UV-visible spectroscopy

Behind Air Strip,Dewas Road, Ujjain, Madhya Pradesh -456664

 Spectroscopy is the tool for study of atomic &
molecular structure.
 It deals with interaction of electronic radiation with
matter involving the measurement & interpretation of
the extension of absorption or emission of
electromagnetic radiation by molecule.
ALOK SHARMA Asst. Professor, Mahakal Institute of Pharmaceutical Studies, Ujjain (M.P.)
UV-Visible Spectroscopy

Most important consequence of such interaction is
the energy is absorbed or emitted by the matter in
discrete amount called as quanta.
UV radiation starts at blue end of visible light(4000Å)
& ends at 2000A.
It divided into two spectral region-
Near UV region- 2000Å-4000Å.
Far or vacuum UV region- below 2000Å.
UV-spectroscopy involved with electronic excitation.

 Absorption And Emission spectra:-
Spectroscopy mainly concerned with interaction of
electromagnetic radiation with matter.
After interaction they may variation in intensity of EMR
with frequency.
Instrument which record this variation in intensity
known as spectrophotometer
Two way in which interaction may observed-
Sample itself emits radiation Called as emission spectra
Absorbs radiation from continuous source Called as absorption spectra

 Wavelength(λ):-
 distance between two successive maxima of one
electromagnetic wave. express in Angstron units or (mu)
 Frequency(ν):-
 Number of wavelength passing through a given point. per
sec.
 Unit:- Hertz or cycles per second
 Wave number:-
 Number of waves per centimeter in vacuum.
 Reciprocal of wavelength, express as per (cm).
 relation between frequency, velocity & wave number
ν=(1/λ)c=(c/v)λ=(v/c)

10910710510310110-110-310-510-710-910-11
gamma
X-rays
Ultra Violet
Infra red
microwave
Radio
waves
500
600
700
Violet, indigo, blue
Green, yellow
Orange, red
Color Wave
length
(nm)
violet 400-435
indigo 435-480
blue 480-500
green 500-560
yellow 560-595
orange 595-610
red 610-750

 Ultra violet absorption spectra arises from transition of electron
or electrons within molecule.
 UV emission spectra arises from reverse type of transition.
 Electron undergoes transition from lower to higher energy level,
this energy difference given by,
E=hν erg
 But actually energy difference between ground & excited states
of electrons
E1-E0=hν
 Total energy of the molecule is sum of electronic, vibrational,
rotational energy.
E=Eele +Evib +Erot

 UV-visible spectroscopy is the measurement of absorbance or
transmittance of radiation in the ultra-violet &visible region of
the spectrum.
 It arises from transition of electron.
 Stage -1
M + hν M*
 Stage -2
M* new species
Excitation of species by absorption of
photon with the limited life time.
Relaxation by converting M* to
the new species by
photochemical reaction

σ-electrons-
Involved in the saturated bonds.
Found in the carbon, hydrogen in the paraffin.
Energy required to excite σ-produced is electron more
than the produced by the UV-light.
π- electrons-
Involved in unsaturated hydrocarbon.
Present in trienes & aromatic compounds.
n-electrons-
It does not evolved in the bonding of the molecules.

transition
σ-σ*
n-σ* usually
allowed
n-π*
π-π* forbidden
Allowed transition:- having ε max 104 or more.This transition due to π-π* transition.
In 1,3-butadiene exhibits absorption at 217 nm & has εmax 21000 represent allowed transition.
Forbidden transition:- transition having εmax less than 104 .Occurs due to n-π*transition.

Transition Region Wavelength
σ-σ*
π-π*
Far UV- region <200 nm
n-σ* Ultra-violet =200 nm
n-π* Near UV &
visible
300- 600 nm
Energy required for various transitions are in the order
σ-σ*> n-σ* > π-π*> n-π*
Thus, n-π*transition required less energy than a π-π* or σ-σ* transition.

Components of spectrophotometer
Source
Monochromator
Sample compartment
Detector
Recorder

RADIANT
SOURCE
WAVELENGTH
SELECTOR
SOLVENT
PHOTO-
DETECTO
R
READOUT
SAMPLE
Fig.-block diagram of instrumentation of UV-spectrophotometer

Light
source
a)D2
Lamp
b)WI
Lamp
Entrance slit
monochromator
sample
Exit slit
Read outamplifierdetector
Fig.- block diagrammatic representation of UV-spectrophotometer

fig.-Schematic representation of
single beam UV-spectrophotometer
Fig.-schematic representation of
double beam UV- spectrophotometer

 Distribution of energy through spectrum is function of
temperature.
For Visible region-
 Tungsten filament lamp
Use for region 350nm to 2000nm.
Problem-
 Due to evaporation of tungsten life period
decreases.
 It is overcome by using tungsten-halogen lamp.
 Halogen gas prevents evaporation of tungsten.

For ultra violet region-
Hydrogen discharge lamp
 consist of two electrode contain in deuterium filled
silica envelop.
 gives continuous spectrum in region 185-380nm.
 above 380nm emission is not continuous.
UV-Vis spectrophotometer have both deuterium &
tungsten lamps.
Selection of lamp is made by moving lamp mounting or
mirror to cause the light fall on monochromator.

 Deuterium lamps:-
 Radiation emitted is 3-5 times more than the hydrogen
discharge lamps.
 Xenon discharge lamp:-
 Xenon stored under pressure in 10-30 atmosphere.
 It possesses two tungsten electrode separated by 8
cm.
 Intensity of UV radiation more than hydrogen lamp.
 Mercury arc:-
 Mercury vapour filled under the pressure .
 Excitation of mercury atom by electric discharge

 Provides a beam of radiant energy of a given nominal
wavelength and spectral bandwidth.
 Parts of a monochramator
1. An entrance slit
2. A collimator
3. A grating
4. A collimator*
5. An exit slit

Filters –
a) Glass filters-
 Made from pieces of colored glass which transmit limited
wavelength range of spectrum.
 Color produced by incorporation of oxide of vanadium,
chromium, iron, nickel, copper.
 Wide band width 150nm.
b) Gelatin filters-
 Consist of mixture of dyes placed in gelatin &
sandwiched between glass plates.
 Band width 25nm.
c) Inter ferometric filters-
 Band width 15nm.

Prisms-
 Prism bends the monochromatic light.
 Amount of deviation depends on wavelength.
 Quartz prism used in UV-region.
 Glass prism used in visible region spectrum.
Function –
 They produce non linear dispersion.

Fig.-mechanism of working of prism.

Grating-
Large number of equispaced lines on a glass blank
coated with aluminum film.
Blaze angle
Normal surface
vector
Normal to
groove face

Spectroscopy requires all materials in the beam path
other than the analyte should be as transparent to the
radiation as possible.
The geometries of all components in the system should
be such as to maximize the signal and minimize the
scattered light.
The material from which a sample cuvette is fabricated
controls the optical window that can be used.

Some typical materials are:
Optical Glass - 335 - 2500 nm
Special Optical Glass – 320 - 2500 nm
Quartz (Infrared) – 220 - 3800 nm
Quartz (Far-UV) – 170 - 2700 nm
•Keep the cuvette clean.
•Don’t clean with paper products.
•Store dry.
•Don’t get finger prints on them.
•Store carefully and gently

 Three common types of detectors are used
Barrier layer cells
Photocell detector
Photomultiplier
Photo voltaic cells or barrier layer cells :-
 They are primarily used for measurement of radiation
in visible region.
 Maximum sensitivity-550nm.
 It consist of flat Cu or Fe electrode on which
semiconductor such as selenium is deposited.
 on the selenium a thin layer of silver or gold is
sputtered over the surface.

Barrier layer cells
 A barrier exist between the selenium & iron which
prevents the electron flowing through iron.
 Therefore electrons are accumulated on the silver
surface.
 These electrons are produced voltage.
- terminal
Silver surface
selenium
+ terminal
fig.-Barrier layer cell

 Photocell detector:-
 It consist of high sensitive cathode in the form of a half
cylinder of metal which is evacuated.
 Anode also present which fixed along the axis of the
tube
 Photocell is more sensitive than photovoltaic cell.
+ -
light
Fig.- photocell detector

Photomultiplier tube:-
It is generally used as detector in UV-spectrophotometer.It
is the combination of photodiode & electron multiplier. It
consist of evacuated tube contains photo-cathode. 9-16
electrodes known as dynodes.
Fig.-photomultiplier tube

 Signal from detector received by the recording system
 The recording done by recorder pan.

 Important to reposition the cuvettes properly
 To clean the cuvettes, lens paper soaked in
spectrograde methanol, which is held by a hemostat is
used.
 When cuvettes are cleaned the methanol film left
evaporates quickly leaving cuvette surface free of
contaminants
 For maximum precision syringes are used to change
the solution leaving the cuvettes in the same place.

Single beam spectrophotometer:-
Double beam spectrophotometer:-
 Advantage of double beam spectrophotometer:-
It is not necessary to continually replace the blank with
the sample or to adjust the auto zero.
The ratio of the powers of the sample & reference is
constantly obtained.
It has rapid scanning over the wide wavelength region
because of the above two factors.

Lamberts Law:-
 Intensity of beam of parallel monochromatic radiation
decreases exponentially as it passes through medium
of homogeneous thickness.
 Absorption is proportional to the thickness (path
length) of solution.
I0/It=KC
k=“absorption coefficient” defined as reciprocal of the
thickness which required to reduced to light to 1/10 of
its intensity

Beers law:-
 Intensity of a beam of parallel monochromatic radiation
decreases exponentially with the number of absorbing
molecule.
 Absorption is proportional to concentration.
 Combination of two law yields beers- lamberts law.
A=Io/It=abc
Where,
A=absorbance
Io-intensity of incident light
It-transmitted light
C-concentration
B-thickness

 Deviation from beer’s law:-
 From the beer’s law plot the absorbance against the
conc.
 A straight line passing through origin is obtained.
Deviation is due to the following factors:-
 A foreign substance having colour particle may affect the
absorption & extinction coefficient.
 Deviation also occur if colored solute ionized or dissociates
in the solution.
for e.g.- benzyl alcohol in chloroform
 Due to the presence of impurities that fluoresce or absorb at
the absorption wave length.
 If monochromatic light is not used deviation may occurs.
 If width of the slit is not proper.
 If the solution species undergoes polymerisation

 Transmittance (T)= Io/It
 % transmittance (%T)= It/Io ˣ 100
 Absorbance (A) = log (It/Io)
 Absorbance also term as,
Extinction coefficient(E)
Optical density (D)
 A= log (Io/It) =abc
when concentration is in moles/lit. the constant called
as molar absorptivity (ε) molar extinction coefficient.
 Specific absorbance-
absorbance of a specific concentration in a cell of
specific pathlength.

 Most common form in p’ceutical analysis A1cm
1% is
absorbance of 1g/100ml (1%w/v) solution in 1cm cell.
ε = A 1cm
1%
ˣ mole.wt.
10

 It is used for calculating the absorption maxima
 Woodward (1941) gives certain rule for correlating λmax with the
molecular structure
 These rules are modified by Scott & Feiser.
 This rule for calculating λmax in conjugated dienes, trienes,
polyenes.
 Homoannular dienes:-
cyclic dienes having conjugated double bonds in the same ring.
e.g. CH3
CH3

 Hateroannuler dienes:-
Cyclic dienes in which double bonds in conjugation are present
in the different ring.
 Endocyclic double bonds:-
It is the double bond present in ring as shown.
 Exocyclic double bonds:-
Double bond in which one of the double bonded atom is the part
of ring system.
CH2
CH2
e.g. Heteroannuler dienes
Endocyclic
double bond
Exocyclic
double bond

a)Parent values-
1. acyclic & Heteroannuler conjugated dienes 215 nm
2.Homoannular conjugated dienes 253 nm
3.Acyclic trienes 245 nm
b)Increments-
1.Each alkyl substituent or ring residue 05 nm
2.Exocyclic double bond 05 nm
3.Double bond extending conjugation 30 nm
4.auxochromes-
-OR 06 nm
-SR 30 nm
-Cl , Br 05 nm
-NR2 60 nm
-OCOCH3 0 nm

Problems:-1) 1,4- dimethyl cyclohex-1,3,-diene
Parent value for Homoannular diene = 253 nm
Two alkyl substituent's 2 ˣ 5 = 10 nm
Two ring residues 2ˣ 5 = 10 nm
Calculated value = 273 nm
Observed value = 265 nm
2)
Parent value for Heteroannuler diene = 215 nm
Four ring residue 4 ˣ 5 = 20 nm
Observed value = 236 nm
CH3 CH3

a)Parent values:-
1.α,β-unsaturated acyclic or six membered ring ketone 215 nm
2.α,β-unsaturated five membered ring ketone 202nm
3.α,β-unsaturated aldehyde 207nm
b)increments:-
1.Each alkyl substituent or ring residue
at α, position 10nm
at β,position 12nm
at γ,position 18nm
2.Each Exocyclic double bond 5nm
3.Double bond extending conjugation 30nm
4.Homoannular conjugatated dienes 39nm
5.Auxochromes. Positions
α β γ
-OH 35 30 50
-OR 35 30 17
-SR - 85 -
-OCOCH3 6 6 6

Problems:- O
CH3-C-C=CH3
CH3
1) Parent value = 215 nm
2) One alkyl substituent in α position = 10 nm
3) Calculated value = 225 nm
4) Observed value = 220 nm
Parent value for α,β- unsaturated 6 membered cyclic ketone=215 nm
One ring residue at α position = 10 nm
2 ring residue at β- position 2* 12 =24 nm
Double bond Exocyclic to 2 ring 2* 5 =10 nm
Calculated value = 259nm
Observed value =
A α,β- unsaturated
acyclic ketone
O

R-C6H4-COG λmax (nm)
parent chromophore:-
G=alkyl/ring residue
G=H & R=H
G=OH or O alkyl
246
250
230
Add for R:
Alkyl or ring residue o,m 3
p 10
-OH, OMe, -O-Alkyl o,m 7
p 25
O- o 11
m 20
p 78
Cl o,m 0
p 10
Br o,m 2
P 15
NH2 o,m 13
p 58
NMe2 o,m 20
p 85

1) Para chloroacetophenone
Basic value = 246 nm
Cl substitution at p- position = 10 nm
Observed value = 254nm
Cl
C CH3
O

 Extent of conjugation
 Distinction between conjugated and non conjugated compound
 Detection of chromophore in an unknown compound
 Identification of a chromophore (functional group)
 Study of strain
 Study of geometric isomerism
 Study of tautomerism
 Study of structural features in different solvents
 As an analytical tool

 Willard H. et.al.; Instrumental Methods Of Analysis.
 Gurdeep R. chatwal; Sham K. Anand; Instrumental Methods Of Chemical Analysis.
 Y. Anjaneyulu; K. Chandrasekhar; Valli Manickam; Text book of analytical chemistry.
 Y. R.Sharma; Elementary organic spectroscopy.
 P.S.Kalsi; Spectroscopy of organic compound.
 B.K.Sharma; Instrumental methods of chemical analysis.

Queries

UV-visible spectroscopy

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