Visible & Ultraviolet Spectroscopy

Spectroscopy

Spectroscopy is the science which deals with the interaction between a matter (atom/molecule)
and an electromagnetic radiation.

Ultraviolet and visible spectroscopy is a type of absorption spectroscopy that uses the ultraviolet
and visible parts of the electromagnetic spectrum.

What is absorption spectroscopy?

Absorption spectroscopy may be defined as the analysis of chemical substances by the

measurement of the amount of radiation absorbed by these substances.

In other sense, it is the study which deals with the measurement of the absorption of radiant
energy by various substances on the basis of light; in the UV, visible and IR regions of the
spectrum.

Its significances are-

- To determine the nature of the chemical compound in drug dosage form

- To determine the amount of the chemical compound in drug dosage form
- To determine the structure of the compound
- To determine the absorption capacity of the drug
- To determine the effectiveness of many pharmaceuticals
- To assess the drugs produced by the industry
 Visible & Ultraviolet Spectroscopy

What is electromagnetic spectrum?

The arrangement of all types of electromagnetic radiation in order to their increasing wavelength
is known as electromagnetic spectrum.
 Visible & Ultraviolet Spectroscopy

Electromagnetic radiation (EMR) can be expressed by various types of units:

1. Wavelength: It is a linear distance from any point of one wave to the corresponding
point of adjacent wave. It is denoted by the letter λ (lambda). In UV and IR spectroscopy, the
common unit is wavelength.
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2. Wave number: It is the reciprocal of wavelength. It is expressed as v̄

Mathematically, v̄ =1/λ. In IR techniques, it is frequently used.

3. Frequency: Frequency is the total number of occurrences of a repeating event per unit of the
given time. There are different frequency formulas to calculate frequency depending upon the
quantities known. The formula for the frequency of a wave is used to find frequency (f), time
period (T), wave speed (V), and wavelength (λ). 1 Hertz refers to one cycle per second. It is
expressed as v (nu)/f.

Mathematically,
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Photon: The emission or absorption of radiation is quantized and each quantum of radiation is
called photon.

Dispersion: When a group of EMR (visible light) passed through a prism, it splits up in to seven
colors which correspond to definite wavelengths. This phenomenon is called dispersion.

Middle UV: 200-300 nm Near UV: 300-400 nm

Brief summary on Visible & Ultraviolet Spectroscopy and Spectrometer:

The ultraviolet and visible spectrum is obtained by passing ultraviolet and visible
radiation through a transparent layer of substances being examined and then recording the
transmitted light.
When a beam of electromagnetic radiation is passed through a substance, the radiation can
be either absorbed or transmitted depending upon its frequency and the structure of the
molecule. The radiation not absorbed by the substance, i.e., the transmitted light can be
measured by means of a suitable instrument, known as ―Spectrometer‖. The result is the
―spectrum‖ of that compound.

If we plot the changes in absorption

against wavelengths, we get certain
absorption bands which are highly
characteristic of a compound and the
technique provides an excellent tool to
ascertain the molecular structure of an
unknown substance.

Figure: UV spectrum
 Visible & Ultraviolet Spectroscopy

Electromagnetic radiation is energy and hence, when a molecule absorbs radiations, it gains
energy. Just how much energy it gains depends upon the frequency of the radiation. ‗The higher
the frequency of the radiation, the greater the gain in energy‘.

The energy gained by the molecule in this way may bring about changes within the molecule,
such as:

 It may bring about increased vibration or rotation of the atom, or

 May raise electrons to higher energy levels.

The region of electromagnetic radiation which causes changes in electronic states (i.e., which
causes electronic transition) exists within 10 nm -780 nm, that is, ultraviolet & visible region.

The ultraviolet and visible spectrometers are commonly used to measure absorption of light in
the visible & near ultraviolet region, that is, 190 nm to 780 nm.

Vacuum ultraviolet region: The UV light below 200 nm is relatively uninformative and difficult
to measure, since, oxygen in the air also absorbs UV light in this region. Therefore, in order to
measure absorption of UV light below 200 nm, air must be removed from the instrument. The
region is known as vacuum ultraviolet region & it ranges from 10 nm to 200 nm.

The UV light above 200nm can be readily measured and gives informative spectra.

Spectroscopy

Spectroscopy is the use of the absorption, emission, or scattering of electromagnetic radiation by

matter to qualitatively or quantitatively study the matter or to study physical processes.

The matter can be atoms, molecules, atomic or molecular ions, or solids. The interaction of
radiation with matter can cause redirection of the radiation and/or transitions between the energy
levels of the atoms or molecules.
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Classes: The study of spectroscopy is mainly divided under four general headings-

 Ultra violet & visible spectroscopy (UV-VIS)

 Infrared spectroscopy (IR)
 Nuclear magnetic resonance spectroscopy (NMR)
 Mass spectroscopy MS)

Absorption: A transition from a lower level to a higher level with transfer of energy from the
radiation field to an absorber, atom, molecule, or solid.

Emission: A transition from a higher level to a lower level with transfer of energy from the
emitter to the radiation field. If no radiation is emitted, the transition from higher to lower energy
levels is called nonradioactive decay.

Scattering: Redirection of light due to its interaction with matter. Scattering might or might not
occur with a transfer of energy, i.e., the scattered radiation might or might not have a slightly
different wavelength compared to the light incident on the sample.

Spectrophotometry: Spectrophotometry is the measurement of the absorption & emission of

radiant energy by various substances depending upon their quality and quantity. The instrument
associated with spectrophotometry is called spectrophotometer.

Absorption spectrophotometry: Absorption spectrophotometry is the study which deals with

the measurement of the absorption of radiant energy by various substances. It includes the
measurement of the absorptive capacity for radiant energy in the UV.

UV Spectroscopy

UV (ultra violet-visible) spectroscopy is the spectroscopic method of analysis where

electromagnetic radiation of ultra violet region (190-800nm) is used to determine the
concentration of an ascertain compound in solution. It is also called electronic spectroscopy,
 Visible & Ultraviolet Spectroscopy

because it involves the electronic transitions means the promotion of electrons from the ground
state to the higher energy state.

Basic Principle of UV Spectroscopy

All atoms and molecules are capable of absorbing energy in accordance with their own structure
variation and so the kind and amount of radiation absorbed by molecule depending upon:

 The structure of the molecule

 The number of molecule interacting with the radiation

The study of these dependencies is called absorption on spectroscopy, when EMR is absorbed by
a molecule; it undergoes transition from a state of lower to a state of higher energy. If the
molecule is monoatomic (H2, N2) the energy absorbed can only be used to raise the energy
levels of electrons. If the molecule consists of more than one atom (polyatomic, CH3OH), the
radiation absorbed may bring about changes in electronic, rotational, vibrational or transitional
energy.

Electronic changes: Associated with the motion of electrons around the nuclei

Rotational changes: Associated with the overall rotation of the molecule

Vibrational changes: Associated with the movement of atoms within the molecule

Transitional changes: Associated with the motion of the molecule as a whole

Electronic transitions give absorption in the visible and UV region of the spectrum whereas;
transitional rotational and vibrational changes give absorption in the IR and near IR spectrum.
 Visible & Ultraviolet Spectroscopy

Principle of Ultraviolet-Visible Absorption

The Principle of UV-Visible Spectroscopy is based on the absorption of ultraviolet light or

visible light by chemical compounds, which results in the production of distinct spectra.
Spectroscopy is based on the interaction between light and matter. When the matter absorbs the
light, it undergoes excitation and de-excitation, resulting in the production of a spectrum.

When matter absorbs ultraviolet radiation, the electrons present in it undergo excitation. This
causes them to jump from a ground state (an energy state with a relatively small amount of
energy associated with it) to an excited state (an energy state with a relatively large amount of
energy associated with it). It is important to note that the difference in the energies of the ground
state and the excited state of the electron is always equal to the amount of ultraviolet radiation or
visible radiation absorbed by it.

It involves the promotion of electrons from HOMO to LUMO (HOMO means Highest Occupied
Molecular Orbital whereas LUMO means Lowest Unoccupied Molecular Orbital).

The HOMO-LUMO gap decrease as the conjugation increase Law of spectroscopy.

If a substance is placed in the path of an EM beam, a number of events may take place:

1. The intensity of the incident light may be identical to the intensity of the transmitted
light. It indicates that no absorption has taken place.
2. The intensity of the transmitted light is less than the intensity of the incident light. It
indicates that some absorption has taken place.
3. Reflection, refraction and/or scattering may occur.

Hafez Mohammad Arman, Dept. of Pharmacy, IIUC Page 9

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Law of spectroscopy

If a substance is placed in the path of an EM beam, a no. of events may take place:

 The intensity of the incident light may be identical to the intensity of the transmitted
light. It indicates that no absorption has taken place.
 The intensity of the transmitted light is less than the intensity of the incident light. It
indicates that some absorption has taken place.
 Reflection, refraction and/or scattering may occur.

The absorption law

 In 1729, Pierre Bouguer discovered the law.

 Later, in 1760, Johann Heinrich Lambert quoted Bouger‘s discovery saying that the
absorbance of a sample is directly proportional to the path length of light. Although
Lambert dint claim the discovery, he was often credited with it.
 In 1852, August Beer discovered a related law which stated that the absorbance is
proportional to the concentration of the sample.

There are two laws that govern absorption of light by the molecules.

i. Lambert‘s law
ii. Beer‘s law

What is Beer’s Law: Beer law states that concentration and absorbance are directly proportional
to each other and it was stated by August Beer.

What is Lambert Law: Lambert law states that absorbance and path length are directly
proportional and it was stated by Johann Heinrich Lambert.
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From these two laws, the following expression is obtained-

log10 𝐈˳ /𝐈 = A=a b c

Here,
Iᵒ = Intensity of incident light
I= Intensity of transmitted light
A= log10 𝐈˳/𝐈 = Absorbance or optical activity

As, absorbance is expressed as ‗A’ so that the expression may be written as

A=a b c

Here,
a= Proportionality constant (also known as absorptivity)
b= Path length of absorbing solution (in cm)
c= Conc. of the absorbing solute (molesL-1)

When c is expressed in mole/L, then absorptivity is called molar absorptivity.

It is also known as molar extinction co-efficient (ϵ).

Then,
A= ϵ b c [when c is in mole/L]
or, ϵ = A/b c
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Verification of Beer lambert law

From Beer Lambert‘s law, we have, A=εCb

This law can be verified by plotting the absorbance (A) against the molar concentration (C) of
the solution. A straight line passing through the origin and having a slope εb is obtained.

Limitations of the Beer-Lambert law

The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of
nonlinearity include:

 Deviations in absorptivity coefficients at high concentrations (>0.01M) due to

electrostatic interactions between molecules in close proximity.
 Scattering of light due to particulates in the sample.
 Fluorescence or phosphorescence of the sample.
 Changes in refractive index at high analyte concentration.
 Shifts in chemical equilibria as a function of concentration.
 Non-monochromatic radiation, deviations can be minimized by using a relatively flat part
of the absorption spectrum such as the maximum of an absorption band.
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 Stray light (any electro-magnetic radiation that is unwanted and interferes with the
performance of an optical system's intended functions).

Deviation of beer lambert law

When a plot of absorbance as a function of concentration at a particular path and wavelength of

monochromatic is drawn, a straight line passing through the origin is obtained. But when
concentration is very high, a plot of absorbance and concentration deviates from linear behavior.
The main causes of such deviation from Beer Lambert‘s law are given as:

 The deviation may occur when the light of a single wavelength is not used.
 Polymerization of solutes during the measurements.
 Association, dissociation, or ionization of solutes causes deviation.
 Presence of some other substance that absorbs at the same wavelength as the solute may
cause deviation.
 The presence of some impurities in the colored compounds may cause deviation

Spectrophotometer Instrumentation

The essential components of spectrophotometer instrumentation include:

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1. A Stable and cheap radiant energy source.

2. A monochromator to break the polychromatic radiation into component wavelength (or)
bands of wavelengths.
3. Transport vessels (cuvettes) to hold the sample.
4. A Photosensitive detector and an associated readout system

1. Radiant Energy Sources: Materials that can be excited to high energy states by a high-
voltage electric discharge (or) by electrical heating serve as excellent radiant energy sources.

i. Sources of Ultraviolet radiation: The most commonly used sources of UV radiation are
the hydrogen lamp and the deuterium lamp. Xenon lamp may also be used for UV
radiation, but the radiation produced is not as stable as the hydrogen lamp.
ii. Sources of Visible radiation: ―Tungsten filament‖ lamp is the most commonly used
source for visible radiation. It is inexpensive and emails continuous radiation in the range
between 350 and 2500nm. ―Carbon arc‖, which provides more intense visible radiation,
is used in a few commercially available instruments.
iii. Sources of IR radiation: ―Nernst Glower‖ and ―Global‖ are the most satisfactory
sources of IR radiation. Global is more stable than the nearest flower.

2. Wavelength selectors: Wavelength selectors are of two types.

I. Filters: ―Gelatin‖ filters are made of a gelatin layer, coloured with organic dyes and
sealed between glass plates.
II. Monochromators: A monochromator resolves polychromatic radiation into its
wavelengths. It isolates these wavelengths into very narrow bands. The essential
components of a monochromator are.
 The entrance slip-admits polychromatic light from the source.
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 Collimating device–Collimates the polychromatic light onto the dispersion

device.
 Wavelength resolving device like a PRISM (or) a GRATING
 A focusing lens (or) a mirror
 An exit slip–allows the monochromatic beam to escape.

The kinds of resolving element are of primary importance

i. PRISMS
ii. GRATINGS
PRISMS: A prism disperses polychromatic light from the source into its constituent
wavelengths under its ability to reflect different wavelengths to a different extent.
The degree of dispersion by the Prism depends on upon
a. The optical angle of the Prism (usually 600)
b. The material of which it is made

Two types of Prisms are usually employed in commercial instruments—namely, 600 cornu
quartz prism and 300 Littrow Prism.

GRATINGS: Gratings are often used in the monochromators of spectrophotometers operating

ultraviolet, visible and infrared regions.

3. Sample Containers: Sample containers are also one of the parts of Spectrophotometer
instrumentation. Samples to be studied in the ultraviolet (or) visible region are usually glasses
(or) solutions and are put in cells known as ―CUVETTES‖.
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Cuvettes meant for the visible region are made up of either ordinary glass (or) sometimes Quartz.
Most of the spectrophotometric studies are made in solutions. The solvents assume prime
importance.

The essential factor in choosing the solvent is that the solvent should not absorb (optically
transparent) in the same region as the solute.

4. Detection Devices: Most detectors depend on the photoelectric effect. The current is then
proportional to the light intensity and, therefore, a measure of it. Essential requirements for a
detector, including

 High sensitivity to allow the detection of low levels of radiant energy

 Short response time
 Long-term stability
 An electric signal which easily amplified for a typical readout apparatus.

5. Amplification and Readout: Radiation detectors generate electronic signals which are
proportional to the transmitted light. These signals need to be translated into a form easy
to interpret. It is accomplished by using amplifiers, Ammeters, Potentiometers and
Potentiometric recorders.

Electronic transitions

When molecule is getting excited by the absorption of electromagnetic radiation in UV-visible

region then its electrons are promoted from ground state to excited state or from bonding orbital
to anti-bonding orbital.
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Types of electronic transitions:

The absorption of UV or visible radiation corresponds to the excitation of outer electrons. There
are three types of electronic transition which can be considered:-

1. Transitions involving p, s, and n electrons

2. Transitions involving charge-transfer electrons
3. Transitions involving d and f electrons (not covered in this Unit)

When an atom or molecule absorbs energy, electrons are promoted from their ground state to an
excited state. In a molecule, the atoms can rotate and vibrate with respect to each other. These
vibrations and rotations also have discrete energy levels, which can be considered as being
packed on top of each electronic level.

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. The
spectrum of a molecule containing these chromophores is complex. This is because the
superposition of rotational and vibrational transitions on the electronic transitions gives a
combination of overlapping lines. This appears as a continuous absorption band.

Possible electronic transitions of p, s, and n electrons are:

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σ→σ* transition: In this transition, the electrons in a molecule move from a bonding (σ) orbital
to its comparable anti-bonding (σ*) orbital. This transition takes place due to the electromagnetic
radiation that gets absorbed. The highest quantity of energy is needed to undergo this transition.
It can be observed in the methane molecule due to the presence of only C-H bonds.

n→σ* transition: In this transition, the electrons from a nonbonding orbital (n) move to an anti-
bonding (σ*) orbital. The lowest quantity of energy is needed to undergo this transition.
Halogens and elements like sulfur, oxygen, and nitrogen display this transition.

n→π* transition: In this transition, the electrons from a non-bonding orbital (n) move to an
anti-bonding (π*) orbital. The lowest quantity of energy is needed to undergo this transition.
Halogens and elements like sulfur, oxygen, or nitrogen display this transition.

π→π* transition: In this transition, the electrons move from a bonding orbital (π) to an anti-
bonding (π*). Its energy requirement ranges in between the energies required for (n→π*) and
(n→σ*). Organic compounds like the aromatic ones, alkenes, alkynes, nitriles, and carbonyl
compounds display this transition.
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Some transitions between energy levels are radiative, and some are nonradiative. The photon
absorption involved between two energy levels is a radiative transition, whereas a transition
involving no photons between two energy levels is a nonradiative transition.

Probability

There is a direct relationship between the area of the chromophore and the absorption intensity.
Depending upon the symmetry and the value of εmax, the transition can classed as-

a) Allowed transitions - more probable - high intensity/ high energy transition

The transitions with values of εmax more than 104 are usually called allowed transition generally
arise from π→π* and n→σ * transition. e.g. In butadiene 1,3, the absorption at 217mμ and εmax
21,000.

b) Forbidden transitions - less probable - low intensity/ low energy transition

The forbidden transition is a result of the excitation of one electron from the lone pair present on
the hetero atom to antibonding π* orbital. i.e. n→π*. Values of εmax for such transition less than
104.

The π to σ∗, n to π∗ transitions usually lie in the UV-Vis region, whereas transitions σ to σ∗, π to
π∗, π to σ∗ lie in the vacuum or far UV region. The lowest energy transition is typically that of
an electron in the highest occupied molecular orbital (HOMO) to the lowest unoccupied
molecular orbital (LUMO). Electronic transitions may be as intense or weak according to the
magnitude of εmax that corresponds to allowed or forbidden transition as governed by the
selection rules of electronic transition, which states that the transitions change in their spin states
 Visible & Ultraviolet Spectroscopy

are not allowed. Thus, S→S, T→T, are allowed, but S→T, T→S, are forbidden (where S=
Singlet state, T= Triplet state). Ultraviolet-visible spectroscopy involves the absorption of
ultraviolet/visible light by a molecule causing the promotion of an electron from a ground
electronic state to an excited electronic state. Although, there are six possible transitions σ to σ∗,
σ to π ∗, π to σ∗, π to π∗, n to π∗ and n to σ∗, the most commonly observed transitions in organic
molecules are π to π∗, n to σ∗ and n to π∗.

Choice of solvent

A most suitable solvent is one which does not itself absorb in the region under investigation.
Always very dilute solutions are prepared for spectral analysis. Therefore, solvents play an
important role in UV-visible spectroscopy. Most commonly used solvent is 95% ethanol. Ethanol
is a best solvent as it is cheap and is transparent down to 210 nm. Commercial ethanol should not
be used as it contains benzene which absorbs strongly in the UV region.

Solvents used in UV-spectroscopy are

Solvents Minimum WL for 1cm cell (nm)

Water 191
Ethanol 204
CHCl3 234
Hexane 201
CCl4 257
Benzene 254
Methanol 203
Ether 215
Hexane and other hydrocarbons can be used as these are less polar and have least interaction
with the molecule under investigation. For, UV spectroscopy, water and cyclohexane serve the
purpose best. In case of water, there is the chance of hydrolysis, so water is not used always.

Effect of solvents on different transitions:

Polar substances are more sensitive to solvent change than non-polar substances.
 Visible & Ultraviolet Spectroscopy

Effect of solvents on n→π* transition (less intense)

 In such a case, the absorption will move to shorter wavelength by increasing the polarity
of the solvent.
 This is due to the fact that, in this case, the ground state is more polar as compare to the
excited state.
 Therefore, polar solvent stabilizes the non-bonding electrons in the ground state more
than the excited state, due to hydrogen bonding.
 More energy is needed & absorption will move to shorter wavelength.
 In other words, dipole-dipole interactions with solvent molecules will lower the energy of
the ground state more than that of the excited state.

Effect of solvents on π→π* transition (intense):

For such a case,

 The absorption band moves to longer wavelength by increasing the polarity of the
solvent.
 This is due to the fact that in this case, the excited state is more polar as compare to the
ground state.
 Therefore, polar solvents will stabilize the excited state more than that of the ground
state.
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 Dipole-dipole interaction with solvent molecules will lower the energy of the excited
state more than that of the ground state.
 That is less energy is needed and absorption will move to longer wavelength

Effect of solvents on n n→ σ* transition:

Alcohols and amines form hydrogen bonding with solvent molecules, such associations occur
due to the presence of non-bonding electrons on the hetero atom and thus, transition requires
greater energy.

Effect of Temperature and Solvent on the Fineness of Absorption Band

It is known that the vibrational and the rotational states depend on temperature. As the
temperature is decreased, vibrational and the rotational energy state of the molecules are also
lowered. Thus, when the absorption of light occurs at a lower temperature, smaller distribution of
excited states result. It produces finer structure in the absorption band than what is noticed at
higher temperature. Consider the UV spectrum of dodecapentaenic acid in ether-alcohol solvent
at 20 ℃ and -195 ℃.
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The solvent used also affects the fineness of absorption band in UV spectrum. If the dielectric
constant of the solvent is high, there will be stronger solute-solvent interactions. Due to this,
vibrational and rotational energy states of molecules increases and thus, the fineness of the
absorption band falls.
Effect of Sample pH
The pH of the sample solution can also have a significant effect on absorption spectra. The
absorption spectra of certain aromatic compounds such as phenols and anilines change on
changing the pH of the solution. Phenols and substituted phenols are acidic and display sudden
changes in their absorptions maxima upon the addition of a base. After the removal of the
phenolic proton, we get phenoxide ion. In the phenoxide ion, lone pairs on the oxygen is
delocalized over the π-system of the aromatic ring and increases the conjugation of the same.
Extended conjugation leads to a decrease in the energy difference between the HOMO and
LUMO orbitals, which results in red or bathochromic shift (to longer wavelength), along with an
increase in the intensity of the absorption.
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Similarly, an aromatic amine gets protonated in an acidic medium which disturb the conjugation
between the lone pair on nitrogen atom and the aromatic π-system. As a result, blue shift or
hypsochromic shift (to shorter wavelength) is observed along with a decrease in intensity.

Effect of Sample Temperature

The effect of temperature is less pronounced. However, simple thermal expansion of the solution
may be sufficient to change band intensity. Therefore to get more accurate results the spectrum
needs to take at a specified or constant temperature.

The following three criteria may be given as general effect of temperature on solution spectra-

1. Band sharpness increases with decreasing temperature.

2. Position of absorption maximum does not move or moves very little towards the longer
wavelength side, with decreasing temperature.
3. The total absorption intensity is approximately independent of the temperature.

Effect of Sample Concentration

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According to Lambert-Beer law, it might be expected that the sample concentration is directly
proportional to the intensity of the absorption. But at high concentrations, molecular interactions
can take place causing changes to the position and shape of absorption bands. Such effects need
to be identified and taken into consideration for quantitative work.

The solvent used also affects the fineness of absorption band in UV-spectrum. It has been
observed that polar solvents give rise to broad bands, non-polar solvents show more resolution,
while completely removing the solvent gives the best resolution. This is due to solvent-solute
interaction. If the dielectric constant of the solvent is high, there will be stronger solute-solvent
interaction. e.g., a polar solvent like water or ethanol has the ability of hydrogen bonding with
the solute if the solute has a hydrogen bonding component, or simply through induced dipole-
dipole interactions.

The non-polar solvents can also interact though polarizability, but the effect is very less.
Depending on the interaction, this can cause the ground state and the excited state of the solute to
increase or decrease, thus changing the frequency of the absorbed photon. Due to this, there are
many different transition energies that overlap in the spectra and cause the broadening of UV-
band.

CONJUGATED DIENES

Conjugated dienes are two double bonds separated by a single bond. Non conjugated (isolated)
dienes are two double bonds are separated by more than one single bond. One of the most
important factors affecting the wavelength of absorption by a molecule is the extent of
conjugation. A conjugated dienes is one that contains alternating double and single bonds. One
characteristic is that they are more stable than their non-conjugated counterparts.

The wavelength of absorption is shifted to higher values (Bathochromic shift), if two or more
chromophoric groups are present in conjugation in a molecule.
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e.g., ethylene (one double bond) absorbs at 170 mμ (π to π* transition) while butadiene (two
double bonds in conjugation) absorbs at 217 mu. The bathochromic shift is more pronounced if
the double bonds are in as compared to the isolated double bonds in which there is a little
interaction between them.

The absorption maximum is usually shifted 15-45 mμ towards higher wavelength in conjugated
system (compared to unconjugated) as the electron density is spread over at least four atomic
centers. The value of extinction coefficient also increases. In conjugated dienes, π to π*
transition results in the formation of a band, called K-band.

Types of Absorption Bands

Following types of bands originate as a result of the possible transitions in a compound.

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Some terminologies

The terms commonly used in UV-Visible spectroscopy are:

 Chromophore
 Auxochrome
 Bathochromic shift (Red shift)
 Hypsochromic shift (Blue shift)
 Hyperchromic effect
 Hypochromic effect
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Chromophores: A chromophore is a group responsible for characteristic absorption of radiation

by a molecule. Most chromophores contain one, two or more double bonds. Ex: carbonyl group
in acetone.

 If a compound absorbs light in the visible region (400-800 nm), only then it appears
colored to our eyes. Therefore a chromophore may or may not impart colour to a
compound depending on whether the chromophore absorbs radiation in the visible or UV
region.
 There are no standard criteria for the identification of a chromophore because the
wavelength and intensity of absorption depend on many factors such as the molecular
environment of the chromophore and on the solvent in which the sample is dissolved.
 Other parameters, such as pH and temperature, also may cause changes in both the
intensity and the wavelength of the absorbance maxima.
 All the compounds having the same functional group will absorb at almost the same
wavelength if the other factors such as conjugation, substituents etc. are absent.

There are two types of chromophores:

a) Chromophores in which the group contains π (bonding) electrons and they undergo π→ π*
transitions (requires smaller energy and hence transition of this type occurs at longer
wavelength). Such chromophores are ethylenes, acetylenes etc.

b) Chromophores which contain both π (bonding) electrons and n (nonbonding) electrons. Such
chromophores undergo two types of transitions i.e. π → π* and n → π* (requires least amount of
energy and hence transition of this type occurs at longer wavelength).

Examples: carbonyl, nitro compounds, azo compounds etc.

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Auxochrome:

An auxochrome is a group which does not itself acts as a chromophore but when attach to a
chromophore, it alters both the position and the intensity of the absorption peak. Such as: -OR, -
OH, -NH2, -NHR, -NR2, -SHCl2 etc.

Example: -CH=CH- shows absorption peak about 190nm

-CH=CH-NR2 shows about 230nm

An auxochrome contains an atom with unshared pair of electrons.

Absorption and intensity shift:

a) Bathochromic effect/shift (Red shift): It is the displacement of the absorption

maximum towards longer wavelength due to the introduction of an auxochrome or by the
change of solvent. It is also called red shift because the absorption shift toward the red
light of the visible light region.
b) Hypsochromic shift/effect (Blue shift): It is the displacement of the absorption
maximum toward shorter wavelength due to the removal of auxochrome or by the effect
of solvent. It is also called blue shift. Ex: absorption maximum of aniline shifts from
230nm to 203nm, if the solvent is changed from hexane (non-polar) to aqueous acid.
c) Hyperchromic effect: It is the effect leading to increase absorption intensity. The
introduction of an auxochrome usually increases intensity of absorption.
 Visible & Ultraviolet Spectroscopy

d) Hypochromic effect: It is the effect leading to decrease absorption intensity. The

introduction of group which distorts the geometry of the molecule causes hypochromic
effect.

Applications of Absorption Spectroscopy (UV, Visible)

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. By
also measuring the absorbance at specific wavelength, the impurities can be detected.
Benzene appears as a common impurity in cyclohexane. Its presence can be easily
detected by its absorption at 255 nm.
2. Structure elucidation of organic compound: UV spectroscopy is useful in the structure
elucidation of organic molecules, the presence or absence of unsaturation, the presence of
hetero atoms. From the location of peaks and combination of peaks, it can be concluded
that whether the compound is saturated or unsaturated, hetero atoms are present or not
etc.
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.
 Visible & Ultraviolet Spectroscopy

Woodward-Fieser rules for calculating absorption maximum in dienes

Woodward formulated certain empirical rules for calculated the λmax for long wavelength π- π*
transition for dienes and polyenes. These rules were later by FIESER in 1959. Accroding to these
rules, each diene has a certain fixed basic value and the value of the λmax upon the following
points:
(I) the alkyl substituents or ring residues present on the diene system.
(II) The number of double bonds extending conjugation beyond the diene system.
(III) The presence of polar groups or auxochromes such as –X (=C1, Br etc.) ,-OR,-SR,-NH2, -
OCOCH3 etc.

Math from reference books

Reference book
1. Sharma, Y.R (2007), Elementary Organic Spectroscopy, 4th ed. S.Chand and Company.
2. Chatten, L.G. (2010) Pharmaceutical Chemistry, Volume 1 and 2. Mercel Dekker/CBS
Publishers.