Instrumental methods of Chemical analysis

1. UV-VISIBLE
SPECTROPHOTOMETRY
Presented By-
ROHIT
M.Pharmacy
(Pharmaceutics)

2. PharmaceuticalAnalysis
Pharmaceutical Industry
Bulk -API
Formulation – Various Dosage Forms
Qualitative analysis
Quantitative analysis

3. Spectral methods Light absorption or
Emission
UV spectroscopy, IR
spectroscopy, Flourimetry
Chromatographic methods Adsorption/Partition Paper chromatography,
TLC and HPLC
Electro analytical
techniques
Electrochemical property Electrophoresis (paper, gel
and capillary)
Radioactive methods Radio immune assay,
ELISA
Thermal methods Physical characteristics Differential thermal
analysis (DTA),
Differential scanning
calorimetry (DSC), Thermo
gravimetric analysis (TGA)
Miscellaneous methods Titrimetric methods/
volumetric methods

4. SPECTROSCOPY
 It is a branch of Science that deals with the
study of interaction of matter with light.
Or
 It is the branch of science that deals with the
study of interaction of electromagnetic
radiation with matter.

5. Introduction to Spectroscopy
Spectroscopy is the measurement and interpretation of
Electromagnetic radiation (EMR) absorbed or emitted when
the molecules or atoms of a sample move from one energy
state to another energy state.
This change may be from ground state to excited state or excited
state to ground state.
At ground state, the energy of a molecule is the sum of total of
Rotational, vibrational and or electronic energies.
Spectroscopy measures the changes in the Rotational, vibrational
and or electronic energies.
EMR is made up of discrete particles called Photons. They can
also travel in Vacuum.
E= hv
E= Energy
h= Planks constant, v= Frequency of radiation

9. Why we use Spectroscopy?
-Detection of Functional groups
-Detection of Impurities
-Qualitative analysis
-Quantitative analysis
-Single component without chromophore
-Drugs with chromophoric agent
-Used to detect conjugation of the compounds

10. UV-Visible Spectroscopy
Spectroscopy is concerned with the study of
absorption of UV radiation which ranges form
200-400nm.
Compounds which are colored absorb radiation
from 400-800nm.
Compounds which are colorless absorb radiation in
UV range.
It involves the absorption of UV-Visible light by a
molecule, causing the electrons of the molecule to
undergo transition from ground state to the
excited state.

18. Ɛ = E1cm
1% X Molecular weight
10
1cmE 1% means absorbance of 1% w/v solution, using
path length of 1cm.
1cmE 1% at a wavelength is a constant value for each
drug and can be seen in Pharmacopoeias.

19. Absorption Curve
Absorption curve or absorption spectrum of a molecule in
the UV-Visible region is a plot between energy absorbed
by the molecule or absorbance (on Y-axis) and
wavelength (on X-axis).
Fig: Absorption spectra

20. From the Absorption curve, the wavelength at which maximum
absorption of radiation takes place is called λmax (lambda
max)
The λmax of every substance is specific and unique, which is
useful in identifying the substance.
λmax is not affected by concentration of the substance, however
absorbance increases with increase in concentration of the
absorbing molecules.

21. Quantitative analysis can be carried out by plotting a curve between
concentration on X-axis and absorbance on Y-axis which is called asthe
calibration or standard curve.
This curve gives a straight line showing a direct relationship between the
absorbance and concentration.
Thus, it obeys Beer’s Law. The concentration of a substance in the given
sample solution can be determined by extrapolation or intrapolation of the
curve.

22. The degree of absorption of the UV-Visible light by the
substance and the wavelength at which absorption occurs are
measured and recorded by the instrument called UV-Visible
spectrophotometer. They operate within the wavelength range
of 800-200nm.

23. If the curve between absorbance and concentration is concave
upwards, it is termed as positive deviation and if the curve is
concave downwards it is a negative deviation.
Positive deviation: occurs when a small change in concentration
leads to a large change in the absorbance.
Negative deviation: occurs when a large change in concentration
produces small change in the absorbance.

24. Deviations from Beer’s law are classified as
1. Real Deviations
2. Chemical Deviations
3. Instrumental Deviations
Real Deviations: Real deviations are related to the concentration
of the absorbing substance.
Beers law holds good only for dilute solutions having
concentration in the range of 10-6 to 10-7 M.
At higher concentrations, molecules of the absorbing species
undergo collisions and interact with each other. Hence, at
higher concentrations, molecules will not absorb radiation in
the same manner as in dilute solutions.

25. Chemical Deviations
a) Chemical deviations arise if the absorbing species undergoes
chemical changes such as association, complex formation,
dissociation, hydrogen bonding, hydrolysis, ionization.
Ex: Benzyl alcohol in chloroform exists as a polymer. Upon
dilution it dissociates into its monomers.
The polymeric form shows positive deviation
The monomeric form shows negative deviation

26. b) Presence of impurities in the solution that fluoresence or
absorb the radiations at the required wavelength also cause
deviation from Beer’s law.
c) Reaction of the absorbing species with each other or with the
solvent results in the formation of new species. These show
different absorbance values which results in deviations.
Chemical deviations can be corrected by using appropriate
wavelength, buffers and selecting suitable solvents.

28. 3. Instrumental Deviations
a) Beer’s law is obeyed only when a monochromatic radiation is
used. Polychromatic radiations lead to negative deviations
from Beer’s law.
b) Any fluctuations in the intensity of the radiation source may
also cause deviations from Beer’s law.
c) Changes in the sensitivity of the detector employed and
defects in the amplication of the radiation also cause
deviations.
d) Improper slit width also contributes towards deviation from
Beer’s law. Improper slit width allows the stray radiations to
reach the detector.

30. These stray radiations are absorbed by the impurities present in thesample
solution and lead to changes in the absorbance value of the sample.
Applications of Beer-Lamberts Law
1. It is used in determining the concentration of unknown solution by
comparing with a solution of known concentration (standard solution)
using colorimeter or spectrophotometer.
Conc. of unknown solution= Absorbance of unknown solution x Conc. of standard solution
Absorbance of Standard solution
2. It is used in quantitative analysis of both single and multicomponent
analysis.
3. It is used for estimating the concentration of chlorophyll in leafcells.

31. Limitations of Beer’s Law:
It is applicable only at low concentrations.
It is not applicable to suspensions.
It is not applicable to coagulated particles which
cause scattering of radiations, which may either
increase or decrease absorbance values
Only monochromatic light should be used.

32. In both UV as well as Visible spectroscopy, only the valence
electrons absorb the energy, thereby the molecule undergoes
transition from Ground state to excited state. This absorption
is characteristic and depends on the nature of electrons
present.

33. The types of electrons present in any molecule may be
conveniently classified as σ, Π and n bonds.
‘σ’ electrons: These are the ones present in saturated
compounds. Such electrons do not absorb near UV,
but absorb vacuum UV radiation (<200nm)
Π electrons: These electrons are present in unsaturated
compounds ex: double or triple bonds ex: c꞊c
‘n’ electrons: These are the non bonded electrons
which are not involved in any bonding ex: lone pair
of electrons like S, O, N and Halogens.

34. A molecule has either n, Π or σ or a combination of these
electrons.
These bonding ( Π or σ ) and non-bonding (n) electrons absorb
the characteristic radiation and undergoes transition from
ground state to excited state.
By the characteristic absorption peaks, the nature of the electrons
present and hence the molecular structure can be elucidated.

35. Various Electronic transitions are

36. Sigma electron (σ electron): Sigma (σ) bonds are the single
bonds in molecules. Also, recall that a double bond is
composed of 1 σ bond and 1 π bond and a triple bond is
composed of 1 σ bond and 2 π bonds. This means in the given
molecule: There are 5 σ bonds and 1 π bond in CH2=CH2.

37. Pi electrons

40. Of all the electronic transitions, this type of transition requires
highest energy.
This is observed with Saturated compounds (especially
hydrocarbons).
The peaks do not appear in UV region, but occur in vacuum UV
region i.e., 125-135nm.
Some of the examples with such transition are :
Methane -122nm
Ethane- 135nm
Cyclopropane-190nm

41. MethaneCH4

42. CyclopropaneC3H6

43. This type of transition gives rise to B,E and K bands.
B bands (Benzenoid bands)-Aromatic and hetero aromatic groups
E bands (Ethylenic bands)- Aromaticcompounds
K bands- Conjugated bonds
Extended conjugation (due to addition of more double/triple
bonds) and alkyl gps shifts the λmax towards longer
wavelength (Bathochromic shift)
Trans isomers absorbs at longer wavelength with more intensity
than cis isomers (Hyperchromic shift)

44. Compounds containing multiple bonds like alkenes, alkynes,
carbonyls, aromatic compounds undergo these type of
transitions.
Ex: Alkenes generally absorb in the region 170-205nm.

45. This transition occurs in Saturated compounds with hetero
atoms like S, O, N or halogens.
These compounds usually detected at wavelength 180-250nm.
Compound λmax
Methylene chloride 173nm
Methanol 203nm
Ether 215nm
Trimethylamine 227nm
Chloroform 237nm

46. Of all the transitions this transition requires the lowest
energy.
The peaks due to this transition are called as R-bands.
Compounds containing double bonds involving hetero atoms
like S,O,N undergo such type of transitions ex aldehydes
and ketones
These are detected at 270nm-350 nm
These all transitions are used in structural elucidation.

47. Terms used in UV-Visible Spectroscopy
Chromophore
Chromophore or chromophoric group is a group or
part of a molecule responsible for characteristic
absorption at a wavelength.
Chromophores are covalently unsaturated.

49. Auxochrome
These are co-ordinately saturated/ unsaturated
groups.
They do not have any characteristic absorption on
their own but can modify or enhance the
absorbing properties of chromophore.

51. Shift in λmax towards longer wavelength is called Bathochromic
shift.
It is also called as Red shift.
It can occur due to increase in conjugation (addition of double or
triple bonds), addition of alkyl substituents in the molecule.
The effect is due to presence of auxochrome or by change of
solvent.
This arises due to substitution of Functional gps like: OH, 10, 20,
30 amino groups or solvent effect.

52. shorter wavelength is calledShift in λmax towards
Hypsochromic shift.
Also called as Blue shift
It may occur due to removal of double or triple bonds by
saturation, dealkylation and also due to change of solvent.

59. Solvent Effect
Polarity of the solvent has a great influence on the
position of λmax as well as on the intensity of absorption
maximum.
λmax for polar compounds usually shifts with a change
in the polarity of the solvent.
However, λmax of non-polar compounds remains same
in polar as well as non-polar solvents.
For ex: absorption transitions of polar bonds like C=O
(but not ethylene) are effected by the solvent polarity.

60. As the polarity of the solvent is increased, π – π*
transition bands undergo bathochromic shift.
This is because, the π* state is more polar than the π
state hence stabilization is greater in excited state than
the ground state in polar solvents.
The excited state is stable due to the formation of
hydrogen bonds with the polar solvents. As the stability
is increased, the energy of the excited state decreases
which eventually causes Bathochromic shift.

61. In case of n – π* transition, by increasing the polarity of
the solvent, the absorption maximum is shifted towards
shorter wavelength because ground state with two n
electrons is more stabilized than the excited state with
only 1 n electron.
In n - π* transition, the ground state is more polar than
the excited state and hence is more stabilized because of
greater extent of hydrogen bonding with the polar
solvents.
Similarly with increasing polarity, the n – σ* bands are
displaced towards the shorter wavelength i.e., blue shift.

62. Therefore, increase in polarity of the solvent shifts the n – π* and
n–σ* absorption bands towards shorter wavelengths
(hypsochromic or blue shift) and π – π* absorption bands towards
longer wavelengths (bathochromic or red shift).
Examples of various solvents used in UV-Visible spectroscopy
are:
 Water
 Ethanol
 n-hexane
 Acetone
 Ether
 Dioxane
 Chloroform
 Benzene etc.

63. Chromogenic agent
Chromogen or chromogenic agents are those which
is capable of forming a chromophore or color by
complexation, chemical reaction, ionisation etc.
Ex: Ferric chloride when added to salicylic acid
produces a violet colour with λmax at 530 nm.
So ferric chloride reagent is called as chromogen.

64. Isobestic point
It is a specific wavelength at which the molar absorption
coefficient λmax of two or more chemical substances in
equilibrium.

65. Instrumentation of UV-Visible spectroscopy
The spectrophotometers used in UV-Visible spectroscopy
measure the ratio of the intensity of light transmitted (It) through
a sample and the intensity of incident light (Io).
The components of UV and visible spectrophotometer are
identical except that they differ in their radiation sources and
material used in Sample holders.
The radiation source used in visible region is tungsten lamp
whereas in UV region :deuterium lamp, hydrogen discharge
lamp, mercury arc and xenon discharge lamp are commonly
used.

67. Components of UV-Visiblespectrophotometer
1. Source of Light/ Radiation source
2. Filters and Monochromators
3. Sample Holder/container to hold sample/Cuvvettes
4. Detectors
5. Suitable amplifier and readout device

68. Components of UV-Visible Spectrophotometer
Components UV region Visible region
Radiation sources Deuterium discharge lamp, Tungsten lamp
Hydrogen discharge lamp,
Mercury arc,
Xenon discharge lamp
Monochromators Prism Monochromators Prism Monochromators
Grating Monochromators Grating Monochromators
Filters Absorption filters Absorption filters
Interference filters Interference filters
Sample cells Made of Quartz or fused Made up of Glass
silica
Detectors Photovoltaic cell Photovoltaic cell
Phototubes Phototubes
Photomultiplier tubes Photomultiplier tubes

69. Source of Light
A UV spectrum ranges from 200-400nm andVisible
spectrum ranges from 400-800nm.
Any lamp source which can give adequate intensity of
radiation can be used.
Ideal characteristics of Light
It should provide continuous radiation.
It should provide adequate intensity.
It should be stable and free from fluctuations.
Should not show exhaustion on continuous usage.

70. For visible Radiation
1.Tungsten lamp: is used mostly in colorimeter and
spectrophotometers.
The lamp consists of a tungsten filament in a vacuum
bulb and filled with an inert gas.
2.Carbon arc lamp: For a very high
intensity, carbon arc lamp is used.
It provides an entire
Range of visible spectrum.

71. ForUVsource of radiation
1.Hydrogen discharge lamp:
It is more stable, robust and widely used.
It gives radiation from 120-350nm.
The lamp consists of hydrogen gas with high
pressure.
2.Deuterium lamp:
It is similar to hydrogen discharge lamp, but it is
filled with deuterium instead of hydrogen.
Offers more intensity than other light sources.
This is most widely used, but is little expensive.

72. 3.Xenon discharge lamp:
In this lamp, xenon at 10-30 atmospheric pressure is
filled in and has two tungsten electrodes.
The intensity is greater than hydrogen discharge
lamp.
4.Mercury arc lamp:
This lamp contains mercury vapour stored under high
pressure and offers bands which are sharp.
 Since the spectrum is not continuous, it is not widely
used.

73. Filters and Monochromators
The source of light gives radiations from 200nm to 800nm.
This is polychromatic in nature (light of several wavelengths)
In colorimeter or spectophotometer, we require only
monochromatic light (light of single wavelength).
Hence a filter or monochromator is used which converts
polychromatic light into monochromatic light.

74. Filters are of two kinds:
1. Absorption Filters
2. Interference Filters
Monochromators are of two types:
1. Prism type (Dispersive type or Littow type)
2. Grating type (Diffraction grating &
Transmission grating)

75. 1. Filters
i) Absorption filters
These filters are made up of glass, coated with pigments
or they are made up of dyed gelatin.
They absorb the unwanted radiation and transmit the rest
of the radiation which is required for colorimetry.

76. These filters can be selected according to the
procedure given below:
1. Draw a filter wheel (circle with 6 parts).
2. Write the colours (VIBGYOR) in clockwise and
anticlockwise manner omitting Indigo.
3. If the colour of the solution is Red, we have to use
Green filter and if the colour of the solution is
Green, we have to use Red Filter. (The colour of
the filter is opposite to the colour of the solution
i.e., complimentary in nature).
4. 4. Similarly, we can select the required filter in a
colorimeter, based upon the colour of the solution.

78. Merits:
 Simple in construction
 Low Cost
 Selection of filter is easy.
Demerits:
 Less accurate since band pass is more (±30nm) i.e., if we have
to measure at 500nm, radiation ranging from 470nm to 530nm
falls on the sample.
 Intensity of radiation becomes less due to absorption by filters.

79. ii) Interference Filters
 This filter is also known as Fabry-Perot filter.
 It has dielectric spacer film made up of CaF2, MgF2, or Sio2
between two parallel reflecting silver films.
 The thickness of dielectric spacer film can be 1/2λ (1st order),
2λ/2 (2nd order), 3λ/2 (3rd order). Etc.
 The mechanism is, the radiation reflected by the 2nd film and
the incoming radiation undergoes Constructive interference to
give a monochromatic radiation, which is governed by the
following equation.
λ= 2դb/m
Where, λ= wavelength of light obtained
դ= dielectric constant of layer material
b= layer thickness
m= order no.

81. Band pass is 10-15nm (i.e., if we select 500nm, the obtained
radiation ranges from 490-510nm).
Maximum transmission is 40%.
Merits
 Inexpensive
 Lower band pass when compared to absorption filters and
hence more accurate.
 Use of additional filter cuts off undesired wavelengths.
Demerits
Peak transmission is low, and becomes so when additional
filters are used to cut off undesired wavelength.
The band pass is only 10-15nm and hence higher resolution
obtained with monochromators or gratings cannot be achieved.

82. b. Monochromators
A monochromator is a device which converts a polychromatic
beam of light into a monochromatic beam.
It consists of the following parts:
Entrance slit: It passes the incoming beam of polychromatic light
into a narrow beam.
Collimator1: It collimates or makes parallel the radiations coming
from the entrance slit.
Prism / Grating: It disperses the radiations with respect to the
component wavelengths.
Collimator2: It reforms the images of the entrance slit.
Exit slit: It selects a narrow band of dispersed spectrum for
observation by the detector.

85. Prism Monochromator
These are usually made up of glass, quartz, or fused
silica.
They disperse the polychromatic light falling on them
into its individual rainbow colours according to their
wavelengths.
They are commonly used in inexpensive instruments.
The band pass is lower than that of the filters and hence
it has better resolution.

86. Prism monochromators are of 2 types:
1. Refractive type 2. Reflective type
Refractive type: Light from the radiation source /
source of light falls on a collimator.
The parallel radiations from collimator are dispersed
into different colours or wavelengths, and by using
another collimator, the images of the entrance slit are
reformed.

87. The reformed ones will be either Violet, Indigo, Blue, Green,
Yellow, Orange or Red.
The required radiation on exit slit can be selected by rotating the
prism or by keeping the prism stationary and moving the exit slit.

88. 2. Reflective type (Littrow prism/ Littrow type mounting)
Its working is similar to refractive prisms.
It consists of a reflective surface on one side so that light does
not pass through the prism on the other side.
The dispersed radiations are reflected and collected on the same
side as the source of light radiation falls.

89. Grating Monochromators
Gratings are made up of glass, quartz or alkyl halides like KBr
and NaBr.
Back surface of the gratings are coated with aluminium to make
them reflective.
These are highly effective than prisms in converting a
polychromatic light into monochromatic light.
They consist of densely arranged parallel lines or grooves.
The no. of grooves per mm in a grating monochromator vary
depending upon the type of spectrophotometer used.
They give a resolution of ±1nm.
Grating monochromators are of two types:
1. Diffraction gratings
2. Transmission Gratings

91. Diffraction gratings: These are used when polychromatic light is
to be separated with high resolution.
It works on the Mechanism of reinforcement (strengthening).
The incident rays upon the grating gets reinforced with the
reflected rays, resulting in a radiation whose wavelength is
expressed by the equation:
λ = d(Sin i + Sin r)
n
Where, n = Order no. (0,1,2,3)
λ = wavelength of the resultant radiation
d = grating space
i = Angle of incidence
r = Angle ofreflection

92. 2. Transmission gratings:
Transmission grating is similar to diffraction grating.
But, Refraction takes place instead of reflection.
Refraction rays produces reinforcement.
When the transmitted radiations reinforce with the refracted
radiations, a resultant radiation is obtained whose wavelength is
given by the equation: λ = d Sin ϴ
n
Where,
λ = Wavelength of the resultant radiation
d = Grating spacing
ϴ = Angle of Diffraction
n = Order no. (0,1,2,3 etc)

95. Thus a light radiation at any angle ϴ or any order can be
collected and used in the instrument by either moving the grating
and fixing the slit or moving the slit and keeping the grating
constant.

96. Sample cells/ Sample Holders:
Sample cells or cuvettes are used to hold the sample
solutions.
Their shape (rectangular or cylindrical) and material of
construction varies depending on the instrument and the
nature of the sample being analyzed.

97. For ex: cuvvettes made up of quartz are used in UV
spectrophotometer, while those of glass are used in visible
spectrophotometers.
The pathlength of the cell is normally 1cm, however cells with
longer pathlengths upto 10cm or shorter pathlengths of 1-2mm
are also available.

98. Before taking the measurements, sample cells should be
thoroughly cleaned to avoid any contamination.
The level of the sample solution must be up to the mark etched on
its surface or above the light beam to avoid reflections from the
upper surface of the liquid.

99. An ideal characteristics of sample cell
 The material used in the construction of the sample
cell should not react (chemically inert) with the
solvent it is holding.
 It must transmit light of the required wavelength.
 It should have uniform thickness.

100. Detectors
Detectors are the devices which convert light energy into
electrical signals, that are displayed on the readout device.
After passing through the sample cell, a part of the radiation is
absorbed by the sample and the remaining is transmitted.
The transmitted radiation falls on the detector which determines
the intensity of radiation absorbed by the sample.
1. Photomultiplier tubes
2. Barrier layer cell / Photo voltaic cell
3. Photo tubes or Photoemissive tubes
4. Silicon Photodiode detector

101. Barrier layer cell (Photovoltaic cell)
Construction
It consists of a photocathode which is a thin metallic
layer coated with gold or silver.
It also contains a metal base (usually iron) which acts
as anode.
Between these two electrodes is a semiconductor layer
of selenium.

102. Working
When light rays falls on the selenium layer,
electrons are generated and taken up by the
photocathode.
Because of the poor electrical conductivity of the
selenium, the electrons get accumulated on the
cathode leading to the development of potential
difference across the two electrodes which results in
the generation of electric current.

104. The current flow causes deflection in the galvanometer which
gives the measure of the intensity of radiation, i.e., greater the
intensity of radiation, greater is the current produced hence
greater the deflection in galvanometer.
Advantage:
It is economical hence commonly used.
Disadvantages:
For signal amplification, the resistance of the external circuit
should be low.
It is less responsive towards light except for blue and red.

105. Phototubes (Photoemissive Tubes)
Construction
It consists of a hollow glass tube with a photocathode and a
collector anode.
The surface of the photocathode is coated with a layer of
elements like cesium, potassium, silver oxide or a mixture of
these.
Working
When light falls on the photocathode, electrons are produced
that travel towards the collector anode and generate current.
The amount of current generated is directly proportional to the
intensity of light radiation. Compared to barrier layer cell,
phototubes are more sensitive and therefore widely used.

108. Photomultiplier Tube
This type of detector is the most sensitive of all
the detectors, expensive and used in
sophisticated instruments.
Principle
The principle employed in this detector is that,
“multiplication of photoelectrons by secondary
emission of electrons”.

109. Construction
It consists of a light sensitive cathode (photocathode) and a
series of 10 anodes (dynodes) maintained at a potential of 75-100
volts.
Photomultiplier tube being sensitive can detect extremely weak
signals also, therefore it is used in intricate instruments.
PMT can detect very weak signals, even 200 times weaker than
that could not be done using Photovoltaic cell. Hence it is useful
in flourescence measurements.
PMT should be shielded from stray light in order to give
accurate results.

111. Silicon photodiode detector
 Silicon photodiodes have become important
recently because 1000 or more can be fabricated
side by side on a single small silicon chip. (the
width of individual diodes is about 0.02mm).
 With one or two of the diode-array detectors placed
along the length of the focal plane of a
monochromator.
 All the wavelengths can be monitored
simultaneously, thus making high-speed
spectroscopy possible.
 Silicon photodiode detectors respond extremely
rapidly, usually in nanoseconds.

112. Silicon photodiodes are solid state semiconductor devices,
sensitive to light in the wide spectral range of 200 –
1200nm, which extends from deep ultraviolet through the
visible to the near infrared region.
Working
Photodiodes are semiconductor light sensors that generate a
current or voltage when the P-N junction in the semi
conductor is illuminated by light.
When a photon of sufficient energy strikes the diode, it excites
an electron, thereby creating a free electron (and a positively
charged electron hole). This mechanism is also known as the
inner photoelectric effect.
This device can be used in three modes: Photovoltaic as a solar
cell, reversed-biased as a photo detector, and forward-biased
as an LED.

113.  A photodiode is a type of photo detector capable of
converting light energy into electrical energy.
 Photodiodes are similar to regular semiconductor
diode except that they may be either exposed (to
detect UV or X-rays) or packaged with a window
or optical fibre to allow light to reach the sensitive
part of the device.
 A photodiode is designed to operate in reverse bias.

114. Reverse Biasing
In reversed bias the negative region is connected to
the positive terminal of the battery and the
positive region is connected to the negative
terminal.
The reverse potential increases the strength of the
potential barrier. The potential barrier resists the
flow of charge carrier across the junction.
It creates a high resistive path in which no current
flows through the circuit.

118. Advantages of Silicon Photodiode
 Excellent linearity with respect to incident light
 Low noise
 Wide spectral response
 Compact and light weight
 Long life

119. Instrumentation
Colorimeters
 These are usually inexpensive and less accurate.
 They measure either Absorbance or Transmittance or both.
 Filters are used for different coloured solutions.
The wavelength used is 400-700nm.
Spectrophotometers
 These are little more expensive than colorimeters.
 They can be used for a wide wavelength range ex 360-900nm.
 The accuracy of instrument is very high since grating
monochromators and photomultiplier tubes are used.
 They can be supported by amplifiers or recorders. Nowadays
microprocessors are used.

120. Different types of Instruments
Colorimeters, Spectrocolorimeters and Spectrophotometers
Colorimeters:
They contain
Tungsten lamp
Absorption filters and
Photovoltaic cell
They are designed to read either %Transmittance or absorbance
Single beam instruments are non-recording type.

121. Spectrocolorimeters
They contain:
Tungsten lamp
Prisms as monochromators
Photovoltaic cell or Phototubes
as detector
They are also single beam, and
non-recording type
Designed to measure %Transmittance orAbsorbance

122. Spectrophotometers
• These are expensive
• Designed to measure %Transmittance orAbsorbance
• Record the absorbance using a plotter or recorder
• These are of double beam type where we can use both
sample and reference solution at a same time.
Advantages
 Storage of Spectrum
 Comparison of Spectra
 Rapid wavelength scanning
 Data Manipulation
 Derivative spectral mode
 Software can be used.
 More accurate and reliable.

123. Single beam UV-Visible 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 the incident light I0 is absorbed by the sample while the
remaining gets transmitted It.
The transmitted light strikes the detector and produces the
electrical signals.
The signal produced by the detector is directly proportional to
the intensity of the light.
The output is measured by a micrometer or galvanometer and
displayed on the readout device.
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 the wavelength is
changed.

126. Advantages:
 Simple in construction
 Easy to operate
 Economical
Disadvantages:
 Any fluctuations in the intensity of the radiation source affects
the absorbance readings.
 It requires adjustment of transmittance to 0% and 100%
whenever the wavelength is changed. Hence, a continuous
spectrum is not obtained.

127. Double beam UV-Visible Spectrophotometer
Double beam spectrophotometer allows direct measurement of
the ratio of intensities of sample and reference beams respectively.
The design of double beam spectrophotometer allows direct
measurement of the ratio of intensities of sample and reference
beams respectively.
The design of a double beam spectrophotometer is similar to
single beam spectrophotometer except it contains a beam splitter.

128. Monochromator selects the required wavelength of light which
is then passed through the exit slit and received by a rapidly
rotating beam splitter.
Beam splitter is a circular disc one third of which is opaque,
one third is transparent and the remaining is mirrored.
The beam splitter splits the monochromatic beam of light into
two beams of equal intensities.
One beam is passed through the sample cell and the other
through the reference cell.

129. After passing through the sample and reference cells, the
transmitted beams reach the detectors and produce a pulsating
current which is proportional to the intensities of the incident
light (I0) and transmitted light (It).
The detectors are connected to an amplifier and readout device
which gives the final result in absorbance log I0 / It or
transmittance log It / I0.
The ratio between the intensities of incident and transmitted
beams gives the direct measure of the absorption or transmittance
by the sample and reference solutions.
A double beam spectrophotometer can be designed using one or
two detectors.

130. Double beam UV-Visible Spectrophotometer

132. Advantages
 It facilitates rapid scanning over wide wavelength
region.
 Fluctuations due to radiation source are minimized.
Disadvantages
 Construction is complicated.
 Instrument is expensive

135. Applications
1. Qualitative analysis
a) Detection of Purity/Impurities:
Impurities present in the sample can be detected from
Absorption spectrum by measuring the absorbance at specific
wavelength and can be compared with that of standard.
a) Identification of the compound:
Compounds containing lone pair of electrons or conjugated
double bonds absorb UV radiations and give characteristic
absorption spectrum.
The unknown compound can be identified by comparing its
absorption spectrum with that of the standard.

136. 2. Quantitative analysis
a)A1% value(Absorbance factor method)
1cm
A1%
1cm values represent the absorbance or extinction value of a
1% solution at a definite wavelength.
This method helps in the estimation from raw materials and
finished products (formulation).
This method is used when reference standard is unavailable.
The percent purity can be determined by the formula:
%Purity = Observed absorbance X 100
A1%
1cm X Concentration

137. b) Reference standard method
This method involves the measurement of average A1%
1cm
value .
The average value can be determined by measuring the
absorbance of different standard solutions and calculating
their average.
The average A1%
1cm value can be utilized to determine the
percentage purity using the formula:
%Purity = Observed absorbance X 100
AverageA1% value X Concentration1cm

138. c) Direct comparison method or single standard method
In this method, the absorbance of a standard solution of
known concentration and a sample solution is measured.
The concentration of can be calculated using the formula
A1 = Ɛ C1 t
A2 = Ɛ C2 t
Where,
A1 ,A2 = Absorbance of standard and sample
C1, C2 = Concentration of standard and sample
Ɛ = Molar ext. coefficient
t = Pathlength (1cm)
Dividing 1st eqn with 2 we get, C2 = C1 X A2
A1

139. d. Calibration curve Method
Calibration curve is a plot of concentration on X-Axis and
absorbance of series of standard solution of known
concentration on Y-axis.
A straight line is drawn through maximum no. of points
coinciding.
This line is called calibration curve
Using this calibration curve-
-Concentration of the drug
-Amount
-% Purity can be determined

140. Overlayspectra or Linearity

141. Difference spectrophotometric method
This method is used for quantification of sample drug in the
presence of interfering ions or substances especially in
biological fluids.
It involves the measurement of differential absorption between
two different chemical forms of the same drug of equal
molarity.
The different forms of drug can be obtained by changing the
pH with the help of buffers or by chemical reactions such as
oxidation, reduction etc.
There is a shift in wavelength with different forms of drug.

142. i) Quantitative Estimation Using UV spectroscopy
The following drugs can be estimated by using UV spectroscopy
Drugs
Ampicillin
Solvents
Water
λmax (nm)
325
Griseofulvin Ethanol 291
Paracetamol 0.1 M NaoH 257
Phenformin Water 520
Verapamil HCl 0.1M HCl 278

143. Estimation of Paracetamol in Tablets using UV- spectroscopy
• Weigh accurately 20 tablets and powder them.
• The powdered drug which is equivalent to 150mg of
paracetamol is weighed accurately.
• To the powdered drug, add 50ml of 0.1M sodium hydroxide
and dilute it with 100ml of water. Shake the solution for
15minutes.
• Make the volume with water upto 200ml. Shake gently and
filter.
• 10ml of this filtrate is diluted to 100ml with water.
• To 10ml of the above solution, add 10ml of 0.1M sodium
hydroxide and dilute with water to get 100ml. Shake
thoroughly.
• The absorbance of the resulting solution is measured at 257nm.

144. • Concentration of paracetamol is calculated by taking A1%1cm
value as 715 at λmax of about 257nm.
• The amount of drug can be estimated in terms of percentage
purity.
%Purity = ObservedAbsorbance X 100
A1%1cm X Concentration
ii) Quantitative Estimation Using Visible Spectroscopy:
Estimation of Metals and Functional groups
Drugs are generally colourless or white in nature. Colourless
drugs are rendered coloured by coupling them with chromogenic
agents, followed by their estimation using visible spectroscopy.

145. Keto-enol Tautomerism
It is a form of Tautomerism in which the keto form (an aldehyde
or ketone ) and enol form of a compound are interconvertible and
exist in chemical equillibrium. Thus, keto-enol tautomerism can
be studied using UV spectroscopy as λmax and molar extinction
coefficient € differ among the keto and enol form.

146. Cis and Trans Isomerism
UV spectroscopy differentiates cis and trans isomers.
Trans-isomer exhibits absorbance at a longer wavelength than
cis- isomer.
Conversion of cis-isomer to trans-isomer form results in
bathochromic shift and hyperchromic effect and viceversa.

147. Conjugation
Conjugation can occur between two or more carbons containing
double or triple bonds and also in carbon –oxygen double bond.
Conjugation helps in determining the presence of an aromatic ring,
the number and position of the substituents present on the carbon
of conjugated system.
Conjugation causes shifting of λmax towards longer wavelength,
as the no. of double bonds increases.
λmax
174nm
217nm
267nm
Compounds
H2C=CH2
Ethylene
H2C=CH-CH=CH2
Butadiene
H2C=C=C=CH2
Butatriene

148.  Functional group determination
 Alkyl substitution
 Presence or absence of Unsaturation
 Identification of Unknown compound
 Structure of Organic and Inorganic compounds
 Determination of Molecular Weight

149. A solvent for UV spectroscopy should meet thefollowing
requirements
1. It should not itself absorb radiation in the region under
investigation.
2. It should be less polar, to minimum interaction with solute
molecule.
3. Spectroscopic (Analytical grade) solvents should be used.
4. The most commonly used solvent is 95% ethanol.
 It is cheap
 Good dissolving power
 Does not absorb radiation above 210nm

150. 5.Some other solvents which can be used above 210nm are
n-hexane, cyclohexane, methanol, water and ether.
6. Hexane and other hydrocarbons are sometimes preferred to
polar solvents. Because they have minimum interaction with
solute molecule.
7. Benzene, chloroform, carbon tetra chloride cannot be used,
because they absorb in the range of about 240-280nm.

151. A solvent is a liquid that dissolves another solid, liquid,or
gaseous solute, resulting in a solution.
Solvents can be broadly classified into two categories:
 Polar
 Non-Polar
 A drug may show varied spectrum at particular wavelength
in one particular condition but shall absorb partially at the
same wavelength in another condition.
 These changes are mainly due to:
1. Nature of the solvent
2. Nature of Absorption Band
3. Nature of theAnalyte

152.  The position and intensity of an absorption band may shift
when the spectrum is recorded in different solvents.
 A dilute sample solution is preferred 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 Ex dienes, conjugated
hydrocarbons no shift

153. Solvent Effects
 A most suitable solvent is one which does not itself
absorb in the region under investigation. A dilute
solution of the sample is always prepared for the
spectral analysis.
 Most commonly used solvent is 95% Ethanol.
Ethanol is a best solvent as it is cheap and is
transparent down to 210 mµ.
 Commercial ethanol should not be used as it
contains benzene which absorbs strongly in the
ultraviolet region. Some other solvents which are
transparent above 210 mµ are n-hexane, methyl
alcohol, cyclohexane, acetonitrile, diethyl ether etc.

155.  Hexane and other hydrocarbons can be used as these are
less polar and have least interactions with the molecule
under investigation. For ultra-violet spectroscopy,
ethanol, water and cyclohexane serve the purpose best.
 The position and the intensity of absorption maximum
is shifted for a particular chromophore by changing the
polarity of the solvent. By increasing the polarity of the
solvent, compounds like dienes and conjugated
hydrocarbons do not experience any appreciable shift.
Thus, in general, the absorption maximum for the non-
polar compounds is the same in alcohol (polar) as well
as in hexane (non-polar).The absorption maximum for
the polar compounds is usually shifted with the change
in polarity of the solvents.

156. α,β-unsaturated carbonyl compounds show two different
shifts.
n-π* transition (less intense): In such a case, the absorption
band moves to shorter wavelength by increasing the
polarity of the solvent.
In n-π* transition, the ground state is more polar as
compared to the excited state. The hydrogen bonding with
solvent molecules takes place to lesser extent with the
carbonyl group in the excited state.
For ex, absorption maximum of
acetone is at 279 nm in hexane
as compared to 264 nm in water.

157. π-π* transition (intense): For such a case, the
absorption band moves to longer wavelength by
increasing the polarity of the solvent.
The dipole interactions with the solvent molecules
lower the energy of the excited state more than that
of the ground state.
Thus, the value of absorption maximum in ethanol
will be greater than that observed in hexane.

161.  The absorption spectra of Ethyl-4-hydroxy-1-(4-
methoxyphenyl)-2-quinolinone-3-carboxylate
(L1) is depicted in attached Figure 1. Two
maximum absorption peaks were observed at
231nm and 288nm in ethanol, while in water at
225 and 296nm. according to solvent polarity,
water more polar, and increasing polarity of the
solvent shifts pi-pi* to higher energy and n-pi* to
lower energy. but the situation is reversed, the
first peak at 225nm (in water) while 231nm (in
Ethanol)
 and the second peak appears as one peak at
296nm (in water) while it is two peaks at 288-
296nm (in Ethanol).