efficient than prisms
in converting a polychromatic light into monochromatic light. They consist of densely arranged parallel
lines (grooves or rulings). A grating for UV visible region typically contain 300 to 2000 grooves/mm.
They give resolution of 0.1 nm.

Reflective-Diffraction (Echellette) grating:

It works on the principle of diffraction of light. It is most commonly used grating, where the grooved
surface is made up of glass with reflective coating (Al, Pt). The ray incident upon the grating gets
reinforced with the reflected ray and hence the resulted radiation has wavelength which is governed
by the equation
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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

nλ = d (sinβ + sinγ)

where, d= distance between 2 grooves, β= Incident angle, γ= diffraction angle and n = 1, 2, 3 positive
integers.

Constructive and deconstructive interference occurs because light travels different distances when
reflected from each grating.

Czerny turner type grating:

This is the reflection (diffraction) type of grating monochromator. Entrance slit that provides a narrow
optical image of the radiation source. A collimator (concave mirrors) that renders the radiation
emanating from the entrance slit parallel. A reflection (diffraction) grating disperses the light. A second
collimator re-forms the image of the entrance slit and focus it onto the exit slit. The exit slit to isolate
the desired spectral band by blocking all of the dispersed radiation except that in the desired range.

Transmission grating:

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Similar to diffraction grating, but refraction takes place instead of reflection. When the radiation is
transmitted through grating, it reinforces with the partially refracted radiation. Wavelength produced
is given by the equation

Where, n= positive integer or order number (1,2,3,4)

d= lines per cm.

θ = Angle of diffraction.

Filters:

Filters operate by absorbing all but a restricted band of radiation from a radiation source. 2 types of
filters are used in spectroscopy

1) Absorption filters
2) Interference filters

Absorption filters:

These filters, which are generally less expensive and more rugged than interference filters, are limited
in use to visible region. This type of filter usually consists of a colored glass plate that removes part of

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the incident radiation by absorption. Absorption filters have effective bandwidth that range from 30
to 250 nm.

Selection of absorption filter is done according to the following procedure:

1) A circle (Filter wheel) is drawn which is divided into six equal parts (as shown in above fig.).
2) Each division is given a colour name i.e. Violet, Blue, Yellow, Orange and red either in clockwise
or anticlockwise direction.
3) The desired colour of the filter will be complementary to the colour of the solution

Advantages:

1) Absorption filters are easy to construct and manufacture.

2) The selection of filter is easy.
3) The cost is appreciably less.

Disadvantage:

1) High band pass makes the absorption filters less accurate.

2) Intensity of radiation decreases due to absorption by filters. Most of the absorption filters
have a transmittance of 1% or less at their band peaks.

Interference filters (Fabry-Perot filters):

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Interference filters are used with UV and Visible radiation. They consist of a middle layer of a dielectric
material such as CaF2, MgF2, SiO. The dielectric film is sandwiched between two semi-reflective silver
films. The thickness of dielectric film can be of 1st order (1λ/2) or 2nd order (2λ/2). Interference filters
have a band pass of 10-15nm and peak transmittance of 40-60%.

When a beam of light falls on an interference filter, a part of it is reflected back while the remaining
gets transmitted by the metallic layer (silver film). The reflected rays undergo interference
simultaneously, thus giving monochromatic radiation of wavelength, which is expressed by the
following equation-

λ = 2εt /n

where,

λ= Wavelength of monochromatic radiation.

Ε= dielectric constant of the film.

t = thickness of the layer.

n= Order number (1,2,3 etc)

Advantages:

1) Interference filters give pure radiation without any overlapping.

2) These are more accurate than absorption filters.
3) They show grater transmittance.
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Disadvantage:
1) Because of the band pass, the resolution is not high as that achieved by prism or grating
monochromators.

Sample cells/Sample holders/Cuvettes:

sample cells (cuvettes) are used to hold sample solutions. Their shape and material of construction
varies depending upon the instrument and nature of the sample being analysed. They may be
rectangular or cylindrical in shape. The thickness (path length) of the cell is normally 1cm, however

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cells with long path-lengths upto 10cm or short path-length upto 1-2 mm are also available. Before
taking the measurement, the sample cell must be thoroughly cleaned to avoid any contamination. The
level of the sample solution must be upto the mark etched on its surface or in absence of any such
indication, at least 3/4th volume of the total cuvette. Materials used to construct sample cells are
quartz, Alkali metal halide and glass.

An ideal sample cell should fulfil the following conditions:

1) The material used in the construction of the sample cell should not chemically react with the
solvent.
2) The material should be optically UV/Visible transparent i.e. it must not absorb the UV
radiation by itself.
3) It should have uniform thickness.

Detectors used in UV spectroscopy:

To obtain spectroscopic information, the radiant power transmitted or emitted must be detected in
some manner and converted into a measurable quantity. A detector is a device that indicates the
existence of some physical phenomenon. Familiar example of detectors includes the mercury level in
a thermometer (indicating temperature). The human eye is also a detector, it converts visible radiation
into an electric signal that is passed to the brain via a chain of neurones in the optic nerve and produce
vision.

Invariably in modern instruments, the information of interest is encoded and processed as an electric
signal. The term transducer is used to indicate the type of detector that converts quantities, such as
light intensity, pH, mass and temperature, into electrical signals that can be subsequently amplified,
manipulated, and finally converted into numbers proportional to the magnitude of the original
quantity. There are 2 general types of detectors, one type responds to photons (photon detectors),
the other to heat (thermal detectors). All detectors used in UV spectroscopy are photon detectors.

Properties of ideal detector:

 The ideal detector for electromagnetic radiation responds rapidly to low levels of radiant
energy over a broad wavelength range.
 It produces an electrical signal that is easily amplified and has a low electrical noise level.
 The electrical produced by the detector be directly proportional to the radiant power P of the
beam as shown in below equation

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

G = KP + K’

Where, G is the electrical response of the detector in units of current, voltage or charge. The
proportionality constant K measures the sensitivity of the detector. Many detectors exhibit a small
constant response K’, known as dark current, ever when no radiation strikes their surface.

 Ideal detector must have low dark current (explained above).

 It must have high signal to noise ratio.

Photovoltaic cell (Barrier layer cell):

• It consists of semiconductor, such as selenium, which is deposited on a strong metal base such
as iron.

• A very thin layer of silver or gold sputtered over the surface of the semiconductor to act as a
second collector electrode

Working:

- light of sufficiently high energy passes through the thin transparent silver layer and hits
selenium causing electrons to be released which move across barrier toward silver layer and
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collected at iron layer to neutralize selenium layer. Current produced is proportional to

photons hitting surface. Maximum response at 550 nm.

Advantage:

 cheap, rugged.

 no external power source required.

 good for portable instruments.

Disadvantage:

 Not very sensitive,

 shows fatigue effects (decrease in response with continued illumination)

 Amplification of the signal is not possible.

Vacuum Phototube/ PhotoCell /Photoemissive cell:

The response of phototube is based on the photoelectric effect. As shown in above figure,
phototube consists of a semi cylindrical photocathode and a wire anode sealed inside an evacuated
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transparent glass or quartz envelope. The concave surface of the cathode supports a layer of photo-
emissive material, such as an alkali metal or a metal oxide, that emits electrons when irradiated with
light of the appropriate energy.

Working:

When a voltage is applied across the electrodes, the emitted photoelectrons are attracted to the
positively charged wire anode. A photocurrent that produces after the completing circuit is then
easily amplified and measured. The number of photoelectrons ejected from the photocathode per
unit time is directly proportional to the radiant power of the beam striking the surface.

Advantages:

• Composite coatings such as Ce/CeO/AgO can be used to increase the sensitivity.

• Used for UV/Visible region.

• The signals can also be amplified.

• Better sensitivity compared to photovoltoic cells hence widely used.

Limitations:

 Not as sensitive as Photo Multiplier Tube (PMT).

Photo Multiplier Tube (PMT):

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

PMT is similar in construction to the phototubes but is significantly more sensitive. Its photocathode
is similar to that of the phototube, with electrons being emitted on exposure to radiation. In place of
a single wire anode, however, the PMT has a series of electrodes called dynodes, as shown in the
figure.

Working:

The electrons emitted from the cathode are accelerated towards the first dynode, which is maintained
90 to 100V positive with respect to cathode. Each accelerated photoelectron that strikes the dynode
surface produces several electrons, called secondary electrons, that are then accelerated to dynode
2, which is held 90 to 100 V more positive than dynode 1. Again electron amplification results. By the
time this process has been repeated at each of the dynode, 105 to 107 electrons have been produced
for each incident photon. This cascade of electrons is finally collected at the anode to provide an
average current that is further amplified electronically and measured.

Advantages:

 very sensitive to low intensity

 Very fast response.

 Even 200 times weaker signals can be processed. Hence useful for fluorescence
measurements.

Limitations:

 Need high voltage power supply.

 PMT should be shielded from stray light in order to have accurate results.

Silicon Diode or Photodiode detectors:

Photodiode: a type of diode that allows current to flow only when light strikes the diode. P type:
positive mobile charge carrier. n Type: negative mobile charge carrier. Photodiodes are constructed
from a layer of p-type silicon on a n-type silicon substrate, creating a p-n junction diode, which is
overlaid with a protective a SiO2layer.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Working:

A reverse bias is applied, drawing electrons and holes away from the junction. There is a depletion
region at each junction, in which there are few electrons and holes. The junction acts as a capacitor,
with charge stored on either side of the depletion region. At the start of each measurement the diode
is fully charged. When radiation hits the semiconductor, free electrons and holes are created and
migrate to regions of opposite charge resulting in increased conductivity which is easily measured and
is directly proportional to the radiant power.

A silicon-diode detector is more sensitive than a simple vacuum phototube but less sensitive that PMT.

Photo Diode Array detectors (PDA):

 A linear photodiode array comprises many small silicon photodiodes formed on a single silicon
chip. There can be between 64 to 4096 sensor elements on a chip, the most common being
1024 photodiodes.
 Advanced technology led to the development and implementation of photodiode detectors,
which when placed closely spaced linear arrays, after rapid and accurate spectrum analysis.

 The primary advantage of linear array detectors is that they permit the simultaneous analysis
of an entire spectrum over a period of a few sec.

 This advantage when performing kinetic studies involving rapidly changing events

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

 It increased wavelength resolution. Precision matching of slit sizes to individual photodiodes

and focusing the spectrum in a focal plane can enhance wavelength resolving power to 1-2nm

Types of UV-Visible spectrophotometers:

 Single beam spectrophotometer

 Double beam spectrophotometer

Single beam spectrophotometer:

In a single beam UV-visible spectrophotometer, light from the radiation source after passing through
a monochromator enters the sample cell containing the sample solution. A part of incident light (I0) is
absorbed by the sample and remaining gets transmitted (It). the transmitted light strikes the detector
and produces electrical signals. the absorbance readings of both the standard and unknown solutions
are recorded after adjusting the instrument to 100% transmittance with a blank solution each time
whenever the wavelength is changed.

Advantages:

 Simple in construction.
 Easy to operate.
 Economical.

Disadvantage:

 Any fluctuations in the intensity of the radiation sources affects the absorbance readings.
 It requires adjustment of transmittance to 100% whenever wavelength is changed. Hence, a
continuous spectrum is not obtained.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Double beam spectrophotometer:

Double beam spectrophotometer (DBS) allows direct measurement of the ratio of intensities of the
sample and reference beams. The design of a double beam spectrophotometer is similar to single
beam spectrophotometer except that it contains a beam splitter (selector mirror) or chopper. In DBS,
the radiations from source are allowed to pass through the entrance slit into the monochromator.
Monochromator selects the required wavelength of light which is then passed through the exit slit
and received by rapidly rotating beam splitter or chopper (in the fig. selector mirror). The chopper
splits the monochromatic beam of light into two beams of equal intensities. One beam is passes
through the sample cell and the other through the reference cell. After passing through reference and
sample cells, the transmitted beams reach the detectors and produce a pulsating current which is
proportional to the intensities of incident (I0) and transmitted light (It). The detectors are connected
to an amplifier and readout device which gives the final results in terms of absorbance (-log It/I0) or
transmittance (It/I0) by combining the output by using algorithm.

Advantages:

 It facilitates rapid scanning over wide wavelength region.

 Fluctuations due to radiation source are minimal.
 DBS makes automatic compensation for variations in the wavelength of the incident light.
 It does not require adjustment of the transmittance to 100 % at each wavelength.
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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Disadvantages:

 Construction is complicated.
 Instrument is expensive compared to Single beam spectrophotometer.

Applications of UV spectroscopy:

1. Qualitative applications:

Detection of extent of conjugation:

Conjugation of a double bond with another double bond or C=O group causes the absorption band to
shift to longer wavelength with greater intensity.

Example:

CH2OH
CH2OH

Vitamin A1 Lambda Max 326 nm Vitamin A 2Lambda Max 351 nm

Qualitative detection of benzene within compound:

The presence of benzene within structure can be confirmed by the absorbance at wavelengths 243,
250 and 245 nm.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Identification of tautomeric forms:

2-pyridone and 2-hydroxy pyridine are tautomeric forms of each other (like phenol red). 2-hydroxy
pyridine shows absorption at 325 nm, while 2-pyridone has exocyclic double bond which
bathochromically shift the absorption maxima to 330 nm.

Identification of configurations of geometrical isomers:

Trans isomer absorbs at longer wavelength with greater intensity than the cis isomer.

Example: Cis-stilbene has λmax at 280 nm, while trans-stilbene absorbs at 300 nm.

Quantitative applications:

1. Single component analysis

 Single point Assay.
 Multipoint Assay.
2. Multicomponent analysis:
 Simultaneous equation method.
 Absorbance ratio method.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

Single component analysis:

The analyte can be quantified by measuring the absorbance and converting it into concentration and
this can be done by 2 ways. First, if absorptivity value of the analyte under consideration is given eg.
715 for paracetamol then the absorbance can directly be converted into concentration using BL law.
this method is called as Single point assay, where only single absorbance is needed for quantification.
Second, if reference standard of the analyte is provided then calibration curve can be prepared by
measuring the absorbance of appropriately prepared calibration standards. The absorbance of
unknown solution can be extrapolated to calculate the concentration with the help of calibration
curve. This method is called as multipoint assay. Eg. Colorimetric estimation of sulpha drug.

Multicomponent analysis:

The spectrophotometric assay of drugs rarely involves the measurement of absorbance of samples
containing only one absorbing component. The pharmaceutical analyst frequently encounters the
situation where the concentration of more than one component is required to be analysed in the
sample known to contain other absorbing substances which potentially interfere in the assay. In such
a scenario, multicomponent analysis techniques come into picture. The basic assumption behind the
all multicomponents analysis methods is that at all wavelengths the absorbance of a solution is the
sum of the absorbances of the individual component.

Simultaneous equation method (Vierodt’s method):

Following conditions must be fulfilled to use this method:

 λmax of two drugs should be reasonably dissimilar.

 Two components should not interact chemically.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

 If a sample contains two absorbing drugs (X and Y) then each of them must absorb at the λmax
of other (i.e λ1 and λ2).

The information required is:

a) The absorptivities of X at λ1 and λ2, ax1 and ax2 respectively

b) The absorptivities of Y at λ1 and λ2, ay1 and ay2 respectively
c) The absorbances of sufficiently diluted samples at λ1 and λ2, A1 and A2 respectively.

Let, cx and cy be the concentrations of X and Y respectively in the diluted samples.

Two equations can be constructed based on the fact that at λ1 and λ2 the absorbance of the mixture
is the sum of the individual absorbances of X and Y.

At λ1,

A1 = ax1.b.cx + ay1.b.cy (1)

At λ2,

A2 = ax2.b.cx + ay2.b.cy (2)

For measurements in 1cm cell, b=1.

Rearrange the equation (2),

Cy = A2 – ax2.cx/ay2 (3)

In above equation there are two unknown variables i.e. cy and cx. So let us find out the term cx.

Multiply the equation (1) by the term ay2

A1.ay2 = ax1.ay2.cx + ay1.ay2.cy (4)

Multiply the equation (2) by the term ay1,

A2.ay1 = ax2.ay1.cx + ay1.ay2.cy (5)

Now, subtract equation (5) from (4),

A2.ay1 – A1.ay2 = ax2.ay1.cx – ax1.ay2.cx

Taking cx common from RHS,

(A2.ay1 – A1.ay2) = (ax2.ay1 – ax1.ay2) .cx

Cx = (A2.ay1 – A1.ay2)/ (ax2.ay1 – ax1.ay2) (6)

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

By substituting the absorptivity and absorbances values, one can calculate cx.

By substituting the calculated cx value in equation (3), cy can be found out.

Absorbance ratio method:

• It is the modified method of simultaneous equation procedure

• It depends on the property that, for a substance which obeys Beer’s law at all wavelengths,
the ratio of absorbance's at any two wavelengths is a constant value independent of
concentration or path length.

eg: two different dilutions of the same substance give the same absorbance ratio A1 /A2, in the USP
this ratio is referred to as a Q value.

The BP also uses a ratio of absorbance's at specified wavelengths in certain confirmatory test of
identity.

eg: cynocobalmine exhibits three λmax , at 278nm, 361 nm and 550nm. The ratio A361 /A550 is required
to be 3.30 ± 0.15and the A361 / A278 to be 1.79 ± 0.09

In the quantitative assay of two components in admixture by the absorbance ratio method,
absorbances are measured at two wavelengths. One being the λmax of one of the components λ2 and
the other being the wave length of equal absorptivity of the two components λ1 i.e an iso-absorptive
point. Two equations are constructed as described above for the method of simultaneous equations.
And further solved to find out the individual concentrations.

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Dept. of Pharmaceutical Quality Assurance, MCOPS Swapnil Dengale

References:

1. Beckett AH, Stenlake JB. Practical Pharmaceutical Chemistry: Part II Fourth Edition [Internet].
Bloomsbury Academic; 1988. (Practical Pharmaceutical Chemistry). Available from:
https://books.google.co.in/books?id=Up3L2dAI7k8C

2. Kalsi PS. Spectroscopy of Organic Compounds [Internet]. New Age International Pvt; 2016.
Available from: https://books.google.co.in/books?id=-gTdjwEACAAJ

3. Kemp W. Organic Spectroscopy [Internet]. Palgrave Macmillan; 1991. Available from:

https://books.google.co.in/books?id=sO5zjwEACAAJ

4. Skoog DA, West DM, Holler FJ, Crouch SR. Fundamentals of Analytical Chemistry [Internet].
Cengage Learning; 2013. Available from:
https://books.google.co.in/books?id=0bBWPgAACAAJ

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