UV-Visible Spectroscopy

Ultra violet and visible spectroscopy:

Principle, Beer-Lambert’s law and determination of concentration,
instrumentation,
electronic transition, transition probability, selection rules,
chromophore, auxochrome,
absorption & intensity shift, absorption bands, solvent effects,
application in food analysis.
 What is Spectroscopy?
Spectroscopy deals with the production, measurement, and
interpretation of spectra arising from the interaction of
electromagnetic radiation with matter.
There are many different spectroscopic methods available for solving a
wide range of analytical problems.
The methods differ with respect to the
• species to be analyzed (such as molecular or atomic spectroscopy),
• the type of radiation–matter interaction to be monitored (such as
absorption, emission, or diffraction), and
• the region of the electromagnetic spectrum used in the analysis.
 Spectroscopic Methods
Spectroscopic methods are very informative and widely
used for both quantitative and qualitative analyses.
Spectroscopic methods based on the absorption or
emission of radiation in the
ultraviolet (UV), visible (Vis), infrared (IR), and radio
(nuclear magnetic resonance, NMR) frequency ranges
are most commonly encountered in traditional food
analysis laboratories.
Interaction of Radiation and Matter
If matter is exposed to electromagnetic radiation, e.g. infrared light, the
radiation can be absorbed, transmitted, reflected, scattered or undergo
photoluminescence. Photoluminescence is a term used to designate a
number of effects, including fluorescence, phosphorescence, and
Raman scattering.
 Interaction of Radiation and Matter
The complement of the absorbed light gets transmitted. The color of
an object we see is due to the wavelengths transmitted or reflected.
Other wavelengths are absorbed. The more absorbed, the darker the
color (the more concentrated the solution). In spectrochemical
methods, we measure the absorbed radiation.
Wave Motion of Electromagnetic Radiation.
The distance of one cycle is the wavelength (λ).
The frequency (ν) is the number of cycles passing a fixed point per unit
time. λ = c/ ν (c = velocity of light, 3 x 1010 cm s-1 ).
The shorter the wavelength, the higher the energy: E = hν.
 Electromagnetic Spectrum
We see only a very small portion of the electromagnetic spectrum . In
spectrochemical methods, we measure the absorption of UV to far IR
radiation.
• UV = 200-380 nm
• VIS = 280-780 nm
• IR = 0.78 mm-300 mm
Electromagnetic Spectrum
Electromagnetic Spectrum
Absorption and Emission
 Selection Rules
• The electron must be promoted without a change in its orientation. ∆s = 0
• When ∆ s ≠ 0 transition is forbidden. It may occur with very low probability
• Some other from quantum mechanics

In physics and chemistry, a selection rule, or transition rule, formally constrains the possible
transitions of a system from one quantum state to another. Selection rules have been derived
for electromagnetic transitions in molecules, in atoms, in atomic nuclei, and so on. The selection rules
may differ according to the technique used to observe the transition. The selection rule also plays a
role in chemical reactions, where some are formally spin-forbidden reactions, that is, reactions where
the spin state changes at least once from reactants to products.
In the following, mainly atomic and molecular transitions are considered.

A selection rule describes how the probability of transitioning from one level to another cannot be zero. It
has two sub-pieces: a gross selection rule and a specific selection rule. A gross selection rule illustrates
characteristic requirements for atoms or molecules to display a spectrum of a given kind, such as an IR
spectroscopy or a microwave spectroscopy. Once the atom or molecules follow the gross selection rule, the
specific selection rule must be applied to the atom or molecules to determine whether a certain transition in
quantum number may happen or not.
Selection rules specify the possible transitions among quantum levels due to absorption or emission of
electromagnetic radiation.
Jablonski diagram:
However, it is more commonly expressed as a percentage transmittance:
The absorbance, A, of the solution is related to the transmittance and incident and transmitted intensities through the
following relations:

The absorbance has a logarithmic relationship to the transmittance; with an absorbance of 0 corresponding to a
transmittance of 100% and an absorbance of 1 corresponding to 10% transmittance. Additional values of transmittance and
absorbance pairings are given in Table 1. A visual demonstration of the effect that the absorbance of a solution has on the
attenuation light passing through it is shown Figure 2, where a 510 nm laser is passed through three solutions of Rhodamine
6G with different absorbance.
0 100%
1 10%
2 1%
3 0.1%
4 0.01%
5 0.001%
The Beer-Lambert law states that there is a linear relationship between the concentration and the absorbance of the solution,
which enables the concentration of a solution to be calculated by measuring its absorbance. To demonstrate this linear
dependence five solutions of Rhodamine B in water were measured using the DS5 Dual Beam Spectrophotometer (Figure 3a)
and from these absorption spectra, a linear calibration curve of the absorbance versus concentration was created (Figure 3b).
Using this calibration curve the concentration of an unknown Rhodamine B solution can be determined by measuring its
absorbance which is the main utility of the Beer-Lambert Law.

λ
 Instrumentation
The typical ultraviolet-visible spectrophotometer consists of a light source, a monochromator and
a detector.
• A light source is usually a deuterium lamp. Which emits electromagnetic radiation in the
ultraviolet region of the spectrum.
• A second light source, a tungsten lamp, is used for wavelengths in the visible region of
spectrum.
• The monochromator is a diffraction grating; its role is to spread the beam of light into its
component wavelengths. A system of slits focuses the desired wave length on the sample cell.
• The light that passes through the sample cell, reaches the detector, which records the intensity
of the transmitted light. The detector is generally a photomultiplier tube, although in modern
instrument photodiodes are also used.
In a typical double beam instrument, the light emanating from the light source is split into two
beams, the sample beam and the reference beam. When there is no sample cell in the
reference beam, the detected light is taken to be equal to the intensity of light entering the
sample.
6. Recording devices
The majority of the time amplifiers are connected to a pen recording device that is linked to a computer. Computers store all the data produced and generates an array of compound.
 Solvents For Ultraviolet Spectroscopy
Choice of solvent to be used in ultraviolet spectroscopy
• Solvent should not absorb ultraviolet radiation in the same region as the
substance whose spectrum is being determined. Solvents those do not
contain conjugated system suitable for this purpose.
• A second criterion for a good solvent is its effect on the fine structure of
the absorption band. There is a effect of polar non-polar solvents on an
absorption band. A non-polar solvent does not form hydrogen bond with
the solute, and the spectrum of the solute closely approximates the
spectrum that would be produced in the gaseous state, in which the fine
structure is often observed. In a polar solvent, the hydrogen bonding
forms a solute-solvent complex, and the fine structure may disappear.
 Solvents For Ultraviolet Spectroscopy
Some common uv spectroscopy solvents and their cutoff points or minimum regions of
transparency. Of the solvents , water, 95% ethanol and hexane are most commonly used.
Acetonitrile 190 nm
Chloroform 240nm
Cyclohexane 195 nm
1,4-Dioxane 215 nm
95% Ethanol 205 nm
N-hexane 201 nm
Methanol 205 nm
Isooctane 195 nm
Water 190 nm
Trimethyl phosphate 210 nm
 Chromophore
They are groups with one element of unsaturation (unsaturated linkages or groups) and cause coloring to
the molecules when they are attached to a non-absorbing hydrocarbon chain

Absorption of ultraviolet radiation results from the

excitation of electrons from ground state to excited
states, the nuclei that the electrons hold together in
bonds play an important role in determining which
wavelengths of radiation are absorbed. The nuclei
determine the strength with which the electrons are
bound and thus influence the energy spacing
between ground and excited states.
The characteristic energy of a transition and the
wavelength of radiation absorbed are properties of a
group of atoms rather than electrons themselves.
The group of atoms producing such an absorption is
called a chromophore.
Chromophore is defined as any isolated covalently
bonded group that shows a characteristic absorption
in the ultraviolet or visible region (200-800 nm).
Chromophores
Chromophores can be divided into two groups-

a) Chromophores which contain p electrons and which undergo pie to

pie* transitions. Ethylenes and acetylenes are the example of such
chromophores.

b) Chromophores which contain both p and nonbonding electrons.

They undergo two types of transitions; pie to pie* and nonbonding
to pie*. Carbonyl, nitriles, azo compounds, nitro compounds etc. are
the example of such chromophores.
Auxochromes
• They are groups that do not confer color but increase the coloring power of a chromophore.
• They are functional groups that have non-bonded valence electrons and show no absorption at l > 220
nm; they absorb in the far UV
• -OH and -NH2 groups cause a red shift
The attachment of substituent groups in place of hydrogen on a basic chromophore structure changes the
position and intensity of an absorption band of the chromophore.
An auxochrome can be defined as any group which does not itself act as a chromophore but whose presence
modifies the absorption of the principal chromophore. Substituents that increase the intensity of the
absorption, and possibly the wavelength, are called auxochromes. Typical auxochromes include –CH3,–OH,-
OR,-NH2,-NHR, -SH, -X etc.
 Absorption & Intensity Shifts in the UV Spectroscopy

Substituents may have any of four kinds of effects on the absorption:

a) Bathochromic effect- This type of shift is also known as red shift.
Bathochromic shift is an effect by virtue of which the absorption
maximum is shifted towards the longer wavelength due to the
presence of an auxochrome or change in solvents.
The nonbonding to pie* transition of carbonyl compounds observes
bathochromic or red shift.
b) Hypsochromic shift- This effect is also known as blue shift.
Hypsochromic shift is an effect by virtue of which absorption
maximum is shifted towards the shorter wavelength. Generally it is
caused due to the removal of conjugation or by changing the polarity
of the solvents.
Absorption & Intensity Shifts in the UV Spectroscopy
c) Hyperchromic effect- Hyperchromic shift is an effect by virtue of which
intensity of absorption maximum increases. The introduction of an
auxochrome in the compound generally results in the hyperchromic effect.

d) Hypochromic effect- Hypochromic effect is defined as the effect by virtue of

intensity of absorption maximum decreases. Hypochromic effect occurs due to
the distortion of the geometry of the molecule with an introduction of new
group.
An absorption band is a range of wavelengths, frequencies or energies in
the electromagnetic spectrum which are characteristic of a particular transition from initial
to final state in a substance.

Absorptions bands in the Earth's atmosphere created

by greenhouse gases and the resulting effects on
transmitted radiation.
 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.
 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.
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