Introduction to Spectroscopy

Spectroscopy is the study of the interaction between matter and electromagnetic radiation. It is
widely used in physics, chemistry, and engineering to analyze material properties. It can also
be defined as the study of matter and the changes it undergoes when it is subjected to a
particular electromagnetic radiation.

Electromagnetic Spectrum
The electromagnetic spectrum consists of all forms of electromagnetic radiation, classified
based on their wavelength and frequency. It includes:
1) Radio Waves: Longest wavelength, used in communication.
2) Microwaves: Used in radar, cooking, and satellite communication.
3) Infrared (IR): Heat radiation used in thermal imaging and remote sensing.
4) Visible Light: The only part detectable by the human eye, ranging from violet (shortest
wavelength) to red (longest wavelength).
5) Ultraviolet (UV): Can cause fluorescence and is used in sterilization.
6) X-rays: High-energy waves used in medical imaging and material analysis.
7) Gamma Rays: Shortest wavelength, produced by nuclear reactions and used in cancer
treatment.
Each region of the spectrum interacts with matter differently, making it useful for various
spectroscopy techniques.

Ionizing and Non-Ionizing Radiation

Electromagnetic radiation can be classified into ionizing and non-ionizing radiation based on
its energy and ability to ionize atoms.
Ionizing electromagnetic radiation refers to radiation that carries sufficient energy to
remove tightly bound electrons from atoms or molecules, thereby ionizing them. This type of
radiation includes high-energy ultraviolet rays, X-rays, and gamma rays. Ionizing radiation is
always high energy carrier waves/photons. Due to its high energy, ionizing radiation is capable
of breaking chemical bonds and causing cellular damage, making it both useful in medical
applications and potentially hazardous to biological systems. It includes higher-energy
ultraviolet, X-rays, and gamma rays. It has enough energy to ionize atoms by removing
electrons, which can damage biological tissues. It is mainly used in medical imaging (X-rays,
CT scans), radiation therapy, and nuclear power generation.
Non-ionizing electromagnetic radiation refers to radiation that does not carry enough
energy to ionize atoms or molecules by removing electrons. This type of radiation includes
radio waves, microwaves, infrared, visible light, and lower-energy ultraviolet radiation. Non-
ionizing radiation is generally considered less harmful than ionizing radiation and is commonly
used in applications such as communication (radio waves, microwaves), heating (infrared
radiation), and medical imaging (MRI). It includes radio waves, microwaves, infrared, visible
light, and lower-energy ultraviolet radiation.

The Wave Energy Equation (Planck’s Equation)

Planck’s equation describes the energy of a photon based on its frequency:
E = h
Where:
E = Energy of the photon (Joules)
h = Planck’s constant = 6.626×10−34 J.s
  = Frequency of the radiation (Hz)
Alternatively, since  = c  , where c is the speed of light and  is the wavelength, the equation
can be written as:

This equation shows that energy is directly proportional to frequency and inversely
proportional to wavelength. It is fundamental in quantum mechanics and spectroscopy,
explaining phenomena such as blackbody radiation and the photoelectric effect.

Principles of Spectroscopy
Absorption Spectroscopy: An analytical technique which concerns with the measurement of
absorption of electromagnetic radiation. e.g. UV (185 - 400 nm) & Visible (400 - 800 nm)
Spectroscopy, IR Spectroscopy (0.76 - 15 μm)
Emission Spectroscopy: An analytical technique in which emission (of a particle or radiation)
is dispersed according to some property of the emission & the amount of dispersion is
measured. e.g. Fluorescence, Phosphorescence.

Scattering Spectroscopy: Light is deflected or redirected. e.g. Raman Spectroscopy.

The UV Spectrum (150-380 nm)

Ultraviolet (UV) radiation is a type of electromagnetic radiation emitted by the sun and
artificial sources. It is categorized into three types based on wavelength:

UV-A (320 - 400 nm): These are long-wavelength UV rays, also known as "black light." They
penetrate the skin deeply, reaching the dermis, and are primarily responsible for premature
aging and wrinkles. UV-A rays account for approximately 95% of the UV radiation that reaches
Earth's surface.

UV-B (280 - 320 nm): Medium-wavelength UV rays that are mostly absorbed by the ozone
layer. UV-B rays affect the outer layer of the skin, causing sunburn and playing a significant
role in developing skin cancer.

UV-C (100 - 280 nm): Short-wavelength UV rays that are entirely absorbed by Earth's
atmosphere, including the ozone layer. UV-C rays are the most harmful type of UV radiation;
however, they do not reach the surface and are used in germicidal applications.

Understanding these categories is essential for assessing UV exposure risks and implementing
appropriate protective measures.
Near UV and Far UV are subcategories within the broader UV spectrum, distinguished by the
wavelength range of UV radiation. Here's a more detailed breakdown:
Near UV (NUV):
Wavelength Range: Approximately 300 to 400 nm. Sometimes referred to as "UV-A" or "long-
wave UV" depending on the context. This region closely borders the visible light spectrum.
Near UV rays are the most common UV rays that reach the Earth from the sun. They are less
energetic compared to shorter wavelength UV radiation (like Far UV), but they still penetrate
the skin and cause damage over time. It is involved in skin aging, wrinkles, and DNA damage.
It can also cause eye damage, such as cataracts, if exposure is prolonged without protection. Its
sources are primarily sunlight, fluorescent lights, and some artificial light sources.
Far UV (FUV):
Wavelength Range: Typically, 100 to 200 nm. Also known as "Vacuum UV" or "short-wave
UV." Far UV radiation has shorter wavelengths and higher energy compared to Near UV. This
range is absorbed by the Earth's atmosphere, specifically the ozone layer, and does not reach
the surface under normal conditions. Due to its high energy, it has the potential to ionize
molecules, which makes it more hazardous. It can break molecular bonds, causing cellular
damage and posing a higher risk for tissue burns or other biological damage. However, because
it doesn't reach Earth naturally, it's typically not a concern for everyday exposure. In laboratory
settings, high-energy sources like mercury vapor lamps can produce Far UV. In certain
specialized applications, Far UV can be used for sterilization, as it has strong germicidal
properties.
Key Differences between Near UV and Far UV:
1) Far UV has higher energy than Near UV and is more capable of causing damage.
2) Near UV penetrates deeper into the skin compared to Far UV, which gets absorbed by
the atmosphere and does not naturally reach the Earth's surface.
3) Both can cause harm, but Far UV's potential for damage is higher due to its ability to
break molecular bonds and its higher energy levels.
4) Near UV (300 - 400 nm) is closer to visible light, more common, responsible for long-
term skin effects (aging, DNA damage). Far UV (100 - 200 nm) has higher energy,
largely absorbed by the atmosphere, has a stronger potential for biological damage but
is not a significant concern at the Earth's surface under normal conditions.
 5) Far UV is mostly used in controlled settings for disinfection purposes, while Near UV
is a natural part of sunlight exposure, influencing our health and environment.

The Visible Spectrum

Beer Lambert’s Law

Lambert’s Statement: “Absorbance of
a material sample is directly proportional
to its thickness”
Beer’s Statement: “Absorbance is
proportional to the concentrations of the
attenuating species in the material
sample”
Mathematically:

Limitations Beer Lambert’s Law

1) High concentrations: Beer Lambert’s law holds good only for dilute solutions because
at high concentrations interactions between molecule gives rise to anomalies.
 2) Non-monochromatic light: The law is only applicable to light with a single
wavelength. In practice, most light sources emit a range of wavelengths, which can lead
to measurement errors.
3) Chemical deviation: Chemical deviations arise if the absorbing species undergo
chemical changes such as association, complex formation, dissociation, hydrogen
bonding, hydrolysis, ionization or polymerization.
4) Fluorescence and phosphorescence: Fluorescence or phosphorescence of the sample
can cause non-linearity.

Types of Electronic Transitions

In U.V. spectroscopy molecule undergoes electronic transition involving   and non-
bonding electrons. Four types of electronic transition are possible:

σ → σ* transition: σ electron from orbital is excited to corresponding anti-bonding orbital σ*.

The energy required is large for this transition. e.g. Methane (CH4) has C-H bond only and can
undergo σ → σ* transition and shows absorbance maxima at 125 nm.
π → π* transition: π electron in a bonding orbital is excited to corresponding anti-bonding
orbital π*. Compounds containing multiple bonds undergo π → π* transitions. e.g. alkenes,
alkynes, carbonyl, nitriles, aromatic compounds. Alkenes generally absorb in the region 170
to 205 nm.
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 require less
energy than σ → σ* transitions. The number of organic functional groups with n → σ* peaks
in UV region is small (150 – 250 nm). e.g. amines, alcohols, thiols, etc.
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, C≡N, N=O)
undergo such transitions. n → π* transitions require minimum energy and show absorption at
longer wavelength around 300 nm
σ → π* transition & π → σ* transition: These electronic transitions show absorption in region
above 200 nm which is inaccessible to UV-visible spectrophotometer. These electronic
transitions are therefore forbidden transitions & are only theoretically possible.
Chromophore
The part of a molecule responsible for imparting color, are called as chromophores. They are
basically functional groups containing multiple bonds capable of absorbing radiations above
200 nm due to n → π* & π → π* transitions. e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc.

Auxochrome
The functional groups attached to a chromophore which modifies the ability of the
chromophore to absorb light, altering the wavelength or intensity of absorption is known as
auxochrome. The functional group with non-bonding electrons that does not absorb radiation
in near UV region but when attached to a chromophore alters the wavelength & intensity of
absorption. e.g. -OH, -NH2, etc.

Bathochromic Shift (Red Shift)

When absorption maxima (λmax) of a compound shifts to longer wavelength, it is known as
bathochromic shift or red shift. The effect is due to presence of an auxochrome or by the change
of solvent. e.g. An auxochrome group like –OH, -OCH3 causes absorption of compound at
longer wavelength. In alkaline medium, p-nitrophenol shows red shift. Because negatively
charged oxygen delocalizes more effectively than the unshared pair of electrons.
- -
O + O O + O
N N

-
OH

Alkaline
medium -
OH O

p-nitrophenol λmax = 265 nm

λmax = 255 nm
Hypsochromic Shift (Blue Shift)
When absorption maxima (λmax) of a compound shifts to shorter wavelength, it is known as
hypsochromic shift or blue shift. The effect is due to the presence of a group which causes the
removal of conjugation. It is also caused by the change of solvent. e.g. Aniline shows blue shift
in acidic medium, it loses conjugation.
+ -
NH2 + NH3 Cl
H
Acidic
medium

Aniline λmax = 265 nm

λmax = 280 nm

Hyperchromic Effect
When absorption intensity (ε) of a compound is increased, it is known as hyperchromic shift.
If auxochrome introduces to the compound, the intensity of absorption increases.

N N CH3
Pyridine 2-methyl pyridine
λmax = 257 nm λmax = 260 nm
ε = 2750 cm-1 ε = 3560 cm-1

Hypochromic Effect
When absorption intensity (ε) of a compound is decreased, it is known as hypochromic shift.

CH3
Naphthalene 2-methyl naphthalene
ε = 19000 cm-1 ε = 10250 cm-1

Applications of UV-Visible spectroscopy

1) It helps in the detection of functional groups.
2) It identifies the extent of conjugation in polyenes.
 3) Chemical identification and quantification: UV-Vis spectroscopy can confirm chemical
identity and quantify the purity of drugs/drug ingredients.
4) It easily elucidates the structure of vitamins A or K.
5) Quantification of impurities: UV-Vis is commonly utilized in pharmaceutical
monographs for quantifying impurities in drug ingredients and drug products.
6) For Protein analysis and measurements.
7) Structure elucidation of organic compounds.
8) To study Chemical kinetics of chemical reaction.

Luminescence & Photoluminescence

Luminescence is the emission of light by a substance. It occurs when an electron returns to the
electronic ground state from an excited state and loses its excess energy as a photon. It is of 4
types:
➢ Fluorescence
Photoluminescence
➢ Phosphorescence
➢ Chemiluminescence
Luminescence
➢ Bioluminescence

Fluorescence
When a beam of light is incident on
certain substances they emit visible light
or radiations. This is known as S5
fluorescence. Fluorescence starts S4
immediately after the absorption of light S3
and stops as soon as the incident light is S2
Internal Conversion
cut off. The substances showing this S1 T5
phenomenon are known as fluorescent T4
T3
Fluorescence

substances.
Energy

T2
Internal Conversion
Phosphorescence T1
Phosphorescence

Then light radiation is incident on certain

substances they emit light continuously
even after the incident light is cut off.
This type of delayed fluorescence is
called phosphorescence. Substances
showing phosphorescence are
S0
phosphorescent substances.
Selection Rule for Electronic Transitions
A molecular electronic state in which all of the electrons are +1/2 +1/2
paired are called singlet state. In a singlet state molecule are
diamagnetic. Most of the molecules in their ground state are
paired. When such a molecule absorbs UV/visible radiation,
one or more of the paired electrons raised to an excited Triplet State
singlet state /excited triplet state.
Singlet +1/2 -1/2
Ground
Spin Multiplicity = (2S+1) State

For singlet: S = (+1/2) + (-1/2) = 0

Spin multiplicity = 2 x 0 +1 = 1 Singlet State

For doublet: S = +1/2 Excited States

Spin multiplicity = 2 x 1/2 +1 = 2

For triplet:
S = (+1/2) + (+1/2) = 1
Spin multiplicity = 2 x 1 +1 = 3
There are two types of selection rule for electronic transitions
1) Spin Selection Rule: Transition may occur between two energy states of same
multiplicity. Forbidden Transition: ΔS≠O.
i.e. Allowed: singlet → singlet, triplet → triplet.
Forbidden: singlet → triplet, doublet → singlet

2) Laporte Selection Rule (Parity Rule): The Laporte Selection Rule states that for the
light to interact with a molecule and be absorbed there should be a change in Dipole
Moment. Transition in which there is no change in parity (i.e. Dipole Moment) is
forbidden. Transitions between states that arise from d orbitals are forbidden (g  g
transitions are forbidden) because d orbitals are symmetric to inversion. Transitions
between d and p orbitals are allowed (g  u transitions are allowed) because p orbitals
are anti-symmetric to inversion. Laporte-allowed transitions involve Δl = ±1.
i.e. Allowed: g u, u  g
Forbidden: g g, u  u

‘g’ stands for gerade, compounds having centre of symmetry e.g. s and d orbitals.
‘u’ stands for ungerade, compounds which don't have centre of symmetry e.g. p and f
orbitals.

Application of Fluorescence
1) Dairy products contain several intrinsic fluorophores, which represent the most
important area of fluorescence spectroscopy. They include the aromatic amino acids
and nucleic acids (AAA+NA) tryptophan, tyrosine, and phenylalanine in proteins;
vitamins A and B2; nicotinamide adenine dinucleotide (NADH) and chlorophyll; and
numerous other compounds that can be found at a low or very low concentration in food
 products. Dufour and Riaublanc (1997) investigated the potential of Front-Face
Fluorescence Spectroscopy (FFFS) to discriminate between raw, heated (70 °C for 20
min), homogenized, and homogenized and heated milks. They applied PCA (Principle
Composite Analysis) to the tryptophan and vitamin A fluorescence spectra. They
concluded that the treatments applied to milk induced specific modifications in the
shape of the fluorescence spectra.

2) Kulmyrzaev et al. (2005) confirmed these earlier findings. In their research, the
emission and excitation spectra of different intrinsic probes (i.e., AAA+NA, NADH,
and FADH) were used to evaluate changes in milk following thermal treatments in the
range of 57-72 °C for 0.5-30 min. The PCA applied to the normalized spectra allowed
good discrimination of milk samples subjected to different temperatures and times.

3) The environment of intrinsic fluorophores recorded on intact food systems contains

valuable information regarding the composition and nutritional values of food products.
The huge potential for the application of fluorescence spectroscopy combined with
multivariate statistical analyses for the evaluation of food quality has also been
demonstrated through literature.