1. A spectrophotometer is an instrument that measures the intensity of light at different wavelengths absorbed by a sample. It uses a monochromator to select specific wavelengths and a detector such as a phototube to measure the intensity of transmitted light. 2. Key components include a radiant light source, monochromator to select wavelengths, a sample cell, and a detector. Common light sources are tungsten lamps and detectors include phototubes and photomultiplier tubes. 3. Spectrophotometers can be used for quantitative analysis using Beer's Law. The absorbance measured is directly proportional to the concentration of absorbing substances and path length. Double beam instruments compensate for fluctuations and noise in

1. Spectrophotometry
The spectrophotometer is an
instrument used to measure the
intensity of wavelengths in a
spectrum of light.

3. Spectroscopy :
• . It is a branch of science which deals with the
study of interaction of electromagnetic
radiation with matter.
• During such interactions the energy is either
absorbed or emitted by the matter in discrete
amount is called as quanta

5. • Electromagnetic radiation may be considered
as a simple harmonic wave propagated from a
source and travelling in a straight line except
when reflected or refracted.

6. Wavelength :
• It is the distance between two successive
maxima on an electromagnetic waves.
• Usually the wavelength is expressed in cm or
in angstrom

7. rad

8. Frequency :
• The number of complete wavelength units
passing through a given point in unit time is
called the frequency of radiation.
• It is generally expressed in cycle per second or
in hertz.

10. Wave number:
• It is the number of waves per centimeter in
vacuum.

11. Interactions of radiation with matter.
• The entire range over which the
electromagnetic radiation exits is known as
electromagnetic spectrum.

12. • 200- 400nm = u.v region.
• 400-750nm= visible region.
• 750-1500nm= IR region.
The colour of the object arises from the
selective absorption of certain wavelength of
incident light.

13. Terminology used in absorption
measurements:
• The ‘JOINT COMMITTEE ON NOMENCLATURE
IN APPLIED SPECTROSCOPY’ has worked to
develop standard nomenclature for various
quantities in spectroscopic measurements.

14. 1. Radiant energy :
• It is defined as the energy transmitted as
electromagnetic radiation.
• It is characterised by the wavelength and
frequency.

17. Radiant power, P:
• Energy per unit time is called as power.
• Radiant power is the rate at which energy is
transported in a beam of radiant energy.
• The radiant power incident upon a cell is given
the symbol Po. And the transmitted by a cell is
P.

19. Transmittance,T:
• It is the ratio of radiant power transmitted by
a sample to the radiant power incident upon
the sample , both being measured at the same
spectral position.
• It is usually expressed as percent
transmittance.

20. Absorbance ,A:
• The absorbance is the logarithm to the base
10 of the reciprocal of the transmittance,T.
• Absorbance is also defined as the logarithm to
the base 10 of the ratio of the radiant power
incident upon the sample to the radiant
power transmitted by the sample.

21. Absorptivity ,a:
Absorptivity is defined as the ratio of the
absorbance to the product of the length of
optical path b, and the concentration of the
sample ,C.
a=A/bC.
• Absorptivity is the measure of the ability of
sample to absorb light.

22. Molar absorptivity
• Molecular absorptivity is the product of
absorptivity and molecular weight of the
material.

23. Path length,b:
• It is the internal thickness of cell in which the
test sample is taken.
• It was formerly denoted by l.

24. LAMBERT
BEER”S L
LAMBERTS
&
BEER”S LAW

25. “When a monochromatic beam
of light is allow to pass through
the transparent medium the rate
of decrease of intensity with the
Thickness (db) of medium is
directly proportional to the
intensity of incident light”.(A =
a.b)
Lamberts law

28. Problems ;

29. Instrumentation.

30. 1. Radiant sources:
• A stable continuous source of radiant energy
covering the region of spectra of visible and
U.V region is used.
• Radiation sources must fulfill the following
requirements:
1. It must be stable.
2. It must produce sufficient intensity of
radiation so that the transmitted intensity
can be detected.
3. It must supply continuous spectrum of
radiation over the entire wavelength.

31. • A tungsten lamp is commonly used as a source of
radiation.
• In the visible region (400-800nm) , a tungsten
filament is most widely used.
• Its construction is similar to house hold lamp.
• It consist of a tungsten wire which is heated in a
controlled atmosphere.
• To avoid fluctuations in readings it is necessary
that the lamp should provide constant radiant
energy .
• ??????
• It is achieved by employing a constant power
supply to the lamp.

32. Filters and monochromators:
• A radiation source is generally emitting a
continuous spectra i.e. it gives all possible
wavelength of the region .
• However, a narrow band of wavelength is always
necessary in calorimetric and spectrophotometric
analysis.
• Therefore , it is necessary to have device which
will select a narrow band from wavelength of
continuous spectra.
• For this purpose , filters and monochromators are
used.

33. • Both filter and monochromator allow the light
of required wavelength to pass through but
absorbs the light of other wavelength .
Filters
are of two types:
1. Absorption filters
2. Interference filters

34. Absorption filters:
• These filters work by selective abbsorption of
unwanted wavelengths i.e. these filters limit
the radiation by absorbing ertain portion of
light spectrum.
• Absorption filters are generally cheaper than
interference filters.

37. Interference filters:

38. • These filters generally provide somewhat
narrower band width than absorption filters.
• When a ray of light is incident upon it , a part
of light reflects back whereas the remaining
light is transmitted.

39. Monochromators:
• A monochromator is a device which converts a
polychromatic radiation to monochromatic
radiation.
• Usually a monochromator consist of ;
1. Entrance and exit slit: to restrict unwanted
radiation.
2. Lenses or mirrors: to focus radiation.
3. The dispersing medium(like prism and
diffracting grating): to isolate the wavelength.

41. Monochromators are of two types:

42. Cuvette or sample container:
• The cell holding the sample should be
transparent to the wavelength region .this cell
is called as cuvette.
• The cuvette is made up of quartz or glass.
• The thickness of cuvette is 1cm and it is used
for sample and solvent.

43. Detector
• A detector is a device which converts radiant
energy into electrical energy.
• Generally , 3 types of detectors are used to
detect radiation:
1. Photovoltaic cells.
2. Phototubes .
3. Photomultiplier tubes.

44. 1. Photovoltaic cell:
• It is also called barrier –layer cell or photronic
cell.
• This cell operates without the use of battery.
• The photovoltaic cell consist of metal base plate
,thin layer of semiconductor and thin layer of
transparent silver and gold metal layer.
• When the radiation are incident the electrons are
generated at transparent and semiconductor
layer junction.

45. • Because of accomodation of
electrons electric voltage is
generated therefore photovoltaic
current flow through the circuit
which is recorded by galvanometer.
• The photocurrent is directly
proportional to the intensity of
incident light.
• It is used only for visible region.

47. 2. Phototube:

48. • A phototube consist of an evacuated glass bulb.
• A light sensitive cathode in the form of half
cylinder or circle.
• The inner surface of cathode is coated with light
sensitive material.
• A metal ring is inserted near the center of bulb
which act as anode.
• When the radiation is
Incident upon the cathode
Photoelectrons are emitted.

49. • Thus electrons are attracted towards anode
and create electric current which flows
through the circuit.
• Te current is measured by read out system.
• Phototube are more sensitive than
photovoltaic cell.

50. 3.Photomultiplier tube.
• It is more sensitive than phototube .
• It is mostly used in spectrophotometer.

51. Single beam photoelectric
calorimeter:
• The basic components of single beam
photoelectric calorimeter are :
1. A source of light such as tungsten filament lamp
with concave reflector and collimating lens.
2. An adjustable diaphraghms or slit
3. A filter
4. A cuvette
5. A detector such as photovoltaic cell
6. A recorder such as galvanometer.

53. SINGLE BEAM SPECTROSCOPY

54. ◼The photometer is an instrument which is
used to measure radinat power of light .
◼Spectrophotometer is an instrument use to
measure the absorbance or tranmittance of a
solution as a function of wavelenght.l
◼T he single beam spectrophotometer are
particularly suitable for the quantitative
analysis that involves an absorbance
measurement at a single wavelength in both
visible and u.v region.

55. ◼A very widely used spectrophotometer primarily
foer the visible region( 340-625nm)is the Bausch
and Lomb Spectronic- 20 fitted with a blue
sensitive phototube.
◼Its range can be extended to 950 nm by the use of
a red-sensitive phototube and a red-transmitting
filter.
◼The reflecting grating has convergent beam but
not in a collimated light beam

56. ◼ The vaccum phototube is used as a radiation dedector
◼ The signal from the dedector is amplified and the meter in
the bridge circuit is graduated in linear scale divisions from 0
to 100 % tranmittance as well as in non-linear absorbance
unit.
◼ The rotation of the grating drum turns the grating which
gives namely wave-length adjustment, 0 adjustment and
100 % adjustment.
◼ This spectro photo meter is an in expensive and easily
operated instrument.

57. ◼The spectro photo meter is an instrument
used to measure absorbance or transmissioin
of a solution as a function of a wave length.
PRINCIPLES

58. INSTRUMENTATION
◼It consist of following instruments
◼1. Source
◼2. Monochromater
◼3. SampleCell i.e cuvette
◼4. Detector i.e phototube

59. RADIANT SOURCE
◼U.V region is used. A stable continious source
of radiant energy covering the region of
spectra of visible and
◼A tungsten lamp is comm0nly used a source
of raditation

60. MONO CHROMATOR
◼ It is device which converts a polychromatic
radiation to monochromatic radiation.
◼ It consist of :
◼ 1.entrance & exit slit
◼ To restrict unwanted radiation and help to
control the spectral purity of the radiation
emitted from the monochromator
◼ 2.Lenses
◼ To make the beam parallel or to focus radiation
and to isolate the wave length from the
polychromatic radiation of the source

61. SAMPLE CELL
◼1.The instrument contain transparent
container called cuvette which is made of
quartz.
◼2.The thickness of cuvette is 1 cm and it may
be rectangular or cylindrical and it used for
sample and solvent

62. DETECTOR
◼It is used for measuring radiant energy transmitted
through the sample in the form of electrical signal
◼Which is passed through the read out system

63. PHOTOTUBE
◼ The cell is also known as photoemissive tubes
◼ A phototube consist of evacuated glass bulb
◼ A light sensitive cathode in the form of half
cylinder or circle
◼ The inner surface of cathode is coated with light
sensitive material
◼ A metal ring is inserted near the centre of bulb
which act a Anode when the radiation is incident
upon the cathod photo electrons are emitted
◼

64. ◼This electrons are attracted towards anode
and create electric current which flow trough
the circuit
◼The current is amplify and measured by read
out device
◼Phototube are more sensitive than
photovoltic cell.

67. Main Difference – Colorimeter
vs. Spectrophotometer
• Colorimeters and spectrophotometers are
both used to measure colour-absorbing
properties of substances. The main
difference between colorimeter and
spectrophotometer is that colorimeter is a
device which measures absorbance of specific
colours,whereas a spectrometer measures
transmittance or reflectance as a function of
wavelength.

68. DOUBLE BEAM
SPECTROPHOTOMETE
R

69. •One part of the monochromatic
beam is made to pass through the
sample solution and them falls on
the working photocell.
•The other part of the
monochromatic beam is made to
pass through the blank or
reference solution and then falls
on the reference photocell.

70. • The two photocells are
connected to the comparison
circuit.
• The compariaon circuit reads
directly the absorbance of the
solution.

71. ADVANTAGES OF THE BEAM
INSTRUMENT:
•1.The instrument automatically
compensate for the absorbance by the
blank solution.
•2.The continuous replacement of the
blank with the sample is not
necessary as required in single beam
instrument.

72. •3.Since two beam of same radiant
power are used at a time for sample
and reference and two identical
balanced photocells are used,readings
are independent of the fluctuation of
the main power supply.
•4.Since the null method is uses for the
instrument readings are not affected
by variations in the sensitivity of the
photocells.

73. •5.It provide rapid scanning over a
wide wavelength region and use a
recorder.
•6.The instrument is very useful for
qualitative analysis in which the total
spectrum is required.

75. colorimeter spectrophotometer
1.Uses filters. 1. Uses monochromator.
2.It uses narrow
band of
wavelength.
2. usese single
wavelength.
Difference between Colorimeter and
spectrophotometer

76. 3.It uses only in
visible region.
3.It uses in U.V and
visible regions.
4.It operates at the
wavelength of the
filter available.
4. It operates at
any wavelength
simply by rotation
of the prism or
grating.

77. 5.It uses photovoltic
cell as detector. It
ssensitivity is less.
5. It uses phototube
as detector.its
sensitivity is high.
6.It gives less
accurate results.
6.It gives accurate
results.

78. 7.It is less
expensive.
7.It is expensive.
8.It is less
informative,since
broad band of
wavelength used.
8.It is much
informative,since
narrow band of
wavelength used.

85. ▶ The absorbance is proportional to the
concentration i.e. to the no. of particales that are
effective in absorbing radiation at specified
wavelength.
▶ This is easily extended to the solution containing
two or more than two components as absorbing
species.
▶ Each component absorb independently.
Additivity of absorbance

86. ▶ If absorption spectrum (S1) of a
system of two chromophores is
available & if absorption spectrum (S2)
of one out of these two chromophores
is known then S2 can be substracted
from S1 , to get pure absorption
spectrum of another chromophore.
▶ The (S1-S2) spectrum is usefull of
identification of unknown chromophore
of the system.

89. Simultaneous
spectrophotometric
determination

90. ▶ This tecnique is used to measure the absorbance
of a mixture of coloured solution without
separating them.
▶ This involves the simultaneous determination of
the conc. Of two or more components in a given
solution by measuring the absorbance at two or
more wavelenghts.
▶ It is based on the following requirements.

91. ▶ 1. There is no chemical interaction between the
absorbing components.
▶ 2. Each component acts independantly i.e. each
one absorbs as if others are not present.
▶ 3.Beer’s laws obeyed by each component at its
wavelenght (λmax).
▶ 4. λmax values of components are differed
widely.

92. ▶ 5. The absorbance of each component in the
mixture is additive i.e. the observed total
absorbance A at any wavelenght is the sum of
absorbances of ‘n’ components present in the
mixture.
▶ 6. The value of molar absorptivities of each
component of the mixture at any fixed wavelenght
are calculated from absorptive measurements of
solution cantaining the single component.

94. SPECTROPHOTOMETRIC
TITRATIONS
▶ Titration in which absorbance of a solution is used
to determine the end point are called
spectrophotometric titrations.

95. ▶ Both filter photometers and spectrophotometers
can be used for photometric titrations.
▶ Titration those carried with the help of
spectrophotometer are called spectrophotometric
titrations.
▶ Titration those carried with a photometer are
called photometric titration.
Instrumentation

96. ▶ Usually , sphotometer or spectrophotometer are
modified to insert the titration vessel or cell in the
light path , or a special titration cell is used.
▶ Since beer’s law is obeyed for monochromatic
radiation, spectrophotometers are preferred over
the filter photometers for these titrations.

97. ▶ There are two method of photometric titration.
▶ In the one method, the titration is carried out in a
titration flask on the usual laboratory bench.
▶ The absorbance is measured by taking the
fraction of the titrand from the titration flask &
placing it in the absorptive cell.
▶ Of coures, the fraction are returned completely
each time to the titration flask.
▶ Though this is the simplest method of titration, it is
inconvenient for carrying out the titration.

98. ▶ In another method the titration is carried out inside
the absorptive cell.
▶ If the titration is carried out inside the cell , a
modified cell of 5 to 100 ml capacity is used
instead of cuvette.
▶ This cell is placed in the cell compartment of the
photometer or spectrophotometer.
▶ The cell is made up of perspex sheet.
▶ Since perspex is not transparent to ultraviolet light.

99. Perspex sheet

100. ▶ The two holes are made in the cell to
accommodate circular quartz windows.
▶ These two window are fitted in such a way that the
beam of light is passes through their center to the
photoelectric cell.
▶ The cell also has two small holes one for
introducing the tip of a microburette & other for
introducing a micristirrer.
▶ The hole arrangement of the cell,except the
windows , is covered with black paper.

101. Arrangement of spectrophotometric
titration

103. Titration curves

104. ▶ A photometric titraion curve is a plot of
absorbance against the volume of titrant
added.
▶ This graph consist of two straight line with
differing slopes.
▶ One line occurs before the end point and the
other line occurs beyond the end point.
▶ In many titrations a marked curvature is
observed in the equivalent point region.
▶ The end point is taken as the intersection of
extrapolated straight-line portion.

105. ▶ The shape of the titration curve varies with the
absorbing species in the solution.
▶ The general shape of many typical photometric
titration curves, representing titration system
without added indicator substances.

106. 1 st graph

107. ▶ It represents a type of titration in which
only the titrand (solution being titrated)
absorbs at certain wavelenght , while
the titrant and reaction product do not
absorb.
▶ In such a case the absorbance does
not change after equivalence point.

108. 2 nd

109. ▶ It represent a type of titration in which
only the titrand absorbs at certain
wavelenght , while the product do not
absorb.
▶ There is no change in absorbance at
the wavelenght where only the titrant
absorbs upto the equivalence point.

110. 3 rd

111. ▶ It represents a type of titration in which only
the reaction products absorb strongly at the
particular wavelenght , while the titrant and
reactant do not absorb at this wavelenght.
▶ During the course of titration there is gradual
increase in the conc. Of the absorbing
product, since it accumulates as the titration
proceeds.
▶ Hence absorbance increases till the
equivalence point is reached.
▶ After the equivalence point the absorbance
remains constant , because nothing is added
which absorbs light.

112. 4

113. ▶ It represent a type of titration in which
bhot the reactant and titrand absorb at
a particular wavelenght ,but titrant
absorbs less than the reactant.
▶ The product does not absorb and
hence there will be decrease in the
absorbance as the titration prpceeds.
▶ Moreover, the coloured analyte (the
substance to be analysed) gets
transformed into a colourless product
by a coloured titrant.

114. Advantages of
photometric titration

115. ▶ The method is useful to solution with lower &
higher ionic strenght or in non aqueous
solvent.
▶ The method can be used to highly coloured
solution which cannot be determined by the
usual visual indicators.
▶ The slight change in colour are readily
detected by the spectrophotometer & hence
end point determination is sharp and accurate.

116. ▶ These titration can be applied to a large no. of
non absorbing constituents since only one
absorber is necessary among the reactant or
reaction product.
▶ This titration technique is particularly suitable
to titration reaction where a relatively large
degree of reaction incompletion exists at the
equivalence point.

117. Application
Quantitative analysis
Qualitative analysis

118. ▶ 1.structure of organic compounds –
▶ Spectrophotometric technique can be used to
determine the structural problem of organic
molecules.
▶ Example-
▶ For isatin two structures of the forms lactim and
lactam have been proposed for the molecular
formula C8H5O2N.
Quantitative analysis

119. ▶ The spectrophotometric study of inorganic
complexes is very important because,
▶ (a) it distinguishes between, cis and trans isomers
of complex.
▶ (b) it help to study the equilibrium between the
octahedral, tetrahedral, square planar and the
five-coordinate configuration.
Structure of inorganic complexes

120. ▶ The geometrical isomerism of complexs can be
easily distinguished from their visible absorption
spectra.
▶ Example-
▶ The cis isomer of [Co(en)2 F2]NO3 is violet where
as the trans isomer is green.
Geometrical isomerism

121. ▶ The change in colour in cis [Ni (trisethylene
diamine) (H2O)2]²+ from blue to yellow on adding
inert salt to the solution has been shown from
absorption spectra to involve an equilibrium
between octahedral and planar configurations.
Octahedral – planar equilibrium

122. ▶ Sacconi has studied the effect of temp on schiff
base complexes.
▶ This study gives information about the equilibrium
between planar and tetrahedral configuration.
▶ The tetrahedral complexes exhibits two
characteristic bands at 7200 (TB1) and 11200cm-
1 (TB2).
▶ The planar configuration exhibits one
characteristic band at 16000cm-1 (PB).
Planar-tetrahedral equilibrium

123. ▶ It is observed that with increase in temp the
intensity of TB1 and TB2 band increases but that
of PB decreases.
▶ This shown that the equilibrium shifts from planar
to tetrhedral complexe with increasing temp.

124. ▶ 1.A] position and intensity of absorption band
and chromophore –
▶ The term chromophore can be define as any
group in the molecule which exhibits absorption of
electromagnetic radiation in the visible or UV
reagion.
Qualitative analysis

125. ▶ Two type of chromophore-
▶ 1.the chromophore In which the group is having π
electron and during absorption of radiation they
undergo π-π* transition & show coresponding
absorption band in the spectrum.
▶ Example-ethylenes, acetylenes etc.
▶ 2.the chromophore in which the group is having π
electron and n (non-bonding) electrons &during
absorption of radiation they undergo π→π* and
n→π* tansition and show coresponding
absorption bands in the spectrum.
▶ Example-carbonyl , azo compounds, nitro
compounds etc.

126. ▶ Auxochrome is a group which it self does not act
as a chromophore but when attached to a
chromophore it shifts the absorption maximum
(λmax) toward longer wavelenght along with an
increase in the intensity of absorption.
▶ Example- group include –OH, -NH2,-OR, -NHR & -
NR2).
Auxochrome

127. ▶ The presence of auxochrome in the molecule that
cantain chromophore which causes the absorptive
maximum to shift or to change wavelenght is
called as bathochrom sheet.
▶ It is related to the electron donating properties of
auxochrome.
Bathochromic or red shift

128. ▶ It involves the shift of absorption maximum
towards shorter wavelenght because of the
removal of conjugation in a system or by change
of solvent.
▶ Example-aniline shows absorption maximum at
280nm whereas in acidic medium it shifts at 200
nm.
▶
Hypsochromic shift or blue shift

129. ▶ The shape of the titration curve varies with the
absorbing species in the solution.
▶ The general shape of many typical photometric
titration curves, representing titration system
without added indicator substances.

130. ▶ This effect shows an increase in the intensity of
absorption and usually brought about by
introduction of an auxochrome in the molecule
under study.
Hyperchromic shift

131. ▶ This effect shows a decrease in intensity of
absorption and usually brought about introduction
of certain group in the molecule under study and
these group distort the geometry of the given
molecule.
Hypochromic shift