Introduction

Spectroscopy is defined as the measurement and interpretation of electromagnetic radiation

absorbed, scattered or emitted by atoms, molecules or other chemical species. This
absorption or emission results in the change in energy states of interacting chemical species.
It is a scientific technique used to study and analyze the interaction between matter and
electromagnetic radiation. By examining the spectrum of light or other forms of
electromagnetic waves emitted, absorbed, or scattered by substances, spectroscopy provides
valuable insights into the properties, composition, and structure of matter. This technique is
foundational in fields such as chemistry, physics, astronomy, and biology, as it allows
scientists to identify elements and compounds, determine concentrations, and explore
molecular dynamics. Spectroscopy encompasses a wide range of methods, including infrared,
ultraviolet, visible, and nuclear magnetic resonance (NMR) spectroscopy, each offering
unique applications and advantages. Through its ability to reveal detailed information about
the atomic and molecular world, spectroscopy plays a crucial role in advancing scientific
understanding and technological innovation. The spectroscopy can be broadly categorized
into two categories:

1. Emission Spectroscopy
2. Absorption Spectroscopy
 Emission Spectroscopy: It involves analyzing the light emitted by atoms or molecules to
determine their composition and properties. When a substance is excited, typically by
heating or applying an external energy source like electricity or light, its atoms or
molecules move to a higher energy state. As they return to their lower energy states, they
emit light at specific wavelengths, creating an emission spectrum. For example Atomic
absorption spectroscopy, Flame emission spectroscopy, X-ray crystallography, ICP-OES.
 Absorption Spectroscopy: It involves measuring the amount of light absorbed by a
sample at various wavelengths to determine the sample's composition and properties.
Unlike emission spectroscopy, which analyzes the light emitted by a substance,
absorption spectroscopy focuses on the wavelengths of light absorbed as the substance
transitions from a lower to a higher energy state. For example UV-spectroscopy, IR-
spectroscopy, NMR. The phenomenon of absorption and emission of electromagnetic
spectrum was given in Figure 1.
 Figure 1. Principle of spectroscopy

Electromagnetic waves and spectrum

Electromagnetic waves are a form of energy propagation that involves oscillating electric and
magnetic fields traveling through space. They are fundamental to understanding various natural
phenomena and are used in a wide range of technologies, from communication systems to medical
imaging. The general diagram of electromagnetic wave was given in Figure 2.

Figure 2. General depiction of electromagnetic wave

Basic terminology in electromagnetic waves includes:

 Wavelength (λ): The wavelength of an electromagnetic wave is the distance between

successive crest and trough of the wave.
 Wavenumber (ῡ): Wavenumber is a measure of the number of wavelengths per unit
distance, typically expressed in reciprocal centimeters (cm -1). It represents the spatial
frequency of a wave, indicating how many wave cycles fit into a given unit of space.
ῡ = 1/ λ
 Frequency (ƒ): It is the number of cycles or oscillations of a wave that pass a
particular point in one second. It is a measure of how often the wave peaks occur and
is expressed in Hertz (Hz), where 1 Hz equals one cycle per second.
ƒ ∝ λ-1 i.e. ƒ = c λ-1
 Amplitude: The amplitude of an electromagnetic wave is a measure of the strength or
intensity of the wave. It represents the maximum displacement of the wave from its
equilibrium position.

The electromagnetic spectrum is the range of all types of electromagnetic radiation, organized
by wavelength or frequency. It encompasses everything from low-energy radio waves to
high-energy gamma rays. Understanding the electromagnetic spectrum is essential for
exploring how different types of electromagnetic waves interact with matter and how they
can be utilized across various fields, from communication to medicine. Figure 3 describes the
electromagnetic spectrum.

Figure 3. Electromagnetic spectrum

UV-Visible spectrophotometry
Absorption spectrophotometry in the ultraviolet and visible region is considered to be one of
the oldest physical methods used for quantitative analysis and structural elucidation. We
know that infrared and NMR techniques are mainly used for structural elucidation and
qualitative analysis. On the other hand, ultraviolet and visible spectrophotometry is mainly
used for quantitative analysis and serves as a useful auxiliary tool for structural elucidation.
The wavelength of UV radiation starts at the blue end of visible light, about 4000 A, and ends
at 2000 A. However, the wavelength of visible radiation starts at 8000 Å and ends at 4000 Å;
Spectroscopically. visible light acts in the same way as UV light.

 The visible rays: Visible rays, also known as visible light, are a type of
electromagnetic radiation that is visible to the human eye. This part of the
electromagnetic spectrum allows us to perceive colours and is responsible for our
ability to see the world around us. The visible range lies from the range of 380 nm to
759 nm. The detailed visible range was given in Table 1.

Table 1. Visible range spectra and range

Colour Wavelength Frequency Photon energy

(nm) (THz) (eV)

Violet 380–450 670–790 2.75–3.26

Blue 450–485 620–670 2.56–2.75

Green 500–565 530–600 2.19–2.48

Yellow 565–590 510–530 2.10–2.19

Orange 590–625 480–510 1.98–2.10

Red 625–750 400–480 1.65–1.98

 Ultraviolet rays: These electromagnetic radiations are high energy beams majorly
emitted by sun and constitutes about 10% of the total EM output from the sun. It can
 also be released from electric arch, tanning lamps, and mercury vapor lamps. It has
the range from 10-400 nm and are not visible from length. It was first discovered by
German physicist Johann Wilhelm Ritter in 1801. The UV spectra was broadly
categorized into 3 categories described in Table 2.

Table 2. Category and range of Ultraviolet radiation

Name Wavelength range Notes

Near-UV 300-400 nm Visible to bird, insects, and fish
Middle-UV 200-400 nm Used in UV spectrometer
Far/Vaccum-UV 10-200 nm Ionizing radiation completely absorbed
by the ozone layer of atmosphere. Also
employed in Vaccum-UV spectroscopy

Figure 4. UV spectra

The vacuum-UV radiation can be propagated by nitrogen molecule or in vacuum and

strongly absorbed by oxygen. The UV-Visible spectrophotometer employed the Middle
UV-region to Visible range of EM spectrum i.e. from 200-800 nm.

Principle of UV-Visible absorbance spectroscopy (Electronic transition):

When continuous radiation passes through a transparent material, a portion of the

radiation may be absorbed. If that occurs, the residual radiation, when it is passed
through a prism, yields a spectrum with gaps in it, called an absorption spectrum. As a
result of energy absorption, atoms or molecules pass from a state of low energy (the
initial, or ground state) to a state of higher energy (the excited state). In the case of
ultraviolet and visible spectroscopy, the transitions that result in the absorption of
 electromagnetic radiation in this region of the spectrum are transitions between electronic
energy levels. In contrast, the UV radiation leads to the excitation of the electrons from
HOMO (Highest occupied molecular orbital) to LUMO. The electronic transition in UV-
Visible absorption spectroscopy was given in Figure 5.

Figure 5. Electronic transition in UV-Visible spectroscopy

For most molecules, the lowest-energy occupied molecular orbitals are the σ orbitals, which
correspond to σ bonds. The π orbitals lie at somewhat higher energy levels, and orbitals that
hold unshared pairs, the nonbonding (n) orbitals, lie at even higher energies. The unoccupied,
or antibonding orbitals (π* and σ*), are the highest energy orbitals. Some of the important
transition in molecules due to exposure of UV-Vis rays are given in Table 3. Different types
of transition occurs in UV-Visible spectroscopy are discussed below:

i. σ to σ*: These transitions can occur in such compounds in which all the electrons are
involved in single bonds and there are no lone pairs of electrons. Examples involving
such transitions are saturated hydrocarbons. As the energy required for σ to σ*
transition is very large, the absorption band occurs in the far ultraviolet region (126-
135 nm). For instance, methane λmax at 121.9 nm and ethane at 135 nm correspond to
this transition. As commercial spectrophotometers do not generally operate at
wavelengths less than 180-200 nm, σ to σ* transition cannot normally be observed.
ii. π to π*: A π to π* transition corresponds to the promotion of an electron from a
bonding π orbital to an antibonding π* orbital. This transition can in principle occur in
any molecule having a π electron system. However, selection rules are known which
 are based on symmetry concepts. These rules determine whether a transition to a
particular π* orbital is allowed or forbidden. For instance, the spectrum of ethylene
exhibits an intense band at 174 nm and a weak band at 200 nm. Both of these are due
to "->" transitions. According to the selection rules, the band at 174 nm represents an
allowed transition. The intensity of absorption by ethylene is essentially independent
of the solvent because of the non-polar nature of the olefinic bond. Alkyl substitution
of the olefins moves the absorption to a longer wavelength. This effect is known as
the bathochromic effect (a red shift). The bathochromic effect is progressive as the
number of alkyl groups increases.
iii. n to σ*: Saturated compounds with lone pair (non-bonding) electrons undergo n to σ *
transitions in addition to σ to σ* transitions. The energy required for an n to σ *
transition is generally less than that required for a σ to σ * transition and the
corresponding absorption bands appear at longer wavelengths in the near ultraviolet
(180-200 nm) region. However, some compounds are known which absorb at slightly
longer wave-length. For example, (CH3),N, λmax =227 nm for n to σ* and σ to σ* for
this molecule occurs at 99 nm. When absorption measurements are made in the
ultraviolet region, compounds such as aliphatic alcohols and alkyl halides are
commonly used as solvents because they start to absorb at 260 nm. However, these
solvents (aliphatic alcohols and alkyl halides) cannot be used when measurements are
to be made in the 200-260 nm region. In such cases, saturated hydrocarbons which
only give rise to σ to σ* transitions must be used. Saturated hydrocarbons suffer from
one drawback that are poor solvating agents.
Similarly, amines are known to absorb at higher wavelengths as compared to alcohols
and hence the extinction coefficients for amines will be larger, n to σ * transitions are
very sensitive to hydrogen bonding. Alcohols as well as amines are known to form
hydrogen bonding with solvent molecules. Such associations occur because of the
presence of non-bonding electrons on the heteroatom and thus, transition needs
greater energy. Hydrogen bonding shifts the ultraviolet absorptions to shorter
wavelengths.
iv. n to π*: These types of transitions are shown by unsaturated molecules which contain
atoms such as oxygen, nitrogen and sulphur. These transitions exhibit a weak band in
their absorption spectrum. In aldehydes and ketones (having no C=C and C=C bands)
the band due to the n to π * transition generally occurs in the range 2700-3000 Å (270-
300 nm). On the other hand, carbonyl compounds having able bonds separated by two
 or more single bonds exhibit the bands due to the n to π * transitions A the range 3000-
3500 Å (300-350 nm). The summary of electronic transitions in molecules are given
in Table 3

Table 3. Summary of the electronic transition due to UV radiation.

Energy level Type of transition Description
σ σ* The highest energy transition occurs in alkanes. The
higher energy requirement of this transition leads to
shifting of these transitions to far UV region (135
nm).
σ π* It occurs in unsaturated carbon compounds with at
least one pair of π electron or π bond for example
carbonyl compounds. They are rare transitions and
have very little significance in UV-visible
spectroscopy due to their occurrence in far UV region
Increasing (175 nm).
π π* Compounds containing multiple bonds like alkenes,
alkynes, carbonyl, nitriles, aromatic compounds, etc
undergo π → π* transitions (170-205 nm).
n σ* Saturated compounds containing atoms with lone pair
of electrons like O, N, S and halogens are capable of
n → σ* transition (150-250 nm).
n π* An electron from non-bonding orbital is promoted to
anti-bonding π* orbital. Compounds containing
double bond involving hetero atoms (C=O, C≡N,
N=O) undergo such transitions (300 nm).

Transition probability: Not all of the transitions that at first sight appear possible are
observed. Certain restrictions, called selection rules, must be considered. One important
selection rule states that transitions that involve a change in the spin quantum number of an
electron during the transition are not allowed to take place; they are called “forbidden”
transitions. The n to π* transition is the most common type of forbidden transition. The
forbidden transition are not favoured by characterized by the less intense or weak absorbance
bands.
Chromophores, auxochromes and Spectral shift

The term chromophore derived from the word “chroma” which means colour, so initially this
term was used to denote a functional group or some other structural feature which is
responsible to give a colour to the compound. The advancement in analytical chemistry leads
a proper understanding that the nuclei determine the strength with which the electrons are
bound and thus influence the energy spacing between ground and excited states. Hence, the
characteristic energy of a transition and the wavelength of radiation absorbed are properties
of a group of atoms rather than of electrons themselves. The group of atoms producing such
an absorption is called a chromophore. In simple terms, chromophore is the main moiety of
the molecule responsible for the absorbing the UV radiation at specific wavelength. Any
change in the chromophore will also leads to the exact energy and intensity of the absorption
are expected to change increasingly.

Some of the important chromophores are ethylenic, acetylenic, carbonyls, acids, esters, nitrile
groups, etc. These chromophores mainly responsible for the electronic transition inside the
molecule under the exposure of UV or visible radiations. The chromophores can be
characterized into 2 subparts on the basis of its ability to produce electronic transition:

a) Chromophores in which the group having π electrons like ethylenes, acetylenes,

vinyls, etc. These π electrons can undergoes π to π* transitions.
b) Chromophores having both π electrons and n (lone pair) electrons like carbonyls, azo
compounds, nitriles, etc. These compounds are able to induce both n to π * and π to π*
transitions.

Spectral shift in the UV: 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. The substituent groups may not give rise to the absorption of the ultraviolet
radiation themselves, but their presence modifies the absorption of the principal
chromophore. Substituents that increase the intensity of the absorption, and possibly the
wavelength, are called auxochromes. These moieties are responsible for the following shift:

i. Bathochromic shift (red shift): A shift towards lower energy or longer wavelength.
It occurs due to -OH and NH2 auxochromes. Increased conjugation can also leads to
the bathochromic shift.
 ii. Hypsochromic shift (blue shift): A shift towards higher energy or shorter
wavelength. It occurs when the conjugation was removed or the polarity of the solvent
was changed.
iii. Hyperchromic shift: An increase in intensity. It is done by adding the auxochrome
which improve the geometry of the molecule increasing the probability of allowed
transitions.
iv. Hypochromic shift: A decrease in intensity. It is usually brought by the auxochrome
which are able to distort the geometry of the molecule.

This spectral shift can arises only when an auxochrome was attached to the chromophore.
Common auxochrome includes methyl, hydroxyl, alkoxy, halogen, and amino groups.
Sometimes these auxochromes are present in environment and may contaminate the
chromophore which leads to loose of characteristic property of the auxochrome. Figure 6
describes the different types of spectral shifts in UV-Visible spectroscopy.

Figure 6. Spectral shift in UV-Visible spectroscopy

Beer-Lambert law of absorbance

This law was first proposed by August Beer and Johann Heinrich Lambert and applied in
chemical analysis. It is applied in analytical chemistry to calculate the concentration of an
analyte present in the solution. This law states that the absorbance of UV radiation by a
compound is directly proportional to the path length of the radiation and solution
concentration

Figure 7. The phenomenon of UV-absorbance

The basic terminology used in Beer-Lamber law includes:

Absorbance: It is the amount of light absorbed (Ia) by the solution containing UV-active
compounds.

Transmittance: It is the amount of light transmitted (It) by the solution. Transmittance is

inversely proportional to absorbance.

Reflectance: It is the amount of light reflected (Ir) by the solution. The absorbance and
transmittance of the solution is inversely proportional to reflectance.

Incident light (I0) = It is the light wave coming from the light source.

I0 = Ia + It + Ir

According to this law, the absorbance of UV light was increased by increasing path length
and concentration of UV-active compound in the solution. However, Beer-Lambert law is
only applicable to small concentrations and deviates when concentration was increased. This
law states a linear relationship between absorbance and concentration of analyte at lower
concentrations. The mathematical expression of Beer-Lambert law was given below:

A=εxbxc

Here, A is absorbance, ε is the molar absorptivity coefficient, c is the concentration of

solution, and b is the path length. This law also gives a mathematical relation between
transmittance and absorbance, which is defined as:

A = log (I0/It) and T = It/I0 i.e. A = -log (T)

In the above equation, It is the intensity of transmitted light from the solution and I 0 is the
intensity of the incoming light from light source and A is absorbance. The analytical
instruments for UV-Vis analysis are based on Beer-Lambert law that is why their application
is restricted to only lower concentration solutions due to deviation of Beer-Lambert law at
higher concentrations.

Deviations from Beer’s Law

According to the Beer-Lamber Law, the intensity of the transmitted UV-Visible radiation was
decreased by increase the concentration and path length of the radiation inside the solution.
However, this relationship applied only for a small concentration range and deviations from
the law can be observed at higher concentrations. The relationship between absorbance and
concentration can be determined by plotting the curve between the absorbance of solution
and the concentration of the analytes. The straight-line plot should be observed, if the
solution follows the Beer-Lambert law, however at higher concentrations the deviations were
observed (at fixed path length of 1 cm) as given in Figure 8.
Figure 8. Deviations from Beer’s Law

there is usually a deviation from a linear relationship between concentration and absorbance
and an apparent failure of Beer's law may ensure. Deviations from the law are reported as
positive or negative according to whether the resultant curve is concave upwards or concave
downwards. Deviations from Beer's law can arise due to following factors:

I. Beer's law will hold over a wide range of concentration provided the structure of the
coloured ion or of the coloured non-electrolyte in the dissolved state does not change
with concentration. If a coloured solution is having a foreign substance whose ions do
not react chemically with the coloured components, its small concentration (foreign
substance) does not affect the light absorption and may also alter the value of the
extinction coefficient.
II. Deviations may also occur if the coloured solute ionises, dissociates or associates in
solution. For example, benzyl alcohol in chloroform exists in a polymeric
equilibrium:

Dissociation of the polymer increases with dilution. The monomer absorbs at 2.750 to
2.765 μ whereas the polymer absorbs at 3.000 μ. Hence absorption at 2.750 μ shows
negative deviation whereas at 3.000 μ positive deviation
III. Deviations may also occur due to the presence of impurities that fluoresce or absorb at
the absorption wavelength. This interference introduces an error in the measurement
of absorption of radiation penetrating the sample.
IV. Deviations may occur if monochromatic light is not used.
V. Deviations may occur if the width of slit is not proper and, therefore, it allows
undesirable radiations to fall on the detector. These undesirable radiations might be
absorbed by impurities present in the sample which would cause an apparent change
in the absorbance of the sample. The magnitude of this deviation becomes appreciable
at higher concentrations.
VI. Deviations may occur if the solution species undergoes polymerisation. For example,
benzyl alcohol in carbon tetrachloride in high concentration exists in polymeric form
[(C6H5CH2OH)4], dissociation of this polymer increases with dilution. Due to this
change, absorbance will change.
VII. Beer's law cannot be applied to suspensions but the latter can be estimated
colorimetrically after preparing a reference curve with known concentrations.

Instrumental deviation from Beer’s law:

Instrumental deviation from Beer's Law may also result. Remember, Beer's Law requires
monochromatic radiation because absorptivity is a constant at a single wavelength and varies
with a change in wavelength. The possibility of error due to the practical impossibility of
obtaining monochromatic radiation may be minimized by the selection of a spectral region
where the change in absorptivity with a change in wavelength is very small. This dictates a
wavelength selection from a broad band rather than from a sharply rising or sharply falling
section of the absorption curve. This may be clarified by reference to Figure 9. The energy
which passes through the sample and activates the detector is a function of the weighted
average of the absorptivities of all the wavelengths which are transmitted by the
monochromator system. At the top (point A) of the absorption band, the absorptivities at λ 1,
and λ2, are close to that of the desired wavelength (λ max ). Minimum error will 0.00 arise from
the use of this wavelength. On the sharply rising (point B) or falling (point C) Choice of
wavelength and slit widths. positions the weighted average yields low results, Point A is
preferred over either B or C. See text for detail. these low results becoming more and more
apparent at increasing concentrations.

In summary, because of the numerous possibilities of an error arising from deviations it is

necessary in practice to prepare a calibrated curve for the absorbance-concentration
relationship at the chosen wavelength. Beer's Law should be verified in each application on
each instrument. It should not be assumed.
Figure 9. Choice of wavelength and slit widths

Solvent effects: A most suitable solvent is one that does not itself absorb in the region under
investigation. A dilute solution of the sample is always prepared for spectral analysis. Most
commonly used solvent is 95%ethanol. Ethanol is a best solvent as it is cheap and is
transparent down to 210 mu. Commercial ethanol should not be used because it is having
benzene which absorbs strongly in the ultra-violet region. Some other solvents which are
transparent above 210 mu are n-hexane, methyl alcohol, cyclohexane, acetonitrile, diethyl
ether etc. Some solvents with their upper wave-length limit of absorption are given in Table
4.
Table 4. Solvents with upper wavelength limit
Solvent λ of absorption (nm)
Ethanol 210
Hexane 210
Methanol 210
Cyclohexane 210
Diethylether 210
Water 205
Benzene 280
Chloroform 245
Tetrahydrofuran 220
Carbon tetrachloride 265
Hexane and other hydrocarbons can be used because these are less polar and have least
interactions with the molecule under investigation. For ultra-violet spectroscopy,
ethanol, water and cyclohexane serve the purpose best.
The position as well as the intensity of absorption maximum get shifted for a particular
chromophore by changing the polarity of the solvent. By increasing the polarity of the
solvent, compounds such as dienes and conjugated hydrocarbons do not experience any
appreciable shift. Hence in general, the absorption maximum for the non-polar compounds is
usually shifted with the change in polarity of the solvents. α, β-unsaturated carbonyl
compounds show two different shifts.
n to π* (less intense). For such a case the absorption band moves to shorter wavelength by
increasing the polarity of the solvent. In n to π* transition, the ground state is more polar as
compared to the excited state. The hydrogen bonding with solvent molecules occurs to lesser
extent with the carbonyl group in the excited state. For example, absorption maximum of
acetone is at 279 nm in hexane as compared to 264 nm in water.
π to π* transitions (intense). In such a case, the absorption band moves to longer wavelength
by increasing the polarity of the solvent. The dipole-dipole interactions with the solvent
molecules lower the energy of the excited state more than that of the ground state. Hence the
value of absorption maximum in ethanol will be greater than that observed in hexane.
In short π orbitals get more stabilised by hydrogen bonding with the polar solvents like water
and ethanol. It is because of greater polarity of π * orbital compared to π-orbital. Thus, small
energy will be needed for such a transition and absorption shows a red shift.
n to σ* transitions are also very sensitive to hydrogen bonding. Alcohols as well as amines
form hydrogen bonding with the solvent molecules. Such associations occur because of the
presence of non-bonding electrons on the hetero atom and thus, transition requires greater
energy. In general, we say that.
a. When a group (say, carbonyl) is more polar in the ground state than in the excited
state than increasing polarity of the solvent stabilises the non-bonding electrons in the
ground state because of hydrogen bonding. Thus, absorption is shifted to lower
wavelength.

b. When the group is more polar in the excited state, then absorption gets shifted to
longer wave- length with increase in polarity of the solvent which helps in stabilising
the non-bonding electrons in the excited state.
The increase in polarity of the solvent generally shifts n to π * and n to σ* bands to
shorter wavelengths and π to π* bands to longer wave-lengths.