Introduction, electromagnetic radiation, units, electromagnetic and absorption spectra, Lambert’s and Beer’s laws, deviations from Lambert’s–Beer’s law, chromophores and auxochromes, absorption and intensity shift, types of electronic transition, effects of solvents, electronic transition in polyenes, instrumentation, colorimetry, Woodward-Fieser rules for calculating absorption maximum, analysis of mixtures, applications of ultraviolet and visible spectroscopy in quantitative analysis of drugs, use of ultra violet and visible spectroscopy in structural analysis.

2. Spectroscopy
Spectroscopy is the study of interaction between matter (mass) and radiated energy.
Such interaction includes –
Absorption of the incident radiant energy.
Emission of radiant energy.
Scattering or reflection of incident radiant energy.
Impedance of radiant energy transmission.
Changing the frequency/wavelength of the transmitted radiant energy.
Causing interaction between molecules in a non-stationary state and sustaining it.
Absorption spectroscopy/spectrometry
Absorption spectroscopy may be defined as the analysis of chemical substances by
measurement of the amount of radiant energy absorbed by the substance.
UV-Visible spectroscopy
UV-visible spectroscopy is a type of absorption spectroscopy which uses the UV and visible part of the EM s
pectrum.
Introduction

3. Electromagnetic (EM) spectrum
Spectrum:
Spectrum is the condition where a characteristic is not limited to a fixed set
of values rather it varies infinitely within a continuum.
By that definition, radiation arranged by wavelength is a spectrum; drug
activity in terms of action against bacteria is a spectrum.
Electromagnetic spectrum:
It is the arrangement of all types of electromagnetic radiation in order of their
increasing or decreasing wavelength (or frequency).
The above diagram shows the electromagnetic spectrum where
radiations are arranged in the decreasing order of wavelength from left
to right.

4. Visible and UV spectrum
Color Wavelength
Violet 400-420
Indigo 420-440
Blue 440-490
Green 490-570
Yellow 570-585
Orange 585-620
Red 620-780
The visible spectrum starts at 400 nm and ends at around 700 nm. The
electromagnetic radiation adjacent to the violet visible radiation is called
ultraviolet radiation. The electromagnetic radiation adjacent to the red visible
radiation is called infrared radiation.
The UV spectrum ranges from 10 nm to 380 nm. The UV radiation has been
classified as UV A (400-315 nm), UV B (315-280 nm) and UV C (280-10 nm)
based on their wavelength.

5. Electromagnetic radiation
Electromagnetic radiation:
Electromagnetic radiation is a form of energy that shows wave-like characteristics
and –
1. It can travel without any medium
2. At vacuum it moves at speed of light
3. Contains both electric and magnetic field components. These two components
oscillate (moving back and forth) in a phase perpendicular to each other. When an
electric field oscillates it causes the oscillation of the corresponding magnetic
field which in turn oscillates the next electric field. Thus the EM radiation moves
forward without any medium.
EM radiation is described by wavelength () or frequency (ν). The relationship
between wavelength and frequency is given in following equation.
c=νλ
Where c is the speed/velocity of the wave. At vacuum c is equal to the speed
of light i.e.3108ms-1 =31010ms-1 Pharmacists rarely use frequency value for analytic
al purposes and wavelength value is of more importance.

7. Effect of electromagnetic radiation on a substance
When a beam of electromagnetic radiation passes through a transparent medium of the
substance (the substance under analysis must be in solution for proper analysis), the radiation
is absorbed and/or transmitted by the substance. The absorption of the radiation depends on
the –
-Chemical nature of the substance
-Wavelength of the radiation
The radiation not absorbed is transmitted by the substance and it is called transmitted light
which can be measured by an instrument called the spectrophotometer. The record is called
the spectrum of that substance.
Transmittance “T”
The amount of light that passes through a particular thickness of solution is known as transmitt
ance T. Transmittance can be expressed as the ratio of the intensity of the transmitted light I to
the intensity of the incident light beam Io
I
I
T 

8. Absorbance “A”
The negative logarithm of the base ten of transmittance is called
absorbance.
It is also called optical density.
Absorbance is directly proportional to the concentration of the
solution when the thickness of the medium is constant.
Laws governing spectrophotometry
As described before, when a substance is placed in the path of light
(EM radiation), a number of events e.g. absorption, transmission,
elastic and nonelastic scattering of light may take place. The
absorption of EM radiation/light by molecules is governed by two
laws – Lambert’s law and Beer’s law.
I
I
A
I
I
A
TA


log
log
log




9. Laws governing spectrophotometry
Lambert described how intensity changes with distance in an absorbing medium.
Lambert’s Law: When a beam of monochromatic radiation is passed through a
solution of an absorbing medium, the decrease in the intensity of radiation with
thickness of the solution is
directly proportional to the
intensity of the incident
light.
Let,
I be the intensity of
incident radiation.
b be the thickness of the
solution.

10. Kdb
I
dI
KI
db
dI
I
db
dI



Thus
Now integrating the equation between
the limit I=Iₒ at b=0
And I=I at b=b
We know that absorbance
Thus A= Eb
A∞b
The amount of light absorbed by the medium
(solution/ sample) at a given wavelength is
proportional to thickness of the absorbing layer i.e. path length of the light. Basically it states
that the absorbance is proportional to the path length i.e.
A∞b

11. Relates the absorption of light to the properties of the material through which the
light is travelling.
Beer’s Law: When a beam of monochromatic radiation is passed through a
solution of an absorbing medium, the decrease in the intensity of radiation with
thickness of the solution is directly proportional to the intensity of the incident light
as well as concentration of the solution.
Let, I be the intensity of incident radiation.
x be the thickness of the solution.
C be the concentration of the solution.
K'=Absorbing co efficient or
Proportionality constant
dxCK
I
dI
ICK
dx
dI
IC
dx
dI
.
.
.




12. Now integrating the equation between the limit I=Iₒ at x=0
And I=I at x=l (l=Path length of the sample which is usually 1 cm)
we get
Thus A=E.C.l

13. From the equation it is seen that the absorbance which is also called as
optical density (OD) of a solution in a container of fixed path length is
directly proportional to the concentration of a solution.
Accordingly The amount of light absorbed by the a medium (solution/
sample) is proportional to the concentration of the absorbing material or
solute present.
This law states that absorption of the incident light is proportional to the
number of absorbing molecules. So according to this law –
A∞c
Where, c is concentration of the substance in solution and A is absorbance.
Thus the concentration of a coloured solute in a solution may be
determined in the lab by measuring the ABSORBANCY OF LIGHT AT A
GIVEN WAVELENGTH

14. Combining Beer’s law and Lambert’s law – Beer-Lambert
law/Equation of absorbance
According to the Lambert’s law –A∞b
According to the Beer’s Law –A∞c
Combining the two laws we have –
A∞bc
A=kbc (k is proportionality constant)
A=abc (Assuming k=a)

15. Absorptivity
Absorptivity is a measure of how easily the absorbing molecules can absorb the
electromagnetic radiation. It is specific for a given substance. It is defined as the
absorbance by a sample of unit concentration and where the path is of unit length.
It is mathematically quotient of the absorbance (A) divided by the concentration of
the solution (gm/L) and the path length (cm).
When c is expressed in moles/litre in the Beer-Lambert law, the absorptivity is
called molar absorptivity or molar extinction coefficient and it is expressed by
When, c is expressed as %w/v, the absorptivity is expressed as .


16. Deviations from Beer-Lambert law
The accuracy of the Beer Lambert law is dependant upon some chemical and instrumental
factors –
1. This law specifically the part concerning concentration is not maintained in high
concentrated solutions. This is because –
– In highly concentrated solutions, degree of interaction between molecules is higher at
higher concentration. Thus the molecules will not absorb radiation in same manner as
when they are in dilute solution.
– The absorptivity of solution doesn’t remain constant as the concentration changes.
– The refractive index of the solution may change in high concentration.
2. If the solution contains particulate matter then scattering of light occurs which interfere with
the absorption process.
3. If the sample is fluorescent or phosphorescent then this law is not obeyed.
4. If the light is non-monochromatic (in practice, monochromatic light is difficult to produce)
i.e. not completely monochromatic

17. Absorption of radiant energy
Effect on molecules upon absorption of radiation:
Electromagnetic radiation is energy. When a molecule absorbs radiation
it gains energy. The higher the frequency the greater will be the gain in energy.
This energy can bring about one or more of the following changes –
1. The absorbed energy may break bonds within molecule. e.g. conversion of
ergosterol to calciferol.
2. The absorbed energy may increase vibration or rotation of atoms within the
molecule. This principle is used in IR spectroscopy.
3. It may change nuclear or electronic spin. This property is used in NMR
spectroscopy.
4. It may cause electrons to rise to higher energy level.
The UV and visible radiation absorption will cause the last effect i.e. transition of
electrons within the molecule.

18. Every molecule has a definite energy state. Energy possessed by a molecule can be
classified in several categories. These are
1. Transitional energy (Etrans) 2. Vibrational Energy (Evib) 3. Rotational Energy (Erota) and
4. Electronic energy (Eelec)
Transitional energy
Molecule as a whole can move from one place to another and the energy associated with this
motion (Velocity) is called transitional energy as well as the movement is called transitional
movement.
Vibrational Energy (Evib)
The movement of a part of a molecule or a group of molecules that move within themself is
called vibrational movement and the energy associated with this vibration is called vibrational
energy.
Rotational Energy (Erota)
Molecule can rotate along its axis and the energy associated with this rotation is called
rotational energy.
Electronic Energy (Eelec)
Electrons of each atom moves around the central nuclei and the energy associated with this
movement is called electronic energy

19. When a molecule absorbs electro magnetic radiation like UV or Visible light its electron gets promoted from
the ground state to the higher energy state or excited state.
(Ground State: The molecules when attained in a state, having lower energy level i.e. normal unexcited
condition or state is called ground state. Excited state: Absorpsion of EMR by the molecule results in the
shipment of the molecule in the higher energy state called excited state)
In the ground state the spin of electrons in each molecular orbital is essentially paired. In the higher energy
state, the spins of electrons are either paired i.e. excited singlet state or parallel excited triplet state.
Normally the absorption of the UV or visible light results in the transition of the singlet ground state to
become singlet excited state.
To get the molecule in the higher energy level the energy difference between the two energy level must be
equal to the energy of photon absorbed, which can be expressed as
Energy required to get in the excited state, ΔE=E2-E1=hν
Here, E1= Lower energy level
E2=Higher Energy Level
h= Planks Constant
ν=Frequency

21. Generally the energy of the triplet excited state is lower than the singlet excited state. Thus
to be stable the molecule quickly returns from one of its higher energy level to lower one by
the emission of radiation.
Therefore an electron that has been raised to the higher energy level by the absorption of
radiation, quickly returns to the ground energy level within 10-8 seconds either directly or by
the way of second excited state and will give molecular absorption spectrum.
(Molecular absorption spectrum: A set of bands or lines generated, due to the
absorption of EMR by the molecules is called MAS. It is actually a graph obtained
by plotting the absorption against wavelength)
As the transmission rates are different for different molecule or structure, so the absorption
spectra will be different also. Greater the number of molecules capable of absorbing the
light of different given wavelength the greater will be the extent of light absorption. MAS is
characteristics for a molecule and there is no similarity in MAS of one molecule with other,
Thus it is used to in the quantitative analysis of the molecules.
Generally the absorption spectra is graphically represented by plotting the absorbance (A)
on the Y axis and the wavelength on the x axis.;
This is the principle or theory of UV or visible molecular absorption spectroscopy.

22. Energy transitions


*
*
n
Occupied
Energy Lev
els
Unoccupied En
ergy Levels
The bonding orbitals with which you are familiar are the -bonding orbitals typified
by simple alkanes. These are low energy (that is, stable).
Next (in terms of increasing energy) are the -bonding orbitals present in all
functional groups that contain double and triple bonds (e.g. carbonyl groups and
alkenes).
Higher energy still are the non-bonding orbitals present on atoms that have lone
pair(s) of electrons (oxygen, nitrogen, sulfur and halogen containing compounds).
All of the above 3 kinds of orbitals may be occupied in the ground state.
Two other sort of orbitals, called antibonding orbitals, can only be occupied by an
electron in an excited state (having absorbed UV for instance). These are the * an
d * orbitals (the * denotes antibonding). Although you are not too familiar with
the concept of an antibonding orbital just remember the following – whilst electron
density in a bonding orbital is a stabilising influence it is a destabilising influence
(bond weakening) in an antibonding orbital.
Antibonding orbitals are unoccupied in the ground state
UV
A transition of an electron from occupied to an unoccupied energy level can be
caused by UV radiation. For the time being be aware that commonly seen
transitions are  to * which correctly implies that UV is useful with compounds
containing double bonds.
A schematic of the transition of an electron from  to * is shown on the left.
Increasingenergy

23. When UV and visible radiation excites the molecule the electrons are temporarily
moved from one orbital (bonding orbital according to MOT) to another orbital
(Anti-bonding orbital according to MOT). These electrons may be σ, π or n (non-
bonding) electrons. (Time requires 10-8 sec)
Thus possible transitions are –
σ → σ*
n → σ*
π → π*
n → π*
(There is no n* orbital as n electrons don’t form bonds).

24. σ → σ* transition:
1. This type of transition takes place in the saturated centre of the molecules.
2. Here, an electron from a stable σ orbital goes to an unstable or anti-bonding
σ orbital (represented by σ*).
3. High energy is required to excite electrons present in the σ orbital as they
are held tightly. Therefore saturated compounds do not absorb radiation
from normal UV-Vis spectrophotometer which generates radiation of 190-
780 nm region.
n → σ* transition:
1. This type of transition occurs in saturated compounds containing heteroatom
which has an unshared electron pair.
2. Electron transits from unshared pair to anti-bonding σ orbital.
3. This type of transition may take place in halogen, sulfur, nitrogen and
oxygen containing compounds.
4. This transition requires high energy and therefore doesn’t occur in presence
of the EM radiation produced by normal UV-Vis spectrophotometer.

25. π → π* transition (K-band):
1. Takes place in unsaturated compounds containing double or triple bonds.
2. Electron transits from stable π orbital to an unstable/anti-bonding π orbital
(represented by π*).
3. It requires less energy as π bonds are sufficiently weaker than σ bonds.
Conjugated double bonds further lowers required energy. For example β-carotene
containing 11 conjugated double bonds absorbs energy at 451 nm (visible region).
n → π* transition (R-band):
1. This type of transition takes place in compounds having heteroatom which
contains unshared electron pair.
2. Electron transits from unshared pair to anti-bonding π orbital.
3. Requires lower energy and therefore respond to the radiation produced by
normal UV-Vis spectrophotometer.
4. Carbonyl compounds, cyanides etc. show this type of transition.

26. UV/VIS
Vacuum UV or Far UV
(λ<190 nm )

27. The polarity of the solvent affect the energy required for electronic
transition. This is described below. We need to remember that polarity
results from unequal distribution of electrons – the greater is the difference
the greater is the polarity.
n → π* transition:
The diagram above illustrates the polarity of the molecule at excited state
and ground state. If the solvent is polar, then dipole-dipole interaction
between the solvent molecule and sample molecule will be greatest at
ground state. Hence, energy of the grounds state is lowered significantly
but that of excited state is raised (or not lowered significantly). The result is
that more energy i.e. radiation of shorter wavelength is required to excite
the electrons.

28. π → π* transition:
Here polarity increases after transition.
Thus the polar solvent molecule will show greater interaction with the
excited state than with ground state. Hence, energy required for excitation is
decreased.
n → σ* transition:
The polarity decreases after transition to excited state. So the polar solvent
prefers the ground state. Hence more energy is required for excitation.
σ → σ* transition:
In saturated compounds (no atom with lone pair of electrons though)
transition doesn’t change polarity much. Thus solvent effect is not seen.

29. Impurities in the solvent affect the result in UV-Vis spectroscopy. The impurity will
absorb light hence the absorbance is exaggerated. This is why commercial
absolute ethanol should not be used to prepare sample solution as it contains
benzene as impurity which strongly absorbs light in the UV region.
Nature of the absorption band
The solvents used in sample preparation will absorb radiation at some specific
wavelength. So, radiation of that wavelength becomes unavailable for spectral
studies. For example, chloroform will strongly absorb radiation of below 245 nm.
Thus this solvent can’t be used in a sample that will be run at a wavelength lower
than 245 nm. The table below shows the minimum wavelength at which the
solvent can be used to make sample solution. For example, methanol should not
be used if the sample is to be run at wavelength lower than 203 nm.

30. Solvents
Minimum
wavelength for
1 cm cell, nm
Solvents
Minimum
wavelength for
1 cm cell, nm
Acetonitrile 190 Ethanol 204
Water 191 Ether 215
Hexane 201 Chloroform 237
Methanol 203 Carbon
tetrachloride
257

31. There are two types of groups present in a molecule which is responsible
for characteristic electromagnetic radiation absorption by the molecule.
These are described below –
Chromophore
A chromophore is a chemical group that absorbs light at a specific
frequency and so imparts color to a molecule.
It is defined as any isolated covalently bonded group that shows a
characteristic absorption in the ultraviolet or visible region.
When a chromophore is attached to a saturated hydrocarbon, produces a
molecule that absorbs a maximum of UV or visible energy at some specific
wavelength. Compounds containing a chropmophoric group called a
chromogen

32. Chromophore Structure Example of molecule max
Acetylene (carbon-carbon triple bond) C C Acetylene 173
Amide CONH2 Acetamide <208
Azo N N Azomethane 347
Carbonyl (ketone)
C
O Acetone 271
Carbonyl (Aldehyde)
C
O
H
Acetaldehyde 293
Ethylene (carbon-carbon double bond)
C C
Ethylene 234
Nitrile C N Acetonitrile <160
A compound may contain two or more chromophores and then the relative
position of the chromophores will determine the absorption of radiation.
Chemical structure of beta-carotene. The eleven conjugated double bonds that
form the chromophore of the molecule are highlighted in red.

33. An auxochrome is a group of atoms attached to a chromophore which modifies
the ability of that chromophore to absorb light. They themselves fail to produce the
colour; but when present along with the chromophores in an organic compound
intensifies the colour of the chromogen.
Auxochrome group contains at least one atom with lone pair of electrons.
Examples include the hydroxyl group (-OH), the amino group (-NH2), the
aldehyde group (-CHO), ─NHR, ─NR2.
Benzene shows absorption maximum at 255 nm whereas aniline at 280 nm.
=255 nm = 280 nm

34. Absorption and intensity shifts
1. Bathochromic shift or Red shift: It is the displacement/shift of max (absorption
maximum) towards longer wavelength. This may occur due to solvent effect, conjugation or
auxochrome addition. It is known as red shift.
2. Hypsochromic shift or Blue shift: it is the displacement of max towards the shorter
wavelength. This is usually due to solvent effect.
3. Hyperchromic effect: It is the effect of increased intensity of radiation absorption by the
molecule.When absorption intensity (ε) of a compound is increased, it is known as
hyperchromic shift.
If auxochrome introduces to the compound, the intensity of absorption increases.
4. Hypochromic effect: It is the effect of decreased intensity of radiation absorption by the
molecule.When absorption intensity (ε) of a compound is decreased, it is known as
hypochromic shift.

35. Wavelength ( λ )
Absorbance(A)
Shifts and Effects
Hyperchromic shift
Hypochromic shift
Red
shift
Blue
shift
λmax

36. Oxygen of atmosphere absorbs UV light of below 200 nm. So it is difficult to analyze a
sample by running it at wavelength below 200 nm. If we want to measure absorption of UV
radiation below 200 nm we must remove all the air from the instrument.
Thus vacuum is used. So the UV radiation below 200 nm is known as vacuum ultraviolet
region.
This region is relatively uninformative as it excites the σ electrons. Above this region, π
electrons are excited. The double and triple bonds are comparatively more informative in the
analysis of a compound.
Explanation of electronic transitions in polyenes

37. As we can see that, ethylene with only one double bond show most absorption
at 175nm, whereas 1,3-butadiene show most absorption at 217 nm. Generally
the longer the conjugated system the longer is the wavelength of maximum
absorption. For example, β-carotene contains 11 double bonds and show
maximum absorption at 451 nm (visible range).
When two atomic orbitals are combined, two molecular orbitals are produced.
One is of high energy. This is the unstable molecular orbital called the
anti-bonding orbital. Another is of lower energy and this is the stable orbital.
Electrons are present in the stable orbital.
Below is the molecular orbitals of 1,3-butadiene. There are two double bonds
in this molecule. Each double bond forms two molecular orbital – one bonding
orbital and one anti-bonding orbital.
The two bonding orbitals of the two double bonds combine to form two
molecular orbitals. Among them one is higher in energy than ethylene
molecule and another is lower. The higher energy molecular orbital is the
HOMO.

38. Similarly, the two anti-bonding orbitals of two double bonds will produce two new
molecular orbitals. One of them will be lower in energy than the excited ethylene
molecule and another will be higher in energy than excited ethylene molecule
(excited is the keyword – in HOMO determination we took the molecular orbital that was
higher in energy than the ground state ethylene molecule, not excited ethylene
molecule). Among the two the molecular orbital with lower energy than the excited
ethylene molecule is the LUMO. One should remember that anti-bonding molecular
orbitals are normally unoccupied and becomes occupied when electrons are excited.
As we can see that the energy difference between LUMO and HOMO of 1,3-butadiene
will lower than that of ethylene. (Ethylene has only one anti-bonding orbital which is the
LUMO for ethylene, 1,3-butadiene has two anti-bonding orbitals where the one which
has lower energy than LUMO of ethylene is the LUMO of 1,3-butadiene).
As the double bonds/conjugation increases, so the energy difference between LUMO
and HOMO decreases (e.g. in ethylene the difference is 176 kcal/mol and in
1,3-butadiene the difference is 131 kcal/mol). Thus increase of double bonds
decrease energy required for absorption.

40. Odd Roll-Write the

41. Spectrometer
Spectrometer is an instrument that is used to measure a physical property over a
specific region of the spectrum of that property.
The physical property is usually the intensity of light but other properties can also
be used. For example in mass spectrometer, relative abundance of particles is
measured over mass-to-charge ratio.
Simply spectrometer is any instrument used to measure spectrum.
Spectrophotometer
A spectrophotometer is an instrument used to measure the intensity of light over
a specific region of the electromagnetic spectrum.
In another words, a spectrophotometer is a spectrometer with a photomultiplier that
is used to detect light and determine its intensity as a function of wavelength.

42. A colorimeter is a spectrophotometer which is designed to detect only
the absorption at the visible region i.e. it will measure the intensity of light
at the visible region of the electromagnetic spectrum.

43. Basic Parts
1. A Stable and cheap radiant energy source.
2. A monochromator, to break the polychromatic radiation into component wavelength (or) band
s of wavelengths.
3. Transport vessels (cuvettes), to hold the sample.
4. A Photosensitive detector and an associated readout system.
The UV-visible spectrometer is a spectrophotometer designed to work in the UV and visible range
of the electromagnetic spectrum.

44. A source for UV and visible light is perquisite. A UV-visible spectrophotometer works at
around 190-800 nm range. Usually two sources are used –
1. Hydrogen or deuterium discharge lamp: It emits electromagnetic radiation of
185-365 nm wavelengths.
2. Tungsten lamp (6V or 12V): It emits electromagnetic radiation of 350-800 nm.
Since, none of them produces the complete range of the target spectrum. They are us
ed simultaneously.
Requirements for radiation source
1. The radiation should be continuous
2. Its spectrum should include all the wavelengths required for the analysis.
3. The power of the beam should be such that, the solution can transmit some or all of
the radiation energy at all wavelengths.
4. The power of the beam must remain constant throughout the measurement.

45. Various UV radiation sources are as follows
a. Tungsten filament lamp
b. Hydrogen discharge lamp
c. Deuterium lamps
d. Xenon discharge lamp
e. Mercury arc lamp

46. The monochromator disperses the polychromatic light by means of a
prism or grating (in grating there is a flat surface having alternating
reflective and non-reflective portions). The desired monochromatic light
is obtained by changing the angular position of prism or grating and this
monochromatic light is allowed to pass to sample.
Monochromatic Light: The light having a single wavelength and
frequency i.e. the light that vibrates at a single wavelength.
Polychromatic Light: The light having several colours i.e. light
having electromagnetic radiation of several wavelengths.

47. Grating

48. The sample is kept in a small tube of square cross section called “cuvette” i.e. the tube
has four sides.
There are two opposite sides through which
the light is supposed to pass (i.e. these two
sides form the optical window). The other two
sides are usually unclear (in some cases, all
four
sides may be clear) and used for handling.
The cuvette (at least the optical window of
the cuvette) is made of glass, plastic or fused
quartz. The glass and plastic cuvettes are
used to measure at visible spectrum while
quartz cuvettes are used to measure at
ultraviolet spectrum. The length of the optical
window is usually 1cm.

49. The detector converts the radiant energy into electrical energy.
Most detectors depend on the photoelectric effect. The current is then proportional to the light intensity and
therefore a measure of it.
Important requirements for a detector include
1. High sensitivity to allow the detection of low levels of radiant energy
2. Short response time
3. Long term stability
4. An electric signal which easily amplified for typical readout apparatus.
In spectrophotometer the detector is a photomultiplier tube. It consists of following parts
a. One cathode (electron emitter): It emits electrons when the photon of radiation strikes it.
b. Several electrodes called dynodes: After the cathode there are a number of electrodes called the
dynodes. The dynode next to cathode is more positive than cathode and produces several electrons for each
electron striking it. The dynode next to the 1st dynode is even more positive and it also produces more
electrons. By the time electrons are collected at anode, for each photon about 106-107 electrons are
generated.
c. One anode: It receives the electrons generated on the way. The resulting current is then measured.

50. Recorder
From the measurement of current obtained from
transmitted light, it plots a graph of absorbance
versus wavelength.
The chart recorder
Chart recorders usually plot
absorbance against
wavelength.
This particular substance has what are
known as absorbance peaks at 255 and 395 nm.

51. Firstly, the machine is started. The machine will run and take a reading of the
intensity of the radiation source. This is the incident light intensity.
The sample is placed inside the instrument in cuvette. Light is generated by the
radiation source and then passed through the monochromator. The
monochromatic light is passed through the sample and then the intensity of the
transmitted light is measured in the detector. From the intensity of the incident
light and intensity of the transmitted light the computer will calculate and show
the absorbance.
Then the blank/reference (sample solution minus the sample molecules) is
placed in the instrument.
The intensity of the transmitted light is measured again. From these two values
the relative intensities light before passing the sample molecules and after
passing the sample molecules can be determined.
Entrance slit
Exit slit
Detector
Cuvette
Red
Violet
Prism
MonochromatorLight source
I0
I
Readout
device

52. The Spectroscopic Process
1. In UV spectroscopy, the sample is irradiated with the broad spectrum
of the UV radiation
2. If a particular electronic transition matches the energy of a certain
band of UV, it will be absorbed
3. The remaining UV light passes through the sample and is observed
4. From this residual radiation a spectrum is obtained with “gaps” at
these discrete energies – this is called an absorption spectrum





53. So basically there are two measurements involved.
a) Single-beam spectrophotometer
b) Double-beam spectrophotometer
Single beam spectrophotometer
A single-beam instrument uses only single beam of radiation through a single cell. The
reference cell is used to set the absorbance scale at zero for the wavelength to be
studied. It is then replaced by sample cell to determine the absorbance of the sample at
that wavelength.
In some single beam spectrophotometer a “zeroing” method is used where the
absorbance by the reference is set as baseline value and then the absorption by the
sample is measured relative to it.
Double beam spectrophotometer
In single beam spectrophotometer there is a necessity to make two separate
measurements. This has been eliminated with the double beam spectrophotometer.

54. A double beam instrument divides the radiation into two beams of equal
intensity which are passed through two separate cells.
In this instrument the blank (the reference cell) and the sample solution
(sample cell) is placed simultaneously in the spectrophotometer. The light
generated by the radiation source is passed through the monochromator and
the monochromatic light is split into two beams (this is done by a rotating disc).
One beam passes through the reference cell and other passes through the
sample cell. Then the transmitted light (whether from reference cell or sample
cell) is sent to the detector. The detector will measure the intensities of the
transmitted light. A computer will analyze the intensities and subtract the
intensity of transmitted light from sample cell from the intensity of the
transmitted light from reference cell.
Thus the absorption by the rest of the part of solution is cancelled.

55. Double-Beam in time spectrophotometers

58. 2 (b).Odd Roll-Write the

59. 1. Structure elucidation of organic compounds: UV spectroscopy is useful in the
structure elucidation of organic molecules,(the presence or absence of unsaturation, the
presence of hetero atoms). From the location of peaks and combination of peaks, it can be
concluded that whether the compound is saturated or unsaturated, hetero atoms are
present or not etc.
2. Identification of an unknown compound- An unknown compound can be identified with
the help of UV spectroscopy. The spectrum of unknown compound is compared with the
spectrum of a reference compound and if both the spectrums coincide then it confirms the
identification of the unknown substance.
3. Quantitative analysis (Measurement of Concentration ): UV absorption spectroscopy
can be used for the quantitative determination of compounds that absorb UV radiation. This
determination is based on law. A = abc
4. Detection of Impurities: Additional peaks can be observed due to impurities in the
sample and it can be compared with that of standard raw material. Benzene appears as a
common impurity in cyclohexane. Its presence can be easily detected by its absorption at
255 nm.

60. 5. Qualitative analysis: In UV absorption spectroscopy identification is done by comparing
the absorption spectrum with the spectra of known compounds. UV absorption spectroscopy is
generally used for characterizing aromatic compounds and aromatic olefins.
6. Chemical kinetics: Kinetics of reaction can also be studied using UV spectroscopy. The
UV radiation is passed through the reaction cell and the absorbance changes can be observed.
7. Molecular weight determination: Molecular weights of compounds can be measured
spectrophotometrically by preparing the suitable derivatives of these compounds.
8. As HPLC detector: A UV/Vis spectrophotometer may be used as a detector for HPLC.
9. Detection of extent of conjugation- The extent of conjugation in the polyenes can be
detected with the help of UV spectroscopy.
10. Determination of configurations of geometrical isomers- It is observed that
cis-alkenes absorb at different wavelength than the trans-alkenes. The cis-isomer suffers
distortion and absorbs at lower wavelength as compared to trans-isomer.

61. In 1941, Robert Burns Woodward first put forward a set of rules to predict the λmax of a given
compound in UV-Vis spectroscopy. These rules were applicable to open chain and 6-membered ring
dienes.
Later the rules were modified by Fischer Scott and Louis Fieser. This is known as the Wood
ward-Fieser rules (This rule works well when the conjugated system is only 4 double bonds long.
When more than four double bonds are present, one must use the Fieser-Kuhn rules instead).
Various types of double bonds in conjugation are:
1. Homoannular dienes: It is a cyclic diene having conjugated doubles bonds in the same ring.
e.g. Toluene, Naphthalene.
2. Acyclic dienes: Diene contained in an open chain system. Where the basic unit is butadiene.
3. Heteroannular dienes: It is a cyclic diene in which double bonds in conjugation are present in
different ring.
4. Endocyclic conjugated double bond: It is the double bond present in the ring.
5. Exocyclic conjugated double bond: It is a double bond is a double bond, part of the conjugated
system and formed by any carbon atom of any ring but present outside of the ring.

62. According to the rules each type of diene or triene system have a fixed
value at which absorption takes place.
This constitutes the basic value or parent value.
The contributions made by various alkyl substances or ring residue,
double bonded extending conjugation and polar group such as –Cl,
-Br, -OR are added to the basic value to obtain λmax for a particular
compound.
Ring residue is a C-C bond, not a part of the conjugated system; but
attached to any one of the carbon atoms of the conjugated poyene
system.

63. The parent values and contribution of different substituents or groups
are given below (Conjugated trienes and dienes/solvent ethanol/pi-pi*
transition)
1. Parent Values:
Cyclic conjugated dien or butadiene 217nm
Acyclic triene 245 nm
Heteroannular conjugated diene 215 nm
Homoannular conjugated diene 253 nm
2. Increment for each substituent:
Alkyl substituent or ring residue 5 nm
Exocyclic double bond 5 nm
Double bond extending conjugation 30 nm
3. Auxochromes
–OR +6 nm
–SR +30 nm
–BR, -Cl +5 nm
-NR2 +60 nm
-OCOCH3 0

65. Class Structure Wavelength value (nm)
Parent/base/core When X = Alkyl group or ring residu
e, 246.
When X = H, 250.
When X = OH or OR, 230.
Alkyl or ring residue on ortho or meta position. 3
Alkyl or ring residue on para position. 10
OH, OR at ortho or meta position. 7
OH, OR at para position. 25
NH2 at ortho or meta position. 13
NH2 at para position. 58
NHAc at ortho or meta position. 20
NHAc at para position. 45
Cl at ortho or meta position. 0
Cl at para position 10
Br at ortho or meta position. 2
Br at para position. 15
C
O
X

67. REFERENCES

68. Reference Books
• Introduction to Spectroscopy
– Donald A. Pavia
• Elementary Organic Spectroscopy
– Y. R. Sharma
• Physical Chemistry
– Puri, Sharma & Pathaniya

69. Thanks to all