UV-visible spectroscopy is a technique that uses light in the visible and adjacent ranges. It works by measuring how much light is absorbed by a sample at each wavelength. The document discusses the basic principles of spectroscopy, including how electromagnetic radiation interacts with matter. It describes the laws of absorption, specifically Beer's law, which states that absorbance is proportional to concentration. The key aspects of instrumentation are outlined, including light sources, wavelength selectors like monochromators, sample holders, and detection devices. Single beam and double beam spectrophotometers are explained as the main types of instruments used in UV-visible spectroscopy.

1. Instrumentation and
application of UV-VISIBLE
SPECTROSCOPY
Keshav Narayan Pai
Msc II
DOS in Botany
Manasagangotri

2. Contents
• History and introduction to spectroscopy
• Basic principles
• The law of absorption
• UV visible spectroscopy
• Instrumentation
• Application
• Conclusion
• Reference

3. 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.

4. Spectroscopy
• Spectroscopy is the branch of science dealing the study
of interaction of electromagnetic radiation with matter.
• 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 s two main two type
• 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.

5. BASIC PRINCIPLES
• Light is supposed to duel characteristic,
corpuscular and waveform
• Thus a beam of light may be understood as
electromagnetic waveform photons of energy
propagated at 3*108 m/s i.e., speed of light
• The term electromagnetic in a precise description
of the radiation in that the radiation is made up
of electrical & a magnetic wave which are in
phase & perpendicular to each other & to the
direction of propagation.

6. • A beam of light form a bulb consists of many
randomly oriented plane polarised component
being propagated in same direction.
• The distance along the direction of propagation
for one complete cycle is known as wavelength.

7. THE LAWS OF ABSORPTION
The absorption of light by any absorbing
material is governed by two laws .
 Bouger-Lambert law
 Beer’s law
Bouger-Lambert law
This law is suggested by Picre Bouguer
in 1729, its often attributed to Johann
Heinrich Lambert .

8. • This law is states that “ The amount
of the light absorbed is proportional
to the thickness of the absorbing
material & is independent of the
intensity of the incident light “

9. 100% 50% 25% 12.5%
b
6.25% 3.125%

10. I – Intensity of transmitted light
- initial intensity of incident light
b – thickness (path –length)
k – linear absorption co-efficient
The power term can be removed by converting to the log
form.
ln(I/ )=-kb
ln( /I )=kb
Changing to common logarithms we get,
2.303 log /I =kb

11. Second law – Beer’s law
It states that, the amount of light absorbed by a material is proportional to the
number of
Absorbing molecules(concentration)
Again it can be represented –
2.303 log( /I) = k’c
K’=absortivity constant
c= concentration
K and k’ merge together = a
Log = /I = a b c
a = k & k’
b = thickness
C = concentration
This combined law states that the amount of light absorbed is proportional to the
Concentration of the absorbing substance & to the thickness of the absorbing
material
(path – length)
The quantity /I it is absorbance (O.D – optical density)
The reverse I / is - transmittance T (the molecule has not used that energy)

12. =
O.D of the unknown x concentration of std
O.D of the std
The two terms are mathematically commutable i.e., one can be calculated from the
other
A=log - log I
= 100%
Log 100 = 2
=2-log I
Or
O.D is dirctly proportional to the concentration if path is constant
So if we know the value of O.D concentration can be calculated
Concentration of the
Unknown(sample)

13. Terms describing UV absorptions
1. Chromophores: functional groups that give
electronic transitions.
2. Auxochromes: substituents with unshared pair e's
like OH, NH, SH ..., when attached to π chromophore
they generally move the absorption max. to longer λ.
3. Bathochromic shift: shift to longer λ, also called red
shift.
4. Hysochromic shift: shift to shorter λ, also called blue
shift.
5. Hyperchromism: increase in ε of a band.
6. Hypochromism: decrease in ε of a band.

15. UV-VISIBLE Spectroscopy:
Uv-vis spectroscopy is also known as electronic
spectroscopy. In which the amount of light
absorbed at each wavelength of Uv and visible
regions of electromagnetic spectrum is
measured. This absorption of electromagnetic
radiations by the molecules leads to molecular
excitation.

16. Electronic Spectroscopy
• Ultraviolet (UV) and visible (VIS)
spectroscopy
• This is the earliest method of molecular
spectroscopy.
• A phenomenon of interaction of molecules
with ultraviolet and visible lights.
• Absorption of photon results in electronic
transition of a molecule, and electrons are
promoted from ground state to higher
electronic states.

17. • The first discovery of electromagnetic waves other than
light came in 1800, when William Herschel discovered
infrared light. He was studying the temperature of
different colors by moving a thermometer through light
split by a prism.
• The types of electromagnetic radiation are broadly
classified into the following classes
• Gamma radiation
• X-ray radiation
• Ultraviolet radiation
• Visible radiation
• Infrared radiation
• Terahertz radiation
• Microwave radiation
• Radio waves

19. • Ultraviolet: 190~400nm
• Violet: 400 - 420 nm
• Indigo: 420 - 440 nm
• Blue: 440 - 490 nm
• Green: 490 - 570 nm
• Yellow: 570 - 585 nm
• Orange: 585 - 620 nm
• Red: 620 - 780 nm

20. VISIBLE LIGHT
Shorter wavelength and higher frequency than infrared rays.
Electromagnetic waves we can see
Longest wavelength= red light
Shortest wavelength= violet (purple) light

21. ULTRAVIOLET RAYS
Shorter wavelength and higher frequency than
visible light
Carry more energy than visible light

22. Electronic transitions
There are three types of electronic transition
which can be considered;
• Transitions involving p, s, and n electrons
• Transitions involving charge-transfer
electrons
• Transitions involving d and f electrons

23. Absorbing species containing p, s,
and n electrons
• Absorption of ultraviolet and visible
radiation in organic molecules is
restricted to certain functional groups
(chromophores) that contain valence
electrons of low excitation energy.

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

25.  Transitions
• An electron in a bonding s orbital is excited to
the corresponding antibonding orbital. The
energy required is large. For example, methane
(which has only C-H bonds, and can only
undergo  transitions) shows an
absorbance maximum at 125 nm. Absorption
maxima due to  transitions are not seen
in typical UV-VIS spectra (200 - 700 nm)

26. n  Transitions
• Saturated compounds containing atoms with
lone pairs (non-bonding electrons) are capable
of n  transitions. These transitions
usually need less energy than 
transitions. They can be initiated by light
whose wavelength is in the range 150 - 250 nm.
The number of organic functional groups with
n  peaks in the UV region is small.

27. n  and  Transitions
• Most absorption spectroscopy of organic
compounds is based on transitions of n or 
electrons to the  excited state.
• These transitions fall in an experimentally
convenient region of the spectrum (200 - 700
nm). These transitions need an unsaturated
group in the molecule to provide the 
electrons.

28. Instrumentation
Light source:
UV - Hydrogen lamp ( hydrogen stored under
pressure) , Deuterium lamp and Xenon lamp-
it is not regularly used becos of unstability and
also the radiation of UV causes the generation
of ozone by ionization of the oxygen molecule.
VIS – Tungston filament lamp , Tungston
halogen lamp and carbon arc lamp.

29. Waveselectors are mainly either filters or
monochrmators.
Filters : Gelatin filters are made using a layer
of gelatin coloured with organic dyes that are
sealed between glassplates. This filters
resolve polychromatic light into a relatively
wide band width of about 40 nm and these
are commonly used in colorimeters since they
have low transmittance i.e. 5 – 20 %.

30. * Monochromators :
Consists of an entrance slit which admits the polychromatic light
from the source.
 A collimating device – lens or mirror which helps in reflecting the
polychromatic light to the dispersion device.
 A wavelength resolving device - prism or grating.
 A focussing lens or mirror
 Exit slit
 Sample holder/ containers :
 Cuvettes – Quarts or fused silica , ordinary glass is known to absorb uv
rad.
 for IR – samples are ground with potassium bromide and pressed into
a pellet, if aqueous solution silver chloride is coated inside the cell.
While preparing samples selection of solvents is imp. , becos they do
absorb light.

31. INSTRUMENTATION:Single and
Double Beam Spectrometer
• Single-Beam: There is only one light beam or
optical path from the source through to the
detector.
• Double-Beam: The light from the source, after
passing through the monochromator, is split
into two separate beams-one for the sample
and the other for the reference.

33. Single beam spectrophotometer
A single beam of radiation pass
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 . This was the earliest design
and is still use in both teaching and
industrial labs.

36. Double beam
spectrophotometer
• The instrument used in ultraviolet-visible
spectroscopy is called a UV/Vis
spectrophotometer. It measures the intensity of
light passing through a sample (I), and compares
it to the intensity of light before it passes through
the sample (I). The ratio is called the
transmittance, and is usually expressed as a
percentage (%T). The absorbance, (A). is based on
the transmittance.
• A= -log(%T/100%)

38. *
• Detection devices :
• UV-VIS detectors –
• 1. Photocells made of cadmium sulphide ,
silicon and selenium. Steel base coated
with silver film then finally thin coating of
selenium. Electrons pass through
selenium to silver and silver acts as the
collecting electrode and steel plate as
another electrode, the current flowing
between two electrodes is then measured
by a micro-ammeter.

39. *
• 2. Phototubes : glass envelop with a quartz
window, centrally situated metal wire acts as
anode and a semi-circle cathode.
• The energy of the photon is transferred to the
loosely bound electrons of the cathode
surface. The electrons excited move towards
anode causing to flow in the circuit.
Phototube currents are quite small and
require amplification, then it is recorded.

40. *
• 3. photomultiplier : these are designed to
amplify the initial photoelectric effect and are
suitable for the use at very low light intensities.
• This consists of an evacuated glass tube into
which are sealed the cathode and anode and
an additional intervening electrodes known as
dynodes. As the radiation strikes the cathode
electrons are liberated and the applied
potential difference accelerates the electrons
towards the first dynode. Each successive
dynode is at higher electrical potential acts as
amplifier.

41. *
• 4. photodiodes : are semiconductors that
charge their charged voltage upon being
striked by the radiation, the voltage is
converted to current and it is measured.

42. Instrumentation
UV-VIS and INFRARED
Spectrophotometer

44. Reference
• Upadhyay.A,Upadhyay.K, and Nath.N. 2012.
Biophysical Chemistry(Principles and Techniques).
Himalaya Publishing House. Pvt. Ltd, Mumbai.
Pp.175-186.