Uv visible spectroscopy- madan

INSTRUMENTAL TECHNIQUES
OF ANALYSIS
MADAN SIGDEL
M. Pharm. (Pharmacology)

UNIT - ONE
Visible and ultraviolet spectroscopy
a. Introduction and elemental theory
b. Instrumentation, measurement and sample handling
c. Applications
I. Chromophores- isolated functional group
II. Quantitative studies concentration, rate
measurements and acid/base dissociation

SPECTROSCOPY
 It is the 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.

ELECTROMAGNETIC RADIATION
Electromagnetic radiation consist of discrete packages
of energy which are called as photons.
A photon consists of an oscillating electric field (E) & an
oscillating magnetic field (M) which are perpendicular to
each other.

ELECTROMAGNETIC RADIATION
Frequency (ν): – It is defined as the number of times
electrical field radiation oscillates in one second.
The unit for frequency is Hertz (Hz).
1 Hz = 1 cycle per second
Wavelength (λ): – It is the distance between two
nearest parts of the wave in the same phase i.e.
distance between two nearest crest or troughs.

The relationship between wavelength &
frequency can be written as: c=νλ
As photon is subjected to energy, so
E= hν= hc/λ

 a) when a group is more polar in ground state than exited state than
increase polarity of the solvent stabilizes the non-bonding electrons
in the ground state because H-bonding. Thus absorption shifted to
lower wave length.
 b) when the group is more polar in the exited state, then absorption
get shifted to longer wave length with increased polarity of the
solvent which helps in stabilizing the non-bonding electrons in the
exited state.
 The increased in polarity of the solvent generally shifts n-π* and n-σ
* bands to shorter wav lengths and π-π* bands to longer wave
lengths.

PRINCIPLES OF SPECTROSCOPY
 The principle is based on the measurement of spectrum
of a sample containing atoms / molecules.
 Spectrum is a graph of intensity of absorbed or emitted
radiation by sample verses frequency (ν) or wavelength
(λ).
 Spectrometer is an instrument design to measure the
spectrum of a compound.

 1. Absorption Spectroscopy:
• An analytical technique which concerns with the
measurement of absorption of electromagnetic
radiation.
• e.g. UV (185 - 400 nm) / Visible (400 - 800 nm)
Spectroscopy, IR Spectroscopy (0.76 - 15 μm)
 2. Emission Spectroscopy:
• An analytical technique in which emission (of a
particle or radiation) is dispersed according to some
property of the emission & the amount of dispersion is
measured.
• e.g. Mass Spectroscopy

INTERACTION OF EMR WITH MATTER
1. Electronic Energy Levels:
 At room temperature the molecules are in the lowest
energy levels E0.
 When the molecules absorb UV-visible light from EMR,
one of the outermost bond / lone pair electron is
promoted to higher energy state such as E1, E2, …En,
etc is called as electronic transition and the difference is
as: ∆E = h ν = En - E0
where (n = 1, 2, 3, … etc) ∆E = 35 to 71 kcal/mole

2. Vibrational Energy Levels:
• These are less energy level than electronic energy
levels.
• The spacing between energy levels are relatively
small i.e. 0.01 to 10 kcal/mole.
• e.g. when IR radiation is absorbed, molecules are
excited from one vibrational level to another or it
vibrates with higher amplitude.

3. Rotational Energy Levels:
• These energy levels are quantized & discrete.
• The spacing between energy levels are even
smaller than vibrational energy levels.
∆Erotational < ∆Evibrational < ∆Eelectronic

THEORY INVOLVED
• When a beam of light falls on a solution or homogenous
media ,a portion of light is reflected ,from the surface of
the media, a portion is absorbed within the medium and
remaining is transmitted through the medium.
• Thus if I0 is the intensity of radiation falling on the media
• Ir is the amount of radiations reflected,
• Ia is the amount of radiation absorbed &
• It the amount of radiation transmitted then
I0 = Ir + Ia + It

ABSORPTION LAWS
 Lambert’s law
 Beer’s law
 Beer-lambert’s law

LAWS GOVERNING ABSORPTION OF RADIATION
 The two laws related to the absorption of radiation are:
Beer’s law ( related to concentration of absorbing
species)
 Lambert’s law (related to thickness/path length of
absorbing species)
 These two laws are applicable under the following
condition:
I = I a + I t
I = Intensity of incident light
I a = Intensity of absorbed light
I t =Intensity of transmitted light and
No reflection/scattering of light takes place

Beer’s law
“The intensity of a beam of monochromatic light decreases
exponentially with increase in the concentration of absorbing
species. arithmetically
Accordingly, - dI / dc α I
(The decrease in the intensity of incident light (I) with
concentration c is proportional to the intensity of incident light
(I))
-dI / dc = kI
(removing and introducing the constant of proportionality ‘k’)
-dI / I = k dc (rearranging terms)
-In I = kc + b ……Equation (1)
(on integration , b is constant of integration)
When concentration = 0, there is no absorbance. Hence I= Io
Substituting in equation 1,
-In Io = k*0 + b
-In Io = b

Substituting the value of b, in equation 1,
-In I = kc –InIo
In Io – In I = kc
In Io/I = kc (since log A-log B = log A/B)
Io / I = e kc (removing natural logarithm)
I / Io = e –kc (making inverse on both sides)
I = Io e -kc ….Equation (2) (equation of Beer’s law)

Lambert’s law
“The rate of decrease of intensity (monochromatic light)
with the thickness of the medium is directly
proportional to the intensity of incident light”
i.e. –dI / dt α I
This equation can be simplified similar to equation 2 to
get the following equation (by replacing ‘c’ with ‘t’)
I = Io e –kt ….. Equation (3)
[equation of Lambert’s law]

BEER – LAMBERT,S LAW
Equations (2) and (3) can be combined to get
I= Io e –kct
I = Io 10 –kct
(converting natural algorithm to base 10)
I / Io = 10 –kct (rearranging terms)
Io / I = 10 kct (inverse on both side Log
Io / I = kct (taking log on both sides) ….. Equation 4
It can be learnt that transmittance (T) = I / Io and Absorbance
(A) = log 1 / T
Hence A = log 1 / I/ Io
A = log Io /I ……. Equation 5

Using Equation 4 & 5 ,
Since A= log Io /I
and log Io /I = Kct
we can infer that,
A= Kct (instead of K, we can use ε)
A= ε ct (Equation of beer – Lambert’s law)
Where:
A – Absorbance or optical density or extinction coefficient.
ε – Molecular extinction coefficient
c – Concentration of the drug (mol/lit)
t – Path length (normally 10mm or 1cm)

 Absorbance of a material is a logarithmic ratio of the
amount of radiation falling upon a material to the amount
of radiation transmitted through the material. A=log Io/I
 transmittance is the fraction of incident light
(electromagnetic radiation) at a specified wavelength that
passes through a sample T=I/Io

DEVIATION FROM THE BEER’S LAW
 Beer’s law: it states that if we plot absorbance A
against concentration C a straight line passing through
origin is obtained, but usually a deviation from a linear
relationship between concentration and absorption
and an apparent failure of beer’s law. There are two
type of deviation :
 POSITIVE DEVIATION : When a small change in
concentration produces a greater change in
absorbance.
 NEGATIVE DEVIATION : When a large change in
concentration produces a smaller change in
absorbance.

REASONS FOR DEVATION FROM BEER’S LAMBERT LAW
 Instrumental deviation
 Physicochemical change in solution
 Instrumental deviation: Factors like stray radiation,
improper slit width, fluctuation in single beam and when
monochromatic light is not used can influence the
deviation.
 Physicochemical change in solution: Factors like
association, dissociation, ionization (change in pH),
faulty development of color (incompletion of reaction),
refractive index at high concentration, can influence
such deviation.

References
 Instrumental Methods of Chemical Analysis by Gurdeep
R. Chatwal ( page no : 108 to 113 )
 Instrumental Methods of Analysis by Scoog and West)
 http://en.wikipedia.org/wiki .
 Instrumental analysis by willard and merit ( page no :
160 to 163 )

PRINCIPLE OF UV-VISIBLE SPECTROSCOPY
 The UV radiation region extends from 10 nm to 400 nm
and the visible radiation region extends from 400 nm to
800 nm.
Near UV Region: 200 nm to 400 nm
Far UV Region: below 200 nm
 Far UV spectroscopy is studied under vacuum
condition.
 The common solvent used for preparing sample to be
analyzed is either ethyl alcohol or hexane.

PRINCIPLES OF UV-VISIBLE SPECTROSCOPY
 UV-visible spectroscopy measure the response of a
sample to ultraviolet and visible range of
electromagnetic radiation.
 Molecules have either n, π or б Electrons.
 These electrons absorb UV radiation & undergoes
transitions from ground state to excited state.

 The absorption of uv radiation brings about the
promotion of an electron from bonding to antibonding
orbital.
 The wavelength of radiation is slowly changed from
minimum to maximum in the given region, and the
absorbance at every wavelength is recorded.
 Then a plot of energy absorbed Vs wavelength is called
absorption spectrum. The significant features:
λmax (wavelength at which there is a maximum
absorption)
єmax (The intensity of maximum absorption)
The UV spectrum depends on
 solvents
 concentration of solution

 σ electrons
 π electrons
 n electrons
 σ -electrons: these electrons are involved in saturated
bonds such as those b/w carbon of hydrogen in paraffin.
These bands are known as σ bonds. As amount of
energy required to excite electrons in σ bonds in much
more than that produced by UV-light.
 Compounds containing σ bonds do not absorb UV
radiations. Therefore paraffin compounds are very use
full as solvents.

 π-electrons: these electrons are involved in unsaturated
hydrocarbons. Typical compounds with π bonds are
trienes and aromatic compounds.
 n-electrons: these are electrons which are not involved in
bonding b/w atoms in molecule.
Eg: organic compounds containing N,O & halogen.
 As n electrons can be excited by UV radiations, any
compound that contain atoms like N,O,S, halogens or
unsaturated hydrocarbons may absorb UV radiation.

1. σ → σ* transition
 σ electron from orbital is excited to corresponding anti-
bonding orbital σ*.
 The energy required is large for this transition.
 e.g. Methane (CH4) has C-H bond only and can
undergo σ → σ* transition and shows absorbance
maxima at 125 nm.

2. π → π* transition
• π electron in a bonding orbital is excited to
corresponding anti-bonding orbital π*.
• Compounds containing multiple bonds like alkenes,
alkynes, carbonyl, nitriles, aromatic compounds, etc
undergo π → π* transitions.
• e.g. Alkenes generally absorb in the region 170 to
205 nm.

3. n → σ* transition
• Saturated compounds containing atoms with lone pair
of electrons like O, N, S and halogens are capable of n
→ σ* transition.
• These transitions usually requires less energy than σ →
σ* transitions.
• The number of organic functional groups with n → σ*
peaks in UV region is small (150 – 250 nm).

 4 • n → π* transition
• An electron from non-bonding orbital is promoted to
anti-bonding π* orbital.
• Compounds containing double bond involving hetero
atoms (C=O, C≡N, N=O) undergo such transitions.
• n → π* transitions require minimum energy and show
absorption at longer wavelength around 300 nm.

5 • σ → π* transition &
6• π → σ* transition
 These electronic transitions are forbidden transitions &
are only theoretically possible.
 Thus, n → π* & π → π* electronic transitions show
absorption in region above 200 nm which is accessible
to UV-visible spectrophotometer
.How does a spectrophotometer work.mp4
 The UV spectrum is of only a few broad of absorption.

TERMS USED IN UV/ VISIBLE SPECTROSCOPY
CHROMOPHORE
The part of a molecule responsible for imparting color, are
called as chromospheres.
OR
The functional groups containing multiple bonds capable
of absorbing radiations above 200 nm due to n → π* & π
→ π* transitions.
e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc

CHROMOPHORE
To interpretate UV – visible spectrum following points
should be noted:
1. Non-conjugated alkenes show an intense absorption
below 200 nm & are therefore inaccessible to UV
spectrophotometer.
2. Non-conjugated carbonyl group compound give a
weak absorption band in the 200 - 300 nm region.

CHROMOPHORE
e.g. Acetone which has λmax = 279 nm
and that cyclohexane has λmax = 291 nm.
When double bonds are conjugated in a compound
λmax is shifted to longer wavelength.
e.g. 1,5 - hexadiene has λmax = 178 nm
2,4 - hexadiene has λmax = 227 nm
CH3
C
CH3
O
O
CH2
CH2
CH3
CH3

CHROMOPHORE
3. Conjugation of C=C and carbonyl group shifts the λmax
of both groups to longer wavelength.
e.g. Ethylene has λmax = 171 nm
Acetone has λmax = 279 nm
Crotonaldehyde has λmax = 290 nm
CH3
C
CH3
O
CH2 CH2
C
CH3
O
CH2

AUXOCHROME
The functional groups attached to a chromophore
which modifies the ability of the chromophore to
absorb light, altering the wavelength or intensity of
absorption.
OR
The functional group with non-bonding electrons that
does not absorb radiation in near UV region but when
attached to a chromophore alters the wavelength &
intensity of absorption.

AUXOCHROME
e.g. Benzene λmax = 255 nm
Phenol λmax = 270 nm
Aniline λmax = 280 nm
OH
NH2

ABSORPTION AND INTENSITY SHIFTS

• When absorption maxima (λmax) of a compound
shifts to longer wavelength, it is known as
bathochromic shift or red shift.
• The effect is due to presence of an auxochrome
or by the change of solvent.
• e.g. An auxochrome group like –OH, -OCH3
causes absorption of compound at longer
wavelength.
• Bathochromic Shift (Red Shift)1

• In alkaline medium, p-nitrophenol shows red
shift.
p-nitrophenol
λmax = 255 nm λmax = 265 nm
• Bathochromic Shift (Red Shift)1
OH
N
+ O
-
O
OH
-
Alkaline
medium
O
-
N
+ O
-
O

• When absorption maxima (λmax) of a compound
shifts to shorter wavelength, it is known as
hypsochromic shift or blue shift.
• The effect is due to presence of an group
causes removal of conjugation or by the
change of solvent.
• Hypsochromic Shift (Blue Shift)2

• Aniline shows blue shift in acidic
medium, it loses conjugation.
Aniline
• Hypsochromic Shift (Blue Shift)2
NH2
H
+
Acidic
medium
NH3
+
Cl
-

• 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.
Pyridine 2-methyl pyridine
ε = 2750 ε = 3560
• Hyperchromic Effect3
N N CH3

• When absorption intensity (ε) of a compound is
decreased, it is known as hypochromic shift.
Naphthalene 2-methyl naphthalene
ε = 19000 ε = 10250
CH3
• Hypochromic Effect4

Wavelength ( λ )
Absorbance(A)
SHIFTS AND EFFECTS
Hyperchromic shift
Hypochromic shift
Red
shift
Blue
shift
λmax

WOODWARD-FEISER RULE
 Woodward (1941) : gave certain rules for
correlating max with molecular structure
 Scott-Feiser (1959): modified rule with more
experimental data, the modified rule is known
as Woodward-Feiser rule
 used to calculate the position of max for a given
structure by relating the position and degree of
substitution of chromophore.

1. HOMOANNULAR DIENE: CYCLIC DIENE HAVING
CONJUGATED DOUBLE BONDS IN THE SAME RING.
2. Heteroannular diene: cyclic diene having
conjugated double bonds in different ring

2. Endocyclic double bond: double bond present in a
ring
3. Exocyclic double bond: double bond in which one of
the doubly bonded atoms is a part of a ring system
Ring A Ring B
Ring A has one exocyclic and endocyclic double bond.
Ring B has only one endocyclic double bond

WOODWARD-FEISER RULE FOR CONJUGATED
DIENES, TRIENES, POLYENES
 Each type of diene or triene system is having a
certain fixed value at which absorption takes
place; this constitutes the BASIC VALUE or
PARENT VALUE
 The contribution made by various alkyl
substituents or ring residue, double bonds
extending conjugation and polar groups such as
–Cl, -Br are added to the basic value to obtain
max for a particular compound

PARENT VALUES AND INCRIMENTS FOR
DIFFERENT SUBSTITUENT/GROUPS
a) Parent value
i. Acyclic conjugated diene and : 215nm
heteroannular conjugated diene
ii. Homoannular conjugated diene : 253nm
iii. Acyclic triene : 245nm

b) INCREMENTS
i. Each alkyl substituents or ring residue : 5 nm
ii. Exocyclic double bond : 5 nm
iii. Double bonds extending conjugation : 30nm
c) Auxochrome : -OR : 6 nm
-SR : 30 nm
-Cl, -Br : 5 nm
-NR2 : 60nm
-OCOCH3 : 0 nm

CALCULATE MAX FOR 1,4- DIMETHYLCYCLOHEX-1,3-DIENE
CH3 CH3 CH3 CH3
Parent value for homoannular ring : 253 nm
Two alkyl substituents : 2 * 5= 10 nm
Two ring residue : 2 * 5= 10 nm
calculated value : =273 nm
observed value : = 263 nm

CALCULATE MAX
 Parent value for heteroannular diene : 215 nm
Four ring residue : 4 * 5 = 20 nm
calculated value : 235 nm
observed value : 236 nm

CALCULATE MAX
 Parent value for heteroannular diene : = 215 nm
 Three ring residue : 3 * 5 = 15 nm
 One exocyclic double bond : = 5 nm
 Calculated value : = 235 nm
 Observed value : = 235 nm

WOODWARD-FEISER RULES FOR ,-
UNSATURATED CARBONYL COMPOUNDS
a) Parent values
i. ,-unsaturated acyclic or six membered ring : 215 nm
ketone
ii. ,-unsaturated five – membered ring ketone : 202nm
iii. ,-unsaturated aldehyde : 207 nm
b) Increments
i. Each alkyl substituent or ring residue
at  position : 10 nm
at  position : 12 nm
at  position : 18 nm

ii. EACH EXOCYCLIC DOUBLE BOND : 5 NM
iii. Double bond extending conjugation : 30 nm
iv. Homoannular conjugated diene : 39 nm
v. Auxochromes position
  
-OH 35 30 50
-OR 35 30 17
-SR - 85 -
-OCOCH3 6 6 6
-Cl 15 12 -
-NR2 - 95 -

CALCULATE MAX CH3-C(O)-C(CH3)=CH2
O
CH3-C-C= CH2
CH3
 Parent value for ,-unsaturated acyclic : 215 nm
ketone
 one alkyl substituent in  position : 10 nm
 calculated value = 225 nm
 observed value = 220 nm

CALCULATE MAX
 Parent value for ,-unsaturated 6 : 215 nm
membered cyclic ketone
One ring residue at  position : 10nm
Two ring residue at  position : 2* 12 = 24 nm
Double bond exocyclic to two ring : 2* 5 = 10nm
calculated value : 259nm
observed value : 256nm

CALCULATE MAX
CH3
C=CH-C-CH3
CH3
C-CH=CH-CH3
CH3
 Parent ,-unsaturated acyclic
ketone 215
 2  alkyl substitute 24
 Calculated value 239nm
 Parent acyclic conjugated
diene 215
2 alkyl subst. 10
2 ring residue 10
Exocyclic double bond 5
 Calculated value 240

SOLVENT EFFECTS
 The most suitable solvent is one which does not
it self absorb in the region under investigation.
 A dilute sample solutions is preferred for analysis
 Most commonly used solvent is 95% ethanol. It
is best solvent as it is cheap, transparent down
to 210µm.

 Commercial ethanol should not be used, because it has
benzene ring which strongly absorb UV region.
 Some other solvents are used which are transparent
above 210µm are n-hexane, methyl alcohol,
cyclohexane, acetonitrile, diethylether.
 Hexane & other hydrocarbons can be used because
these are less polar and have least interactions with the
molecular under investigations.

 For UV spectroscopy, ethanol, water and cyclohexane serve the
purpose best.
 The position as well as the intensity of absorption maximum get
shifted for a particular chromophore by changing the polarity of the
solvent.
 By increasing the polarity of the solvent, compounds such as dienes
and conjugated hydrocarbons do not experience any appreciable
shift.
 Hence, in general the absorption maximum for the non-polar
compounds is usually shifted with the change in polarity of the
solvents. α,β-unsaturated carbonyl compounds show two different
shifts.

INSTRUMENTATION
Various components of UV spectrometers are as
follows:
 Radiation source:
 Monochromators
 Sample cells
 Detectors
 Readout device

80
AMPLIFIER
DETECTOR
RECORDER
Radiation
source
Sample
holder
Monochromator
BASIC COMPONENTS IN UV-VISIBLE
SPECTROPHOTOMETERS
analysisHow does a spectrophotometer
work.mp4

SOURCE OF RADIANT ENERGY:
REQUIREMENTS OF AN IDEAL SOURCE
 It should be stable and should not allow
fluctuations.
 It should emit light of continuous spectrum of
high and uniform intensity over the entire
wavelength region in which it’s used.
 It should provide incident light of sufficient
intensity for the transmitted energy to be
detected at the end of optic path.
 It should not show fatigue on continued use.

FOR VISIBLE RADIATION
TUNGSTEN HALOGEN LAMP
 Its construction is similar
to a house hold lamp.
 The bulb contains a
filament of Tungsten
fixed in evacuated condition
and then filled with inert gas.
 The filament can be heated up to 3000 k, beyond
this Tungsten starts sublimating .

 To prevent this along with inert gas some amount
of halogen is introduced (usually Iodine).
 Sublimated form of tungsten reacts with Iodine to
form Tungsten –Iodine complex.
 Which migrates back to the hot filament where it
decomposes and Tungsten get deposited.
DEMERIT:
It emits the major portion of its radiant energy in
near IR region of the spectrum.

SOURCE FOR UV RADIATION:
I.HYDROGEN DISCHARGE LAMP:
 In Hydrogen discharge lamp pair of electrodes is
enclosed in a glass tube (provided with silica or
quartz window for UV radiation to pass trough)
filled with hydrogen gas.
 When current is passed trough these electrodes
maintained at high voltage, discharge of electrons
occurs which excites hydrogen molecules which in
turn cause emission of UV radiation.

II.DEUTERIUM LAMP:
 It’s similar to Hydrogen discharge lamp but instead of
Hydrogen gas, Deuterium gas is used.
MERIT:
 Intensity of radiation is more as compare to Hydrogen
discharge lamp.
DEMERIT:
 Expensive.

III. XENON DISCHARGE LAMP:
 It possesses two tungsten electrodes separated
by some distance.
 These are enclosed in a glass tube with quartz or
fused silica and xenon gas is filled under
pressure.
 An intense arc is formed between electrodes by
applying high voltage. This is a good source of
continuous plus additional intense radiation.
DEMERIT:
 The lamp since operates at high voltage becomes
very hot during operation and hence needs
thermal insulation.

MERCURY ARC LAMP:
 In mercury arc lamp, mercury vapour is stored under
high pressure and excitation of mercury atoms is done
by electric discharge.
DEMERIT:
Not suitable for continuous spectral studies, because it
doesn’t give continuous radiations.

COLLIMATING SYSTEM
The radiation emitted by the source is
collimated (made parallel) by lenses, mirrors
and slits.
LENSES:
 Materials used for the lenses must be
transparent to the radiation being used.
 Ordinary silicate glass transmits between 350
to 3000 nm and is suitable for visible and near
IR region.
 Quartz or fused silica is used as a material for
lenses to work below 300nm.

MIRRORS
 These are used to reflect, focus or collimate light beams
in spectrophotometer.
 To minimize the light loss, mirrors are aluminized on
their front surfaces.

SLITS:
 Slit is an important device in resolving polychromatic
radiation into monochromatic radiation.
 To achieve this, entrance slit and exit slit are used.
 The width of slit plays an important role in resolution of
polychromatic radiation.

MONOCHRMATORS :
It’s a device used to isolate the radiation of the desired
wavelength from wavelength of the continuous spectra.
Following types of monochromatic devices are used:

A. FILTERS:
 Selection of filters is usually done on a
compromise between peak transmittance and
band pass width; the former should be as high as
possible and latter as narrow as possible.
1. Absorption filters
2. Interference filter

I) Absorption filters:
Absorption filters works by selective absorption of
unwanted radiation and transmits the radiation which
is required.

Selection of absorption filter is done
according to the following procedure:
 Draw a filter wheel.
 Write the color VIBGYOR in clockwise or
anticlockwise manner, omitting Indigo.

 If solution to be analyzed is BLUE in color a filter having a
complimentary color ORANGE is used in the analysis.
 Similarly, we can select the required filter in colorimeter,
based upon the color of the solution.

 An Absorption glass filter is made of solid sheet
of glass that has been colored by pigments which
is dissolved or dispersed in the glass.
 The color in the glass filters are produced by
incorporating metal oxides like ( Cr, Mn, Fe,
Ni, Co, Cu etc.).

 Gelatin filter is an example of absorption filter
prepared by adding organic pigments; here
instead of solid glass sheets thin gelatin sheets
are used. Gelatin filters are not use now days.
 It tends to deteriorate with time and gets affected
by the heat and moisture. The color of the dye
gets bleached.

MERITS:-
 Simple in construction
 Cheaper
 Selection of the filter is easy
DEMERITS:-
 Intensity of radiation becomes less due to
absorption by filters.
 Band pass (bandwidth) is more (±20-30nm) i.e. if
we have to measure at 400nm; we get radiation
from 370-430nm. Hence less accurate results are
obtained.

II) Interference filter
 Works on the interference
phenomenon, causes rejection
of unwanted wavelength by
selective reflection.
 It’s constructed by using two
parallel glass plates, which are
silvered internally and separated
by thin film of dielectric material
of different (CaF2, Sio, MgF2)
refractive index. These filters
have a band pass of 10-15nm
with peak transmittance of 40-
60%.

MERITS:-
 Provide greater transmittance and narrower band pass
(10-15nm) as compare to absorption filter.
 Inexpensive
 Additional filters can be used to cut off undesired
wavelength.

b) PRISM:-
 Prism is made from glass, Quartz or fused
silica.
 Quartz or fused silica is the choice of material
of UV spectrum.
 When white light is passed through glass
prism, dispersion of polychromatic light in
rainbow occurs. Now by rotation of the prism
different wavelengths of the spectrum can be
made to pass through in exit slit on the sample.

 The effective wavelength depends on the
dispersive power of prism material and the
optical angle of the prism.
 There are two types of mounting in an
instrument one is called ‘Cornu type’ and its
adjusted such that on rotation the emerging
light is allowed to fall on exit slit.

 The other type is called “Littrow type”, in which
one surface is aluminized with reflected light
back to pass through prism and to emerge on the
same side of the light source i.e. light doesn’t
pass through the prism on other side.

c) DIFFRACTION GRATINGS:
 More refined dispersion of light is obtained by means of
diffraction gratings.
 These consist of large number of parallel lines ( grooves)
about 1500-3000/ inch is ruled on highly polished surface
of aluminum.

 These acts as scattering centers for light
beam impinging on it. Because of
constructive interference, the separation of
desired wavelength is accomplished.
 The resolved power of grating depends on
the number of lines. Generally resolving
power of grating is better than that of prism
and hence grating is used and is preferred.

Comparison Prism Grating
Made of
Glass-: Visible
Quartz/fused
silica-: UV
Alkali halide:-IR
Grooved on highly
polished surface
like alumina.
Working
Principle
Angle of Incident Law of diffraction
nλ= d (sini±sinθ)
Merits/demerits
Prisms give non-
liner dispersion
hence no overlap
of spectral order
Grating gives
liner dispersion
hence overlap of
spectral order.

Merits/
demerits
Prisms are not moisture
resistant
Prisms are not sturdy
long lasting.
Expensive
Moisture resistant
Grating are sturdy
and long lasting.
Economical.

 SAMPLE HOLDERS/CUVETTES:-
 The cells or cuvettes are used for handling
liquid samples. The cell may either be
rectangular or cylindrical in nature. The cells
that may hold the sample must be made of
substances which are transparent in the
spectral region of interest.
 For study in UV region; the cells are
prepared from quartz or fused silica.
 The internal diameter of the cells is 0.5cm,
1cm, 2cm, or 4cm.

 The cuvettes with lid are used for handling volatile type
solvents and solutions.
 The surfaces of absorption cells must be kept
scrupulously clean. No fingerprints or blotches should be
present on cells. Cleaning of the cells is carried out
washing with distilled water or with dilute alcohol,
acetone.

5. DETECTORS:-
 The light or the intensity of transmitted radiation by a sample is
collected on a detector device. Most modern detectors
generate an electrical current after receiving the radiation. The
generated currents are often amplified and pass on to a meter
(a galvanometer or recorder).
Requirements of an ideal detector:-
 It should give quantitative response.
 It should have high sensitivity and low noise level.
 It should have a short response time.
 It should provide signal or response quantitative in wide
spectrum of radiation received.
 It should generate sufficient signal or electrical current, which
can be measured or easily amplified for detection by meter.

The following types of detectors are employed in
instrumentation of absorption spectrophotometer:-
(a)Barrier layer cell/Photovoltaic cell
(b)Phototubes/Photoemessive tube
(c)Photomultiplier tubes

a)Barrier layer cell/Photovoltaic cell
CONSTUCTION:
It consist of a metallic base plate (A) of Iron or Aluminum (act
as one of the electrodes) over which a thin layer of Selenium
(B) is deposited (act as semi conducting surface), upon that a
fine layer of silver or gold (D) is spread (act as another
electrodes) upper layer of which act as collecting ring (E),
between the layer of Selenium (B) and Silver (D) is
hypothetical barrier layer(C).

WORKING:
When a radiant energy is made incident on the selenium
layer (B) it results in the excitation of electrons from the
same, which passes through hypothetical barrier layer
and are collected on collector ring (E), this causes a
potential difference between electrodes and if external
circuit is complete the current flows, which is the
measure of radiant energy falling on the Selenium layer.
The current can be amplified and measured.

 (b)Photo Tubes/Photoemessive Tubes:
CONSTRUCTION:
This consist of spherical
shaped vacuum bulb
photoemessive cathode and an
anode .The inner surface of
semi-cylindrical cathode
mounted inside the bulb is
coated with a photosensitive
material like Cesium oxide,
Potassium oxide and silver
oxide, which emits electrons
when irradiated with radiant
energy. High potential is
maintain across electrodes.

WORKING:
 When transmitted radiant energy is made incident upon
cathode it causes generation of electrons which flows
towards anode via external circuit and photo current
results. The number of electrons ejected from the
surfaces is directly proportional to the amount of radiant
energy striking the anode surface.

(c)Photomultiplier Tube:
CONSTRUCTION:
In a vacuum tube a
photocathode is fixed which
receives radiant energy
transmitted from the samples.
Some 8 to 10 dynodes are
fixed each with increasing
potential of about 90 volts.
Near the last dynode is fixed
an anode or an electron
collector electrode.

 The transmitted radiant energy strikes the
cathode causes generation of electron which get
attracted towards dynodes maintained at higher
potentials which further causes the generation of
electrons more than the previous ones, these
steps is continued for 8 to 10 times depending
upon the number of dynodes used.
 The electron generated by the last dynode gets
collected over anode which result in potential
imbalance and hence current flows which can be
amplified and measured.

119
SINGLE BEAM
SPECTROPHOTOMETER

Radiation
Source
Entrance
Slit
Exit
Slit
Monochromator
Sample
Cell
Detector Galvanometer
Readout
Device

 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 (Io) is absorbed by the sample
and remaining gets transmitted (It).
121

 The transmitted light strikes the detector and produces
electrical signals.
 The signals produce by the detector is directly
proportional to the intensity of the beam striking its
surface.
 The output is measured by a micrometer or galvanometer
and displayed on the readout device.
122

 The absorbance readings of both the standard and
unknown solutions are recorded after adjusting the
instrument to 100% transmittance with a blank
solution each time whenever the wavelength is
changed.
123

 Advantages;
 Simple in construction
 Easy to operate
 Economical
124

126
Monochromator
C
H
O
P
P
E
R
Amplifer
Entrance
slit
Radiation
Source
Exit
Slit
Reference
cell
Sample
cell
Mirror
Mirror
Detector
Detector
Readout
Device

 Double beam spectrophotometer allows direct
measurement ratio of intensities of sample and reference
beams respectively.
 The design of a double beam spectrophotometer is
similar to single beam spectrophotometer except that it
contains a beam slitter or chopper.
127

 Chopper or beam slitter is a device consisting of a circular
disc. One third of the disc is opaque, one third is
transparent and the remaining one third is mirrored.
 The chopper splits the monochromatic beam of the light
into two beams of equal intensities.
129

 A double beam spectrophotometer can be designed
using one or two detectors.
132
Advantages;
•It facilitates rapid scanning over wide
wavelength region.
•Fluctuations due to radiation source are
minimized.

 It makes automatic compensations for variations in the
wavelength of the incident light.
 It does not required adjustment of the transmittance at
0% and 100% at each wavelength.
 It gives the ratio of the intensities of the sample and
reference beams simultaneously.
133

 Disadvantages;
 Construction is complicated.
 Instrument is expensive.
134

1. Detection of Impurities
 UV absorption spectroscopy is one of the best methods for
determination of impurities in organic molecules. Additional
peaks can be observed due to impurities in the sample and it
can be compared with that of standard raw material. By also
measuring the absorbance at specific wavelength, the
impurities can be detected.
APPLICATIONS OF U.V. SPECTROSCOPY:

U.V. SPECTRA OF PARACETAMOL (PCM)

2. Structure elucidation of organic compounds.
 UV spectroscopy is useful in the structure elucidation of
organic molecules, the presence or absence of
unsaturation, the presence of hetero atoms.
 From the location of peaks and combination of peaks, it can
be concluded that whether the compound is saturated or
unsaturated, hetero atoms are present or not etc.

3. QUANTITATIVE ANALYSIS
 UV absorption spectroscopy can be used for the quantitative
determination of compounds that absorb UV radiation. This
determination is based on Beer’s law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc
Where :
ε -is extinction co-efficient,
c- is concentration, and
b- is the length of the cell that is used in UV spectrophotometer.

4. QUALITATIVE ANALYSIS
 UV absorption spectroscopy can characterize those
types of compounds which absorbs UV radiation.
Identification is done by comparing the absorption
spectrum with the spectra of known compounds.

5. 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.

6. DETECTION OF FUNCTIONAL GROUPS
 This technique is used to detect the presence or
absence of functional group in the compound
 Absence of a band at particular wavelength
regarded as an evidence for absence of particular
group

8. EXAMINATION OF POLYNUCLEAR HYDROCARBONS
 Benzene and Polynuclear hydrocarbons have characteristic spectra
in ultraviolet and visible region. Thus identification of
Polynuclear hydrocarbons can be made by comparison with the
spectra of known Polynuclear compounds.
 Polynuclear hydrocarbons are the Hydrocarbon molecule with two
or more closed rings; examples are naphthalene, C10H8, with two
benzene rings side by side, or diphenyl, (C6H5)2, with two bond-
connected benzene rings. Also known as polycyclic hydrocarbon.

9. MOLECULAR WEIGHT DETERMINATION
 Molecular weights of compounds can be measured
spectrophotometrically by preparing the suitable derivatives of
these compounds.
 For example, if we want to determine the molecular weight of
amine then it is converted in to amine picrate. Then known
concentration of amine picrate is dissolved in a litre
of solution and its optical density is measured at λmax 380 nm.

 After this the concentration of the solution in gm
moles per litre can be calculated by using the
following formula.

10. AS HPLC DETECTOR
 A UV/Vis spectrophotometer may be used as a
detector for HPLC.

REFERENCES:
1. Sharma. Y.R. Elementary Organic Spectroscopy. First
edition .S.Chand Publisher; 2010.
2. Chatwal G.R. Instrumental methods of chemical
analysis. First edition. Himalaya Publisher; 2010.
3. Instrumental analysis by willard and merit

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