This document outlines a training on UV-Visible Spectroscopy conducted by CRCL Group A Officers at IICT. It introduces UV-Visible spectroscopy, covering topics like instrumentation, principles, spectral interpretation and applications. The instrumentation section describes components like light sources, wavelength selectors, sample compartments and detectors. Common applications discussed are determination of beta carotene, denaturants, and elements in various materials. Calibration procedures for the UV-Vis instrument are also provided.

1. CRCL Group A Officer’s Training at IICT
Welcome to All
Distinguished Faculty Members of IICT
& Officers of Revenue Laboratories.

2. CRCL Group A Officer’s Training at IICT
Topic: UV Visible Spectroscopy
Group-III
Team Members:
1.T.R Suresh
2.A.J Aleyamma
3.Ajay Kumar Singh
4.H S Bhandarkar
5.K Thambidurai

3. CRCL Group A Officer’s Training at IICT
Topic to be Covered
1. Introduction
2. Basics of UV-Visible Spectroscopy
3. Instrumentation
4. Spectral Data Interpretation
5. Application

4. CRCL Group A Officer’s Training at IICT
Introduction
1. What is Spectroscopy?
It is the study of interaction of Electromagnetic radiation
with Matter.
2. Electromagnetic Radiation:
It is the waves of Electromagnetic fields propagating
through space. It includes Radio, Microwave, IR, UV,
Visible, X-Ray and Gamma Rays.
UV Region 100 – 380 nm
Visible Region 380 – 750 nm

5. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
 In UV Visible the sample is irradiated with broad
spectrum of UV Radiation.
 The Radiation is passed through the sample solution, the
particular electronic transition of molecular orbitals
matches with the energy band of UV-Vis Region and it
will be absorbed.
 Remaining light is transmitted and detected by the
detector.

6. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Electromagnetic Radiation:

7. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Principle:
UV – Visible Spectroscopy is based on the Beer-Lambert’s
Laws, that is the absorbance of the radiation is directly
proportional to the concentration of the solution.
A = € x L x C
A - Absorbance of incident radiation
€ - Molar absorbtivity Coefficient
L - Path length of Solution and
C - Concentration of the Solution

8. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Type of Electronic transition in Molecular Orbital
There are four electronic transitions are :
(1) δ – δ* transition :
Its very strong and occurs in saturated hydrocarbons such as in Alkanes.
(2) n – δ* transition :
This type of transition occurs in saturated compounds which containing one
hetero atom has unshared pair electron such as O, N, S and Helogens.
(3) π– π * transition :
This type of transition occurs in unsaturated compounds such as
Alkenes ,alkynes and carbonyl compounds
(4) n– π * transition :
This type of transition occurs in unsaturated compounds which containing
hetero atom has unshared pair electron.
Among these four transitions π– π * and n– π * are subjected to study in this section and
other such as δ – δ* & n – δ* are forbidden due to high energy differences.

9. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Instrumentation

10. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Instrumentation
Components of UV Visible Spectrophotometer:
1. Light Source
2. Wave Length Selector
3. Sample Compartment
4. Detector
5. Read-out Device (Recorder / Computer System)

11. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Instrumentation
Light Source: Contineous source of light which emits radiation of
all wavelength within the UV – Vis Spectral Region.
A. Deuterium Lamp: Which Emits the Radiation from 196 - 380 nm
B. Tungsten Lamp: Which emits the radiation from 380 – 2500 nm
( For Visible Region 380 – 750 nm)
2. Wave Length Selector:
It is used to select a required single wave length
(monochromatic light). Filter or Monochromator is used for this
purpose.

12. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Instrumentation
Filter: The Particular colour of the filter absorbs band portion of the
complementary colour and transmitted the remaining colour.
Monochromator: Prism or Grating Monochromator.

13. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Instrumentation
Grating Monochromator:

14. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
3. Sample Compartment: Consists of Cell Holder and the Cell
generally called “Cuvette”.
Cuvette – made of Glass:
Suitable for Visible Region and could not be used for
UV Region since Glass will absorb radiations at UV Region.
Cuvette – made of Quartz:
Suitable for both UV and Visible Region.
Length of Cuvette : 1 cm, 5 cm etc depends upon concentration of
the sample.
4. Detector:
It converts radiant energy into electrical energy as signals.
It should be sensitive and Fast Response over a considerable range of
Wave Length.
(i) Photo Tube: Which emits electron from negatively charge
cathode when stuck by Uv or Visible radiation which flows
through vacuum to anode where to produce current, which is
directly proportional to intensity of radiation.

15. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
(i) Photo Multiplier Tube:
It is a very sensitive device in which electrons emitted from the
photosensitive cathode strike a second surface called dynode
which is positive with respect to the original cathode.
Above process is repeated several times 106
electrons are finally
collected for each photon striking the first cathode.

16. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Schematic Diagram of Single Beam Spectrophotometer:

17. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Schematic Diagram of Double Beam Spectrophotometer:

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UV – Visible Spectroscopy
4. Interpretation of Spectrum
Absorbance Vs Wavelength has been
plotted and we will get the following
graph.
From the graph we can get Absorption
Value, Lamda(Max) and type of
Molecular transition taken place by
Calculating Molar Absorbtivity coefficient.
For Example: If absorbtion of 0.01 M solution is about 0.13 with a path length
of 1 cm.
A = E x l x C
E = 0.13 / ( 1 x 0.01 x 10-3
) = 13000
That means there may be π– π * transition and the sample may be alkenes, alkynes
and carbonyl compounds

19. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
5. Application of UV Visible Spectroscopy:
(a) Applicable for qualitative & Quantitative
determination of both Organic and In-organic
compounds.
(b) High Sensitivity upto 10-6
to 10-4
M
(c) Good Accuracy for the sample concentration in
the range from 1% to 3%.

20. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Application of UV Visible Spectroscopy
In Revenue Laboratories at present the UV Visible Spectroscopy is
used for
(a) Determination of Beta Carotene in Crude Palm Oil
(b) Determination of Denaturant content in Denatured Alcohol.
(c) Biuret content in Urea samples.
(d) Determination of P, Si, Mn & Mo in Stainless Steels.
(e) Titanium Content in Rutile, Anatase, Bauxite etc
(f) Absorbance range determination of Pharma & Drug samples.
(g) Traces of Fe, Silica in Calcium Carbonate, Lime samples.

21. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Determination of Beta carotene content in CPO: (Ref: BS-684)
Procedure:
0.5 to 1.0 gm of homogenised sample weighed accurately and desolved in cyclohexane and
made upto 100ml in a standard measuring flask.
Measure the absorbance of the prepared sample using 1 cm cuvette at 445 nm
wavelength by making zero baseline with the blank (cyclohexane).
Beta carotene (mg/kg) = (383 * Abs) / (path length * wt. in gm for 100ml)
Determination of Theoritical Value of Absorbance of Beta Carotene:
Fieser-Kuhn Rule for Conjugated Polyenes for more than 4 conjugated dienes:
According to the Fieser-Kuhn rule the following equation can be used to solve for the wavelength
of maximum absorption λmax.
λmax = 114 + 5M + n (48.0 – 1.7 n) – 16.5 Rendo – 10 Rexo
Where λmax is the wavelength of maximum absorption
M is the number of alkyl substituents / ring residues in the conjugated system
n is the number of conjugated double bonds
Rendo is the number of rings with endocyclic double bonds in the conjugated system
Rexo is the number of rings with exocyclic double bonds in the conjugated system.

22. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Determination of Theoritical Value of Absorbance of Beta Carotene:
Sample Problem 1: β-Carotene
β-carotene is a precursor of vitamin A which is a terpenoid derived from several isoprene units.
The observed λmax of β-carotene is 452 nm.
Let us therefore use Fieser-Kuhn rules to calculate the λmax .
λmax = 114 + 5M + n (48.0 – 1.7 n) – 16.5 Rendo – 10 Rexo
Base Value = 114 nm
M (number of alkyl substituents) = 10
n (number of conjugated double bonds) = 11
Rendo (number of endocyclic double bonds) = 2
Rexo (number of exocyclic double bonds) = 0
Substituting in equation
λmax = 114 + 5M + n (48.0 – 1.7 n) – 16.5 Rendo – 10 Rexo
= 114 + 5(10) + 11 (48.0-1.7(11)) – 16.5 (2) – 10 (0)
= 114 + 50 + 11 (29.3) – 33 – 0
= 114 + 50 + 322.3 – 33
Calc. λmax = 453.30 nm
λmax observed practically = 452nm

23. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
TiO2 & V2O5 (Hydrogen Peroxide method)
(Ref: Analysis of Ores & Minerals, Indian Bureau of Mines)
Titanium and vanadium both forms yellow coloured complex with hydrogen peroxide. Chloride ion and chlorine affects
the colour of the complex, therefore all chlorides must be expelled before the colour is developed.
Procedure: Take 25 ml. aliquot in 250 ml beaker, add 1-2 ml. dil. Sulphuric acid and fume off the content on hot plate.
Moisten the mass with dilute sulphuric and 50 ml. DM water. Boil the content. Filter if there is any turbidity. Add 5 ml.
dilute sulphuric acid and 5 ml. ortho-phosphoric acid. Add 10 ml. (1:1) H2O2. Yellow colour of titanium complex will
appear. Make up the volume to 100 ml. in volumetric flask. Measure the absorbance at 410 nm.
Note: This measurement will give the quantity of TiO2 and V2O5 together. Add a pinch of sodium fluoride in the
volumetric flask of titanium-peroxide complex, shake vigorously for few minutes filter through dry filter paper. Measure
the absorbance at 410 nm. This colour will correspond to V2O5 content. Find TiO2 by difference. If V2O5 is absent or
negligible, the original reading (before adding sodium fluoride) of absorbance should be taken for finding equivalent of
TiO2 content.
Graph for TiO2 estimation: Fuse 0.1 g of pure TiO2 with potassium pyrosulphate (approx. 1 g.) in silica crucible. Extract
the mass in 5 ml (1:1) sulphuric acid and 50 ml. DM water. Cool this and make up to 100 ml. in volumetric flask. (OR)
0.1 gm of Pure TiO2, 5.0 gm of Ammonium Sulphate Reagent Grade and 20ml Concentrated Sulphuric acid heated in a
Hot plate till the clear solution obtained. Cool, Filter and make upto the 100 ml volumetricflask.
Now each ml. of this solution will be equivalent to 1 mg. of TiO2. Take out 1 ml, 2ml, 5 ml, 6 ml, 7 ml, 9 ml, 10 ml,
aliquot and develop colour of Titanium-hydrogen peroxide complex at described above. Measure the absorbance and
plot the graph.

24. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
TiO2 & V2O5 (Hydrogen Peroxide method)
(Ref: Analysis of Ores & Minerals, Indian Bureau of Mines)
Calibration Curve
0.10 gm of TiO2weighed and dissolved in 20ml Con. Sulphuric acid with 5.0 g Ammonium Sulphate and made upto
100 ml Std. Volumetric flask.
From the above solution 1ml, 2ml 3ml, 4ml and 5ml solution taken in 100 ml SMF and 5 ml of 10% Sulphuric acid & 5
ml Ortho Phosphoric acid added and add 10ml 1:1 H2O2madeupto the volume. Absorbance measured in 410 nm in
Thermo UV1 UV-Visible Spectrophotometer.
STD ABSORBANCE mg Obtained
from Graph
Mg RUN-1 RUN-2 RUN-3
1.0
mg
0.0679 0.0719 0.0807 0.930
2.0
mg
0.1637 0.1596 0.1514 1.997
3.0
mg
0.2407 0.2503 0.2582 3.057
4.0
mg
0.3317 0.3453 0.3461 4.104
5.0
mg
0.4057 0.4070 0.4145 5.081

25. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Comparision of analysis of Ti in Rutile by Calourimetry & Gravimetry
method:
Seven samples of Synthetic Rutile were analysed in UV-Visible Spectrophotometer
and the obtained results are comparable with those obtained with gravimetric method
usingCubferron.
SNo Sample Details
% TiO2
(H2O2 Method)
% TiO2
(Cubferron
Method)
Difference
01 Sample No. 1 94.1 94.8 + 0.7
02 Sample No. 2 95.9 94.5 - 1.4
03 Sample No. 4 94.0 94.4 + 0.4
04 Sample No. 5 93.9 94.7 + 0.8
05 Sample No. 6 95.0 94.5 - 0.5
06 Sample No. 7 96.0 94.5 - 1.5
07 Sample No. 8 95.4 94.6 - 0.8

26. CRCL Group A Officer’s Training at IICT
UV – Visible Spectroscopy
Calibration of UV-Vis Instruments:
It performs the regular performance checks.
It ensures that the testing performed on the respective instrument give the accurate results and meets the
standards of GMP and GLP. For Ultra violet region:
Calibration for UV Region:
Dilute 0.6 ml of Sulphuric acid to 2000 ml with purified water (0.005 M Sulphuric acid).
Weigh accurately 57.0 to 63.0 mg of Potassium dichromate primary standard (previously dried at 130°C for
constant weight) and transfer it to 1000 ml volumetric flask. Dissolve in 0.005M sulphuric acid and make up to
the mark with the same acid.
Measure the absorbance at 235nm, 257nm, 313nm and 350nm using 0.005M sulphuric acid as reference
solution.
For visible region:
Weigh accurately 57.0 to 63.0 mg of Potassium dichromate primary Standard (previously dried at
130°C for constant weight) and transfer it to 100 ml volumetric flask.
Dissolve in 0.005M sulphuric acid and make up to the mark with the same acid. Measure the
absorbance at 430nm using sulphuric acid as reference solution.
Calculate the value of A(1% , 1 cm ) using the expression given below :
A (1%, 1 cm) = Absorbance x 1000 / wt. in g x 100
Check the value of A (1%, 1 cm ) at each wavelength against the acceptance criteria given below :
Wavelength Maximum Tolerance
235 nm 122.9 nm to 126.2 nm
257 nm 142.8 nm to 146.2 nm
313 nm 47.0 nm to 50.3 nm
350 nm 105.6 nm to 109.0 nm
430 nm 15.7nm to 16.1nm

27. CRCL Group A Officer’s Training at IICT