1. Chapter one:
Spectroscopic Techniques
Topic: Spectroscopy
Learning objectives:
• Introduction
• History
• Principles of spectroscopy
• Applications of spectroscopy

2. Teaching and learning methods: Lectures, visual aid, interactive
forms, questions-answers session and group discussion.
At end of the lecture student should be able to answer the
following questions.
• Define spectroscopy.
• Explain the principle of spectroscopy.
• Write the applications of spectroscopy in microbiology and
biochemistry.
Reference books:
1. Principles and Techniques of Biochemistry and Molecular
Biology; Keith Wilson and John Walker; 7th edition, chapter 12,
page: 482-486

3. Analytical Techniques in Microbiology & Biochemistry
Introduction:
In analytical microbiology uses instruments and used to separate,
identify and qualify biomolecules.
Definition:
An analytical technique is a method that is used to determine the
concentration of a chemical compound.

4. Analytical Techniques
• Chromatography
• Electrophoresis
• Spectrophotometer
• Centrifugation
• Enzyme Linked Immuno Sorbant Assay (

5. Spectrophotometer

6. History of spectroscopy
• Spectroscopy began with Isaac Newton's optics
experiments (1666–1672). Newton applied the
word "spectrum" to describe the rainbow of
colors .
• During the early 1800s, Joseph von Fraunhofer
made experimental advances with dispersive
spectrometers that enabled spectroscopy to
become a more precise and quantitative
scientific technique.
• Since then, spectroscopy has played and
continues to play a significant role in chemistry,
physics and astronomy.

7. Spectrum and Spectroscopy
• Spectrum:
(a) Different colors observed when the white light was dispersed
through the prism
(b) The changing of light intensity as a function of frequency
• Spectroscopy: Study of spectrum, to identify substances.
- Spectroscopy is the study of the interaction between matter and
electromagnetic radiation for identifying the substances through
the spectrum emitted from or absorbed by them.
- Spectroscopy is the most powerful tool available for the study of
atomic & molecular structure and is used in the analysis of a wide
range of samples. it is main two type-

8. • Atomic Spectroscopy: This Spectroscopy is concerned with the
interaction of electromagnetic radiation with atoms are commonly
in the lowest energy state called as grown state.
• Molecular Spectroscopy: This Spectroscopy deals with the
interaction of electromagnetic radiation with molecule.
• Spectrometry: An analytical technique in which emission (of
particle/radiation) is dispersed according to some property of the
emission and the amount of dispersion is measured.
 Eg. Mass spectrometry.
• Spectrophotometry: A quantifiable study of electromagnetic
spectra.
• Spectrography : Another name for spectroscopy.

9. The electromagnetic spectrum
180 –380 nm Ultraviolet
380 –800 nm Visible
3000 nm –8000 nm Mid-Infrared
1nm = 10-9meter.

10. b) Double-beam
Spectrophotometer
a)Single-beam

12. Introduction to UV spectroscopy
• UV spectroscopy is type of absorption spectroscopy in which light of ultra-
violet region (200-400 nm.) is absorbed by the molecule.
• Absorption of the ultra-violet radiations results in the excitation of the electrons
from the ground state to higher energy state.
• The energy of the ultra-violet radiation that are absorbed is equal to the energy
difference between the ground state and higher energy states (∆E = hf).
• Generally, the most favored transition is from the highest occupied molecular
orbital (HOMO) to lowest unoccupied molecular orbital (LUMO). For most of the
molecules, the lowest energy occupied molecular orbitals are s orbital, which
correspond to sigma bonds. The p orbitals are at somewhat higher energy levels,
the orbitals (nonbonding orbitals) with unshared paired of electrons lie at higher
energy levels. The unoccupied or antibonding orbitals (pie and sigma ) are the
highest energy occupied orbitals.
• In all the compounds (other than alkanes), the electrons undergo various
transitions. Some of the important transitions with increasing energies are:
nonbonding to pie*, nonbonding to sigma*, pie to pie*, sigma to pie* and sigma
to sigma*.

14. Principle of UV spectroscopy
• UV spectroscopy obeys the Beer-Lambert law, which states that: “when a
beam of monochromatic light is passed through a solution of an absorbing
substance, the rate of decrease of intensity of radiation with thickness of the
absorbing solution is directly proportional to the intensity of the light”
• The expression of Beer-Lambert law is-
A = log (I0/I) = ECL
Where, A = absorbance
I0 = intensity of light incident upon sample cell
I = intensity of light leaving sample cell
C = molar concentration of solute
L = length of sample cell (cm.)
E = molar absorptivity
• From the Beer-Lambert law it is clear that greater the number of molecules
capable of absorbing light of a given wavelength, the greater the extent of
light absorption. This is the basic principle of UV spectroscopy.

15. Instrumentation and working of UV spectroscopy
Instrumentation and working of the UV spectrometers can be studied
simultaneously. Most of the modern UV spectrometers consist of the following
parts-
1. Light Source:- Tungsten filament lamps and Hydrogen-Deuterium lamps are
most widely used and suitable light source as they cover the whole UV region.
Tungsten filament lamps are rich in red radiations; more specifically they emit
the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls
below 375 nm.
2. Monochromator:- Monochromators generally composed of prisms and slits.
The most of the spectrophotometers are double beam spectrophotometers.
The radiation emitted from the primary source is dispersed with the help of
rotating prisms. The various wavelengths of the light source which are
separated by the prism are then selected by the slits such the rotation of the
prism results in a series of continuously increasing wavelength to pass through
the slits for recording purpose. The beam selected by the slit is monochromatic
and further divided into two beams with the help of another prism.

16. Figure: Instrumentation of UV spectroscopy

17. Instrumentation and working of UV spectroscopy (cont….)
3. Sample and reference cells:- One of the two divided beams is passed through the
sample solution and second beam is passed through the reference solution. Both
sample and reference solution are contained in the cells. These cells are made of either
silica or quartz. Glass can't be used for the cells as it also absorbs light in the UV region.
4. Detector:- Generally two photocells serve the purpose of detector in UV
spectroscopy. One of the photocell receives the beam from sample cell and second
detector receives the beam from the reference. The intensity of the radiation from the
reference cell is stronger than the beam of sample cell. This results in the generation of
pulsating or alternating currents in the photocells.
5. Amplifier: - The alternating current generated in the photocells is transferred to the
amplifier. The amplifier is coupled to a small servo-meter. Generally current generated
in the photocells is of very low intensity, the main purpose of amplifier is to amplify the
signals many times so we can get clear and recordable signals.
6. Recording devices:- Most of the time amplifier is coupled to a pen recorder which is
connected to the computer. Computer stores all the data generated and produces the
spectrum of the desired compound.

18. Applications of UV spectroscopy
1. Detection of functional groups- UV spectroscopy is used to
detect the presence or absence of chromophore in the compound.
This is technique is not useful for the detection of chromophore in
complex compounds. The absence of a band at a particular band
can be seen as an evidence for the absence of a particular group. If
the spectrum of a compound comes out to be transparent above
200 nm than it confirms the absence of: a) Conjugation b) A
carbonyl group c) Benzene or aromatic compound d) Bromo or iodo
atoms.
2. Detection of extent of conjugation- The extent of conjugation in
the polyenes can be detected with the help of UV spectroscopy.
With the increase in double bonds the absorption shifts towards the
longer wavelength. If the double bond is increased by 8 in the
polyenes then that polyene appears visible to the human eye as the
absorption comes in the visible region.

19. Applications of UV spectroscopy
3. 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.
4. Determination of configurations of geometrical isomers- It is
observed that cis-alkenes absorb at different wavelength than the trans-
alkenes. The two isomers can be distinguished with each other when one
of the isomers has non-coplanar structure due to steric hindrances. The
cis-isomer suffers distortion and absorbs at lower wavelength as
compared to trans-isomer.
5. Determination of the purity of a substance- Purity of a substance can
also be determined with the help of UV spectroscopy. The absorption of
the sample solution is compared with the absorption of the reference
solution. The intensity of the absorption can be used for the relative
calculation of the purity of the sample substance.

20. Applications of UV Spectroscopy
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.
UV Spectra of Paracetamol (PCM)

21. 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
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.

22. 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. 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.
6. As HPLC detector
• The most ubiquitous use of UV is as a detection device for HPLC

23. 7. Detection of functional groups
UV spectroscopy is used to detect the
presence or absence of chromophore in
the compound. This is technique is not
useful for the detection of
chromophore in complex compounds.
The absence of a band at a particular
band can be seen as an evidence for
the absence of a particular group. If the
spectrum of a compound comes out to
be transparent above 200 nm than it
confirms the absence of: a) Conjugation
b) A carbonyl group c) Benzene or
aromatic compound d) Bromo or iodo
atoms.
Benzene

24. 8. 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.
9. 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.

25. APPLICATIONS:
APPLICATIONS IN ORGANIC COMPOUNDS
1.It is helps to show the relationship between different groups, it is
useful to detect the conjugation of the compounds
2.Detection of geometrical isomers, In case of geometrical isomers
compounds, that trans isomers exhibits λmax at slightly longer
wavelength and have larger extinction coefficient then the cis
isomers .
3.Detection of functional groups, it is possible to detect the
presence of certain functional groups with the help of UV Spectrum.

26. GENERAL APPLICATIONS:
1.Qualitative analysis, UV absorption spectroscopy can characterizes those type of compounds
which absorb UV radiation. Identification is done by comparing the absorption spectrum with
the spectra of known compound.
2. It is useful in Quantitative analysis of the compounds.
3. Detection of impurities, UV absorption spectroscopy is the one of the best method for
detecting impurities in organic compounds.
4. Tautomeric equilibrium, UV spectroscopy can be used to determine the percentage of
various keto and enol forms present in tautomeric equilibrium.
5. Chemical kinetics, UV spectroscopy can be used to study the kinetics of reactions.
6. Molecular weight determination, molecular weights of compounds can be measured by
spectroscopy.
7. Analysis of inorganic compounds.
8. Measuring concentration of solution, absorption band can also used to determine the
concentration of compounds in a solution.
9. Inorganic chemistry, absorption spectra have been used in connection with many problems
in inorganic chemistry.
10. It is useful to deermine the structure of the chloral.