UV VISIBLE

SPECTROSCOPY
Spectroscopy

 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 consist of an oscillating electric field (E) and an oscillating
magnetic field (M) which are perpendicular to each other
Electromagnetic radiation

 as photon is subjected to energy, so

ℎ𝑐
𝐸=
λ
Electromagnetic radiation
 Electromagnetic radiation

Visible Wavelength
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
Principles of spectroscopy

 The principle is based one 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
Principles of spectroscopy

 Absorption spectroscopy
 An analytical technique which concerns with the measurement of absorption of
electromagnetic radiation
 Example
 UV spectroscopy  185-400 nm
 Visible spectroscopy  400-800 nm
 IR Spectroscopy  0.76-15 µm
Principles of spectroscopy

 Emission spectroscopy
 An analytical technique in which emission (of a particle or radiation) is dispersed
according to some property of the emission and the amount of dispersion is
measured
 Mass spectroscopy
Interaction of electromagnetic
radiation with matter

 Electronic energy levels

 At room temperature the molecules are in the lowest energy levels Eo
 When the molecules absorb UV-visible light from electromagnetic radiation, 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 = ℎν = 𝐸𝑛 − 𝐸𝑜

𝑘𝑐𝑎𝑙
∆𝐸 = 35 𝑡𝑜 71
𝑚𝑜𝑙𝑒
Interaction of electromagnetic
radiation with matter

 Vibrational energy levels

 These are less energy level than electronic energy levels
 The spacing between energy levels are relatively small, 0.01 to 10 kcal/mole
 When IR radiation is absorbed, molecules are excited from one vibrational level
to another or it vibrates with higher amplitude
Interaction of electromagnetic
radiation with matter

 Rotational energy levels

 These energy levels are quantized and discrete
 The spacing between energy levels are even smaller than vibrational energy
levels
 Δerotational < Δevibrational < Δeelectronic
Lambert’s Law

 When a monochromatic radiation is passed through a solution, the

decrease in the intensity of radiation with thickness of the solution is
directly proportional to the intensity of the incident light
 When a monochromatic radiation is passed through a solution, the
decrease in the intensity of radiation with thickness of the solution is
directly proportional to the intensity of the incident light as well as
concentration of the solution
𝐴 = 𝜀. 𝑏. 𝑐
Principles of UV VIS
Spectroscopy
Principle

 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 – 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
Electronic transitions

 The possible electronic transitions can graphically shown as:

The possible electronic transitions are

 σ  σ* Transition
 σ electron from orbital is excited to corresponding anti-bonding orbital σ*
 The energy required is large for this transition
 Methane (CH4) has C-H bond only and can undergo σ  σ* transition and
shows absorbance maxima at 125 nm
 The organic compounds in which all the valence sheel electrons are involved in
the formation of σ bond do not show absorption in normal UV region (200-400
nm)
The possible electronic transitions are

 π  π* transition
 π electron in a bonding orbital is excited to corresponding antibonding orbital
π*
 compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles,
aromatic compounds, etc undergo π  π* transitions.
 Alkenes generally absorb in the region 170 to 205 nm
The possible electronic transitions are

 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)
The possible electronic transitions are

 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), N=O)
undergo such transitions
 n  π* transition require minimum energy and show absorption at longer
wavelength around 300 nm
 σ  π* transition and π  σ* transition
 These electronic transition are forbidden transitions and are theoretically
possible
 Thus, n  π* and π  π* electronic transitions show absorption in region above
200 nm which is accessible to UV-Vis spectrophotometer
 The UV spectrum is of only a few broad of absorption
Absorption and intensity shifts

 Bathochromic shift (red shift)

 when absorption maxima of a compound shifts to longer wavelength
 The effect is due to presence of an auxochrome or by the change of solvent
 Hypsochromic shift (Blue Shift)
 When absorption maxima of a compound shifts to shorter wavelength
 the effect is due to presence of an group causes removal of conjugation or by the
change of solvent
 Hyperchromic effect
 When absorption intensity of a compound is increased
 Hypochromic effect
 When absorption intensity of a compound is decreased
Shift and effect
Application of UV/Vis spectroscopy

 Qualitative and quantitative analysis

 It is used for characterizing aromatic compounds and conjugated olefins
 It can be used to find out molar concentration of the solute under study
 Detection of impurities
 It is one of the important method to detect impurities in organic solvents
 Detection of isomers are possible
 Determination of molecular weight using Beer’s law
Molecular spectroscopy
The main transition that occur in
organic molecules
Chromatic colours
Instrumentation (Spectrophotometers)
29
Wavelength selector

A single beam spectrophotometer

The above essential features of a spectrophotometer shows that
polychromatic light from a source separated into narrow band of wavelength
(nearly monochromatic light) by a wavelength selector, passed through the
sample compartment and the transmitted intensity, P, after the sample is
measured by a detector

In a single beam instrument, the light beam follows a single path from
the source, to the monochromator, to the sample cell and finally to the
detector
1- Sources of light
30
Sources used in UV-Vis Spectrophotometers are continuous sources.
• Continuous sources emit radiation of all wavelengths within the
spectral region for which they are to be used.
• Sources of radiation should also be stable and of high intensity.

Continuous
Sources

Visible and
Ultraviolet
near IR
radiation
radiation

Tungsten Deuterium
Lamp Lamp
320-2500 nm 200-400 nm
2. Wavelength Selectors
31
Ideally the output of a wavelength selector would be a radiation of a single
wavelength.

No real wavelength selector is ideal, usually a band of radiation is obtained.

The narrower this bandwidth is , the better performance of the instrument.

Wavelength
selectors

Filters Monochromators
i- Filters
32
• Filters permit certain bands of wavelength (bandwidth of ~ 50 nm) to pass
through.
• The simplest kind of filter is absorption filters , the most common of this
type of filters is colored glass filters.
• They are used in the visible region.
• The colored glass absorbs a broad portion of the spectrum
(complementary color) and transmits other portions (its color).

Disadvantage
• They are not very good wavelength selectors and can’t be used in
instruments utilized in research.
• This is because they allow the passage of a broad bandwidth which
gives a chance for deviations from Beer’s law.
• They absorb a significant fraction of the desired radiation.
ii- Monochromators
They are used for spectral scanning (varying the wavelength of 33
radiation over a considerable range ).

They can be used for UV/Vis region.

All monochromators are similar in mechanical construction.

All monochromators employ slits, mirrors, lenses, gratings or

prisms.
What are the advantages and disadvantages of decreasing
monochromator slit width?

Bandwidth Choice
 Selection of wavelength
Absorbance measurements are always carried out at fixed
wavelength (using monochromatic light). When a wavelength is
chosen for quantitative analysis, three factors should be considered

Wavelength should be chosen to give the highest possible sensitivity.

This can be achieved by selecting max or in general the wavelengths at
which the absorptivity is relatively high.

λmax

λmax - wavelength where maximum

absorbance occurs
 10-2 M
By performing the analysis at
10-3 M

Absorbance
such wavelengths, it will be 10-4 M
5x10-5 M
sure that the lowest sample
10-5 M
concentration can be
measured with fair accuracy.

For example, the lowest

max 1
sample concentration (10-5 M) wavelength
can be measured with good
accuracy at max, while at other
wavelength (1), it may not be
detected at all.
2. It is preferable to choose the wavelength at which the absorbance will not
significantly change if the wavelength is slightly changed,
i.e., A /  is minimum.
At a wavelength corresponding to broad horizontal band on the spectrum
(band A), the radiation is mainly absorbed to the same extent (A /  zero).
However on a steep portion of the spectrum (band B), the absorbance will
change greatly if the wavelength is changed (A /  is large) . Thus on
repeating the absorbance measurements, you might get different readings
and the precision of the measurements will be poor.
Band A

A Broad horizontal
bands
Band B
Absorbance

A shoulder

  m wavelength
3- If the solution contains more than absorbing species, the 38
wavelength should be chosen, whenever possible, in region at which
the other species does not absorb radiation or its absorbance is
minimum. By this way, the second species does not interfere in the
determination.

Absorbance sample
Interfering
species

m wavelength
 3- Sample compartment (cells)
39
 For Visible and UV spectroscopy, a liquid sample is usually
contained in a cell called a cuvette.
 Glass is suitable for visible but not for UV spectroscopy because it
absorbs UV radiation. Quartz can be used in UV as well as in visible
spectroscopy

Long pathlength 1 cm pathlength cuvet

Opaque
Face

Transparent
Face

1 cm 1 cm
Short pathlength (b)
4- Detectors
 The detectors are devices that convert radiant energy into electrical 40
signal.
 A Detector should be sensitive, and has a fast response over a
considerable range of wavelengths.
 In addition, the electrical signal produced by the detector must be
directly proportional to the transmitted intensity (linear response).

i- Phototube

Phototube emits electrons h

from a photosensitive,
negatively charged cathode
when struck by visible or
UV radiation
The electrons flow through
e- amplifier
anode
vacuum to an anode to
produce current which is -V
proportional to radiation Photosensitive cathode
intensity.
ii. Photomultiplier tube
 It is a very sensitive device in which electrons emitted from the photosensitive
41
cathode strike a second surface called dynode which is positive with respect to
the original cathode.
 Electrons are thus accelerated and can knock out more than one electrons from
the dynode.
 If the above process is repeated several times, so more than 106 electrons are
finally collected for each photon striking the first cathode.

light dynodes

anode
electrons
e-
photochathode

voltage divider network

high voltage
 The components of a single beam
spectrophotometer 42
Light source - white light of constant intensity

slits

Grating
slits Separates white light
Phototube
into various colors
detects light &
measures intensity Rotating the grating
Sample
changes the wavelength
When blank is the sample going through the sample
Po is determined,
otherwise P is measured
 Double Beam Spectrophotometer 43

Beam Chopper Semi-transparent

Mirror
Sample

Tungsten Slit Quartz Photo-

Grating
Lamp Cuvette multiplier
Reference
(Blank)
Mirror Mirror

Mirror
 44
Double Beam Spectrophotometer

B B B B B represents P0

S
Signal %T   100
B
S S S S represents P

Time
 45

Schematic diagram of a double beam scanning spectrophotometer

 In double beam arrangement, the light alternately passes through

the sample and reference (blank), directed by rotating half-sector
mirror (chopper) into and out of the light path.

 When light passes through the sample, the detector measures the
P. When the chopper diverts the beam through the blank solution, the detector
measures P0.

 The beam is chopped several times per second and the electronic
circuit automatically compares P and P0 to calculate absorbance
and Transmittance.
Advantages of double beam instruments over single beam instruments

Single beam spectrophotometer is inconvenient because

1. The sample and blank must be placed alternately in the light path.
2. For measurements at multiple wavelengths, the blank must be run at each
wavelength.

In double beam instruments

1. The absorption in the sample is automatically corrected for the absorption
occurring in the blank, since the readout of the instrument is log the
difference between the sample beam and the blank beam.
2. Automatic correction for changes of the source intensity and changes in the
detector response with time or wavelength because the two beams are
compared and measured at the same time.
3. Automatic scanning and continuous recording of spectrum (absorbance
versus wavelength).