Introduction to Spectroscopy

1. The Interaction between Electromagnetic Radiation and Matter

We know from our observation of rainbows that visible light (white light) is composed of a
continuum of colors from violet to red. If a beam of white light is passed through a beaker of
water, it remains white. If potassium permanganate is added to the water, the white light
appears purple after it passes through the solution. The permanganate solution allows the red
and blue components of white light to pass through but absorbs the other colors from the
original beam of light. This is one example of the interaction of electromagnetic radiation, or
light, with matter. In this case, the electromagnetic radiation is visible light and we can see the
effect of absorption of some of the light with our eyes. However, interactions between
electromagnetic radiation and matter take place in many ways and over a wide range of radiant
energies. Most of these interactions are not visible to the human eye but can be measured with
suitable instruments. Interaction of electromagnetic radiation and matter is not haphazard but
follows well-documented rules with respect to the wavelengths of light absorbed or emitted
and the extent of absorption or emission. The subject of spectroscopy is the study of the
interaction of electromagnetic radiation and matter.

1.1. What is Electromagnetic Radiation?

The nature of electromagnetic radiation baffled scientists for many years. At times light appears
to behave like a wave; at other times it behaves as though it were composed of small particles.
While we now understand the “wave–particle duality” of all matter, including
electromagnetic radiation, in terms of quantum mechanics, it is still convenient to consider
electromagnetic radiation as having the properties of waves in many cases.
Light waves can be represented as oscillating perpendicular electric and magnetic fields. The
fields are at right angles to each other and to the direction of propagation of the light. The
oscillations are sinusoidal in shape, as shown in Fig. 1. We can easily and accurately measure
the wavelength λ defined as the crest-to-crest distance between two successive maxima. The
standard unit of wavelength is the SI unit of length, the meter (m), but smaller units such as the
centimeter (cm), micrometer (mm), and nanometer (nm) are commonly used. The amplitude
of the wave is defined as the maximum of the vector from the origin to a point displacement of
the oscillation. An example of the electric field portion of a light wave propagating along only
one axis is shown in Fig. 1. Such a wave, confined to one plane, is called plane-polarized light.
The wave shown represents only a single wavelength. Light of only one wavelength is called

1
monochromatic light. Light that consists of more than one wavelength is called polychromatic
light. White light is an example of polychromatic light.

Figure 1 (a) A plane-polarized light wave in the x-direction, showing the mutually perpendicular
electric and magnetic field components. (b) The wavelength and amplitude of a wave.

The frequency n of a wave is the number of crests passing a fixed point per second. One crest-
to-crest oscillation of a wave is called a cycle. The common unit of frequency is the hertz (Hz)
or inverse second (s-1); an older term for frequency is the cycle per second (cps). One hertz
equals one cycle per second.
The wavelength of light, l, is related to its frequency, n by the equation:
c = λν (1:1)
where c is the speed of light in a vacuum, 2.997 × 108 m/s, ν is the frequency of the light in
inverse seconds (Hz), and λ is the wavelength in meters. In a vacuum, the speed of light is a
maximum and does not depend on the wavelength. The frequency of light is determined by the
source and does not vary. When light passes through materials other than vacuum, its speed is
decreased. Because the frequency cannot change, the wavelength must decrease. If we calculate
the speed of light in air, it only differs by a very small amount from the speed of light in
vacuum; in general, we use 3.00 × 108 m/s (to three significant figures) for the speed of light
in air or vacuum.

2
In some cases, it is more convenient to consider light as a stream of particles. We call particles
of light photons. Photons are characterized by their energy, E. The energy of a photon is related
to the frequency of light by the equation:
E=hν (1:2)
where E is the energy in joules (J), h is Planck’s constant, 6.626 × 10-34 J s, and ν is the
frequency in inverse seconds (Hz). From Eqs. (1.1) and (1.2) we can deduce that
E= h c/ λ (1:3)
We can see from the relationships in Eqs. (1.2) and (1.3) that the energy of electromagnetic
radiation is directly proportional to its frequency and inversely proportional to its wavelength.
Electromagnetic radiation ranges from very low energy (long wavelength, low frequency)
radiation, like radiowaves and microwaves, to very high energy (short wavelength, high
frequency) radiation, like X-rays. The major regions of the electromagnetic spectrum of interest
to us as analytical chemists are shown in Fig 2. It is clear from this figure that visible light, that
portion of the electromagnetic spectrum to which the human eye responds, is only a very small
portion of all radiant energy. Table 1 presents some common units and symbols for various
types of electromagnetic radiation.

Figure 2 The electromagnetic spectrum. The visible light region is expanded to show the colors associated with
wavelength ranges.

3
 Table 1 Common Wavelength Symbols and Units for Electromagnetic Radiation
Unit Unit Symbol Length (m) Type of radiation
Angstrom A˚ 10-10 X-ray
Nanometer nm 10-9 UV, visible
Micrometer mm 10-6 IR
Millimeter mm 10-3 IR
Centimeter cm 10-2 Microwave
Meter m 1 Radio

1.2. How does Electromagnetic Radiation Interact with Matter?

Spectroscopy is the study of the interaction of radiant energy (light) with matter. We know
from quantum mechanics that energy is really just a form of matter, and that all matter exhibits
the properties of both waves and particles. However, matter composed of molecules, atoms, or
ions, which exists as solid or liquid or gas, exhibits primarily the properties of particles.
Spectroscopy studies the interaction of light with matter defined as materials composed of
molecules or atoms or ions. In a gas, atoms or molecules are widely separated from each other;
in liquids and solids, the atoms or molecules are closely associated. In solids, the atoms or
molecules may be arranged in a highly ordered array, called a crystal, as they are in many
minerals, or they may be randomly arranged, or amorphous, as they are in many plastics.
Whatever their physical state or arrangement atoms, molecules, and ions are in constant
motion. For molecules, many types of motion are involved. Molecules can rotate, vibrate, and
translate (move from place to place in space). Interaction with radiant energy can affect these
molecular motions. Molecules that absorb IR radiation vibrate with greater amplitude;
interaction with UV or visible light can move bonding electrons to higher energy levels in
molecules. A change in any form of motion or electron energy level involves a change in the
energy of the molecule. Such a change in energy is called a transition; we have the possibility
of vibrational transitions, rotational transitions, electronic transitions, and so on in molecules.
We have some of the same kinds of motion in atoms and ions; atoms can move in space, and
their electrons can move between energy levels, but atoms or monoatomic ions cannot rotate
or vibrate. The chemical nature of matter (its composition), its physical state, and the
arrangement of the atoms or molecules in the physical state with respect to each other affect
the way in which any given material interacts with electromagnetic radiation. Table 2 lists some
of the important types of transitions studied by spectroscopy.
When light strikes a sample of matter, the light may be absorbed by the sample, transmitted
through the sample, reflected off the surface of the sample, or scattered by the sample. Samples
can also emit light after absorbing incident light; such a process is called luminescence. There

4
are different kinds of luminescence, called fluorescence or phosphorescence depending on the
specific process that occurs. Emission of light may also be caused by processes other than
absorption of light. There are spectroscopic methods based on all of these interactions.
Table 2 Some Types of Transitions Studied by Spectroscopy
Type of transition Spectroscopic method Wavelength
range
Spin of nuclei in a magnetic field NMR spectroscopy 0.5–10 m
Rotation and vibration of molecules Raman and IR spectroscopy 0.8–300 µm
Bonding electron energy, valence UV/VIS spectroscopy 180–800 nm
electron energy
Core electron energy X-ray spectroscopy 0.1–100 A˚

Table 3 Some Interactions of Light and Matter

Interaction Radiation measured Spectroscopic method
Absorption and Incident light, I0 Atomic absorption
transmission Transmitted light, I Molecular absorption
Absorption then emission Emitted light, I’ Atomic fluorescence
Molecular fluorescence
Molecular
phosphorescence
Scattering Scattered light, Is Turbidimetry
Nephelometry
Raman
Reflection Reflected light, IR Attenuated total reflection
or relative Diffuse reflection IR (the
reflected IR term
reflectance is also used for
these
methods)
Emission Emitted light, Ie Atomic emission
Molecular emission
Chemiluminescence

Table 3 summarizes the major types of interaction of light with matter and gives examples of
the common spectroscopic techniques based on these interactions. For the moment, we will
focus on the absorption, transmission, and emission of light by matter. If we pass white light
(i.e., visible light) through blue glass, the emerging light is blue. The glass has absorbed the
other colors, such as red and yellow. We can confirm this absorption by shining red light
through the blue glass. If the absorption is strong enough, all of the red light is absorbed; no
light emerges from the glass and it appears black. How can this be explained?
The interaction of electromagnetic radiation and matter conforms to well established quantum
mechanical laws. Atoms, ions, and molecules exist only in certain discrete states with specific
energies. The same quantum mechanical laws dictate that a change in state requires the
absorption or emission of energy, ΔE, exactly equal to the difference in energy between the

5
initial and final states. We say that the energy states are quantized. A change in state (change
in energy) can be expressed as:
ΔE = Efinal - Einitial = hν (1:4)
Since we know that c = λν, then:
ΔE = hν = hc/λ (1:5)
These equations tell us that matter can absorb or emit radiation when a transition between two
states occurs, but it can absorb or emit only the specific frequencies or wavelengths that
correspond to the exact difference in energy between two states in which the matter can exist.
Absorption of radiation increases the energy of the absorbing species (i.e., Efinal > Einitial).
Emission of radiation decreases the energy of the emitting species (i.e., Efinal < Einitial). So the
quantity ΔE can have either a positive sign or a negative sign, but when using ΔE to find the
wavelength or the frequency of radiation involved in a transition, only the absolute value of ΔE
is used. Wavelength, frequency, and the speed of light are always positive in sign.
A specific molecule, such as hexane, or a specific atom, such as mercury, can absorb or emit
only certain frequencies of radiation. All hexane molecules will absorb and emit light with the
same frequencies, but these frequencies will differ from those absorbed or emitted by a
different molecule, such as benzene. All mercury atoms will absorb the same frequencies of
incident light, but these will differ from the frequencies of light absorbed by atoms of lead or
copper. Not only are the frequencies unique, but also the degree to which the frequency is
absorbed or emitted is unique to a species. This degree of absorption or emission results in light
of a given intensity. The uniqueness of the frequencies and amount of each frequency absorbed
and emitted by a given chemical species is the basis for the use of spectroscopy for
identification of chemicals. We call the set of frequencies and the associated intensities at
which a species absorbs or emits its spectrum.
The lowest energy state of a molecule or atom is called the ground state. All higher energy
states are called excited states. Generally, at room temperature molecules and atoms exist in
the ground state.

2. Atoms and Atomic Spectroscopy

An atom consists of a nucleus surrounded by electrons. Every element has a unique number of
electrons, equal to its atomic number for a neutral atom of that element. The electrons are
located in atomic orbitals of various types and energies and the electronic energy states of
atoms are quantized. The lowest energy, most stable electron configuration of an element is its
ground state. The ground state is the normal electron configuration predicted from the “rules

6
” for filling a many-electron atom, which you learned in general chemistry. These rules are
based on the location of the atom in the periodic table, the aufbau principle, the Pauli exclusion
principle, and Hund’s rule. For example, the ground state electron configuration for sodium,
atomic number 11, is 1s22s22p63s1 based on its position in the third row, first group of the
periodic table and the requirement to account for 11 electrons. The ground state electronic
configuration for potassium is 1s22s22p63s23p64s1, vanadium is 1s22s22p63s23p64s23d3, and so
on. If energy of the right magnitude is provided to an atom, the energy may be absorbed and
an outer (valence) electron promoted from the ground state orbital it is into a higher energy
orbital. The atom is now in a higher energy, less stable, excited state. Because the excited state
is less stable than the ground state, the electron will return spontaneously to the ground state.
In the process, the atom will emit energy; this energy will be equivalent in magnitude to the
difference in energy levels between the ground and excited states (and equivalent to the energy
absorbed initially). The process is shown schematically in Fig. 3. If the emitted energy is in the
form of electromagnetic radiation, Eqs. (1:4) and (1:5) directly relate the wavelength of
radiation absorbed or emitted to the electronic transition that has occurred:
ΔE = Efinal - Einitial = hν =hc/λ. (1:6)

Figure 3 Energy transitions in atoms. Atoms may absorb energy and move a ground state
valence electron to higher energy excited states. The excited atom may relax back to the ground
state by emitting light of a wavelength equal to the difference in energy between the states.

Each element has a unique set of permitted electronic energy levels because of its unique
electronic structure. The wavelengths of light absorbed or emitted by atoms of an element are
characteristic of that element. The absorption of radiant energy by atoms forms the basis of
AAS. The absorption of energy and the subsequent emission of radiant energy by excited atoms
form the basis of absorption and emission spectroscopy (AES) and atomic fluorescence
spectroscopy.
In practice, the actual energy level diagram for an atom is derived from the emission spectrum
of the excited atom. Figure 4 shows an energy level diagram for mercury atoms. Notice that
there are no rotational or vibrational sublevels in atoms! A free gas phase atom has no rotational

7
or vibrational energy associated with it. When an electron is promoted to a higher atomic
excited state, the change in energy is very well defined and the wavelength range absorbed (or
emitted on relaxation to the ground state) is very narrow. The wavelengths of light involved in
valence electronic transitions in atoms fall in the visible and UV regions of the spectrum. This
region is often called the UV/VIS region for short. The energy level diagrams for all elements
have been determined, and tables of wavelengths absorbed and emitted by atoms are available.
Knowing what wavelengths of light are absorbed or emitted by a sample permits qualitative
identification of the elements present in the sample. Measuring the intensity of light absorbed
or emitted at a given wavelength gives us information about how much of a given element is
present (quantitative elemental analysis).

Figure 4 Energy levels in mercury atoms

All of the atomic spectroscopy methods—absorption, fluorescence, and emission are extremely
sensitive. As little as 10-12–10-15 g of an element may be detected using atomic spectroscopy.
It is possible for atoms to absorb higher energy radiation, in the X-ray region; such absorption
may result in the inner shell (core) electrons being promoted to an excited state, with the
subsequent emission of X-ray radiation. This process forms the basis for qualitative and
quantitative elemental analysis by XRF spectroscopy, as well as other X-ray techniques.

3. Molecules and Molecular Spectroscopy

The energy states associated with molecules, like those of atoms, are also quantized. There are
very powerful spectroscopic methods for studying transitions between permitted states in
molecules using radiation from the radiowave region to the UV region. These methods provide
qualitative and quantitative information about molecules, including detailed information about
molecular structure.
3.1. Rotational Transitions in Molecules

8
The ability of a molecule to rotate in space has associated rotational energy. Molecules may
exist in only discrete (quantized) rotational energy states. Absorption of the appropriate energy
causes transitions from lower energy rotational states to higher energy rotational states, in
which the molecule rotates faster. This process gives rise to rotational absorption spectra. The
rotational energy of a molecule depends on its angular velocity, which is variable. Rotational
energy also depends on the molecule’s shape and weight distribution, which change as bond
angles change. While a change in shape is restricted in diatomic molecules such as O2,
molecules with more than two atoms, such as hexane, C6H14, have many possible shapes and
therefore many possible rotational energy levels. Furthermore, the presence of more than one
natural isotope of an atom in a molecule generates new sets of rotational energy levels. Such is
the case with carbon, where a small percentage of the carbon atoms in a carbon-containing
molecule are 13C instead of 12C.
Consequently, even simple molecules have complex rotational absorption spectra. The energies
involved in rotational changes are very small, on the order of 10-24 J per molecule.
The radiation absorbed is therefore in the radiofrequency and microwave regions of the
spectrum. Microwave spectroscopy has been largely unexploited in analytical chemistry
because of the experimental difficulties involved and the complexity of the spectra produced.
The technique is limited to the gas phase and has been used by radioastronomers to detect the
chemical species in interstellar clouds.
3.2. Vibrational Transitions in Molecules
For the purposes of basic understanding of this branch of optical spectroscopy, molecules can
be visualized as a set of weights (the atoms) joined together by springs (the chemical bonds).
The atoms can vibrate toward and away from each other or they may bend at various angles to
each other as shown in Fig. 5.

Figure 5 Some possible vibrations of bonded atoms in a molecule.

9
Each such vibration has characteristic energy associated with it. The vibrational energy states
associated with molecular vibration are quantized. Changes in the vibrational energy of a
molecule are associated with absorption of radiant energy in the IR region of the spectrum.
While absorption of IR radiation causes changes in the vibrations of the absorbing molecule,
the increase in vibrational energy is also usually accompanied by increased molecular rotation.
Remember, the rotational energy levels are sublevels of the vibrational energy levels, as we
saw in Fig. 6 So in practice, absorption of IR radiation corresponds to a combination of changes
in rotational and vibrational energies in the molecule. Because a molecule with more than two
atoms has many possible vibrational states, IR absorption spectra are complex, consisting of
multiple absorption bands. Absorption of IR radiation by molecules is one of the most
important techniques in spectroscopy. Through IR absorption spectroscopy, the structure of
molecules can be deduced, and both qualitative identification of molecules and quantitative
analysis of the molecular composition of samples can be performed. (IR spectroscopy).

Figure 6 Schematic energy level diagram for molecules. Each electronic energy level,
E0,1,2,. has associated vibrational sublevels V1,2,3, . . . and rotational sublevels J1,2,3,

3.3. Electronic Transitions in Molecules

10
A free gas phase atom has no rotational or vibrational energy associated with it, which results
in the absorption or emission of very narrow wavelength ranges. When atoms combine to form
molecules, the individual atomic orbitals combine to form a new set of molecular orbitals.
Molecular orbitals with electron density in the plane of the bonded nuclei, that is, along the
axis connecting the bonded nuclei, are called sigma (s) orbitals. Those molecular orbitals with
electron density above and below the plane of the bonded nuclei are called pi (p) orbitals. Sigma
and pi orbitals may be of two types, bonding orbitals or antibonding orbitals. Bonding orbitals
are lower in energy than the corresponding antibonding orbitals. When assigning electrons in
molecules to orbitals, the lowest energy bonding orbitals are filled first. Under normal
conditions of temperature and pressure, the electrons in the molecule are in the ground state
configuration, filling the lowest energy molecular orbitals available. Absorption of the
appropriate radiant energy may cause an outer electron to be promoted to a higher energy
excited state. As was the case with atoms, the radiant energy required to cause electronic
transitions in molecules lies in the visible and UV regions. And as with atoms, the excited state
of a molecule is less stable than the ground state. The molecule will spontaneously revert (relax)
to the ground state emitting UV or visible radiant energy. Unlike atoms, the energy states in
molecules have rotational and vibrational sublevels, so when a molecule is excited
electronically, there is often a simultaneous change in the vibrational and rotational energies.
The total energy change is the sum of the electronic, rotational, and vibrational energy changes.
Because molecules possess many possible rotational and vibrational states, absorption of UV
or visible radiation by a large population of molecules, each in a slightly different state of
rotation and vibration, results in absorption over a wide range of wavelengths, called an
absorption band. The UV/VIS absorption spectra of molecules usually have a few broad
absorption bands and are usually very simple in comparison with IR spectra. Molecular
absorption and emission spectroscopy are used for qualitative identification of chemical
species, especially for inorganic and organometallic molecules with metal atoms at their
centers. This technique used to be one of the major methods for structural determination of
organic molecules but has been replaced by the more powerful and now commonly available
techniques of NMR, IR spectroscopy, and MS. UV/VIS absorption spectroscopy is most often
used for quantitative analysis of the molecular composition of samples. Molecular fluorescence
spectroscopy is an extremely high sensitivity method, with the ability to detect single
molecules! We will learn the laws governing absorption, which permit quantitative analysis by
UV/VIS spectroscopy.

11
UV/Visible Absorption Spectroscopy-I
We see a lot of colorful things around us. What exactly is the color and what make the things
exhibit these colors? We know that the color we see is the visible region of the electromagnetic
spectrum. We also know that matter can absorb the electromagnetic radiation of different
energy (or wavelengths). The region of electromagnetic energy that is not absorbed is simply
reflected back or gets transmitted through the matter. The colored compounds are colored
because they absorb the visible light. The color that is perceived is called the complement color
to the absorbed wavelength and is represented by a color wheel (Figure 7).

Figure 7 A simplified color wheel showing complementary colors. Green is interesting as

it can arise from the absorption of radiation to either end of the visible spectrum.

Absorption of ultraviolet (UV) and visible radiation is one of the most routinely used analytical
tools in life sciences research. The simplest application of UV/Visible radiation is to quantify
the amount of a substance present in a solution.UV region of electromagnetic radiation
encompasses the wavelengths ranging from ~10 nm – ~400 nm while visible region
encompasses the wavelengths from ~400 nm – ~780 nm. For the sake of convenience in
discussing the observations, UV region is loosely divided into near UV (wavelength region
nearer to the visible region, λ ~ 250 nm – 400 nm), far UV region (wavelength region farther
to the visible region, λ ~ 190 nm – 250 nm) and vacuum UV region (λ< 190 nm). The
wavelength ranges defined for these regions are not strict and people use slightly different
ranges to define these regions. We shall, however, stick to the wavelengths defined here. As
has been discussed in the previous lecture, the absorption of UV and visible light is through
the transition of an electron in the molecule from lower to a higher energy molecular orbital.
The various electronic transitions observed in organic compound are shown in Figure 8.

12
 Figure 8 Schematic diagram showing energy levels of different orbitals and possible
absorption transitions

As shown in figure 8, σ → σ* transition is a high energy process and therefore lies in the
vacuum UV region. Alkanes, wherein only σ → σ* transition is possible show absorption bands
~150 nm wavelength. Alkenes haveπ and π* orbitals and can show several transitions; the
lowest-energy transition, π → π* gives an absorption band ~170-190 nm for non-conjugated
alkenes (effects of conjugation on electronic transitions are discussed later). The presence of
nonbonding electrons in a molecule further expands the number of possible transitions. The
entire molecule, however, is not generally involved in the absorption of the radiation in a given
wavelength range. In an aliphatic ketone, for example, the absorption band around 185 nm
arises due to the π → π* transition in the carbonyl group. Atoms that comprise the molecular
orbitals involved in the electronic transitions constitute the molecular moiety that is directly
involved in the transition. Such a group of atoms is called a chromophore. A structural
modification in a chromophore is generally accompanied by changes in the absorption
properties.
When molecules are electronically excited, an electron moves from the highest occupied
molecular orbital to the lowest unoccupied orbital, which is usually an antibonding orbital.
Electrons in p bonds are excited to antibonding p* orbitals, and n electrons are excited to either
s* or p* orbitals. Both organic and inorganic molecules may exhibit absorption and emission
of UV/ VIS radiation. Molecular groups that absorb visible or UV light are called
chromophores, from the Greek word chroma, color. For example, for a π – π* transition to
occur, a molecule must possess a chromophore with an unsaturated bond, such as C=C, C=O,
C=N, and so on. Compounds with these types of chromophores include alkenes, amides,

13
ketones, carboxylic acids, and oximes, among others. The other transition that commonly
occurs in the UV/VIS region is the n – π* transition, so organic molecules that contain atoms
with nonbonded electrons should be able to absorb UV/VIS radiation. Such atoms include
nitrogen, oxygen, sulfur, and the halogen atoms, especially Br and I. Table 4.

Table 4 Organic Functional Groups that can Absorb UV/VIS Radiation

Instrumentation:
Figure 9 A shows a schematic diagram of a single-beam spectrophotometer. The light enters
the instrument through an entrance slit, is collimated and focused on to the dispersing element,
typically a diffraction grating. The light of desired wavelength is selected simply by rotating
the monochromator and impinged on the sample. The intensity of the radiation transmitted
through the sample is measured and converted to absorbance or transmittance (discussed later).
Double beam spectrophotometers overcome certain limitations of the single beam
spectrophotometers and are therefore preferred over them. A double beam spectrophotometer
has two light beams, one of which passes through the sample while other passes through a
reference cell (Figure 8 B). This allows more reproducible measurements as any fluctuation in
the light source or instrument electronics appears in both reference and the sample and
therefore can easily be removed from the sample spectrum by subtracting the reference
spectrum.

14
 Figure 9 Schematic diagram showing a single beam (A) and a double beam (B)
spectrophotometer

Modern instruments can perform this subtraction automatically. The most commonly used
detectors in the UV/Visible spectrophotometers are the photomultiplier tubes (PMT). Modern
instruments also use photodiodes as the detection systems. These diodes are inexpensive and
can be arranged in an array so that each diode absorbs a narrow band of the spectrum.
Simultaneous recording at multiple wavelengths allows recording of the entire spectrum at
once. The monochromator in these spectrophotometers is placed after the sample so that the
sample is exposed to the entire spectrum of the incident radiation and the transmitted radiation
is dispersed into its components.
2.4. Absorption Laws
When light passes through an absorbing sample, the intensity of the light emerging from the
sample is decreased. Assume the intensity of a beam of monochromatic (i.e., single
wavelength) radiation is I0. This beam is passed through a sample that can absorb radiation of
this wavelength, as shown in Fig. 10. The emerging light beam has an intensity equal to I,
where I0 ≥ I. If no radiation is absorbed by the sample, I = I0. If any amount of radiation is
absorbed, I < I0. The transmittance T is defined as the ratio of I to I0:
𝐼
T=
𝐼0

15
The transmittance is the fraction of the original light that passes through the sample. Therefore,
the range of allowed values for T is from 0 to 1. The ratio I/I0 remains relatively constant even
if I0 changes; hence, T is independent of the actual intensity I0. To study the quantitative
absorption of radiation by samples it is useful to define another quantity, the absorbance A
where
𝐼0 1
A = log ( ) = log ( ) = -log T
𝐼 𝑇
When no light is absorbed, I = I0 and A = 0. Two related quantities are also used in
spectroscopy, the percent transmittance, %T, which equals T × 100, and the percent absorption,
%A, which is equal to 100 - %T.
Suppose we have a sample of an aqueous solution of an absorbing substance in a rectangular
glass sample holder with a length of 1.0 cm. Such a sample holder is called a sample cell, or
cuvette, and the length of the cell is called the path length b. The incident light has intensity,
I0, equal to 100 intensity units. If 50% of the light passing through the sample is absorbed, then
50% of the light is transmitted. The emerging light beam has an intensity denoted as I1 = 50
intensity units. So, the %T = 50, and therefore:
%𝑇 𝐼1
T = 100 = 𝐼0
50
T = 100 = 0.5

From T we calculate that absorbance equals:

A = - log T = - log (0.50) = 0.30

Figure 10 Absorption of radiation by a sample.

The Beer-Lambert law or Beer’s law.

This law shows mathematically, based on observed experimental facts, that there is a linear
relationship between A and the concentration of an absorbing species if the path length and the
wavelength of incident radiation are kept constant. This is an extremely important relationship
in analytical spectroscopy. It forms the basis for the quantitative measurement of the
concentration of an analyte in samples by quantitative measurement of the amount of absorbed

16
radiation. The quantitative measurement of radiation intensity is called spectrometry. Beer’s
Law is used in all quantitative absorption spectrometry—IR absorption spectrometry, AAS,
UV/VIS absorption spectrometry, and so on.

Mathematically, we can write the following equation:

A = abc
The term “a” is a proportionality constant called the absorptivity. The absorptivity is a
measure of the ability of the absorbing species in the sample to absorb light at the particular
wavelength used. Absorptivity is a constant for a given chemical species at a specific
wavelength. If the concentration is expressed in molarity (mol/L or M), then the absorptivity
is called the molar absorptivity and is given the symbol 1. The usual unit for path length is
centimeters, cm, so if the concentration is in molarity, M, the unit for molar absorptivity is M-
1
cm-1. The units for absorptivity a when concentration is expressed in any units other than
molarity (for example, ppm, mg/100 mL, etc.) must be such that A, the absorbance, is
dimensionless. Commonly ε ≈ 104–105L mol-1 cm-1 for an allowed transition and is on the order
of 10–100 for a forbidden transition. The magnitude of the absorptivity is an indication of the
probability of the electronic transition. High values of ε give rise to strong absorption of light
at the specified wavelength; low values of ε result in weak absorption of light. Both a and ε are
constants for a given wavelength and are physical properties of the molecule. The molar
absorptivity may be specified for any wavelength but is usually tabulated for the wavelength
at which maximum absorption of light occurs for a molecule. The wavelength of maximum
absorption is symbolized by λmax and the associated ε is symbolized as λmax.
Deviations from Beer’s Law
Beer’s Law is usually followed at low concentrations of analyte for homogeneous samples.
Absorbance is directly proportional to concentration for most absorbing substances when the
concentration is less than about 0.01 M.
Deviations from linearity are common at high concentrations of analyte. There are several
possible reasons for deviation from linearity at high concentrations. At low concentrations in a
solution, the analyte would be considered the solute. As the solute concentration increases, the
analyte molecules may begin to interact with each other, through intermolecular attractive
forces such as hydrogen bonding and other van der Waals forces. Such interactions may change
the absorptivity of the analyte, again resulting in a nonlinear response as concentration
increases. At extremely high concentrations, the solute may actually become the solvent,
changing the nature of the solution. If the analyte species is in chemical equilibrium with other

17
species, as is the case with weak acids or weak bases in solution, changes in concentration of
the analyte may shift the equilibrium (Le Chatelier’s Principle). This may be reflected in
apparent deviations from Beer’s Law as the solution is diluted or concentrated.
Another source of deviation from Beer’s Law may occur if the sample scatters the incident
radiation. Solutions must be free of floating solid particles and are often filtered before
measurement. The most common reason for nonlinearity at high analyte concentrations is that
too little light is available to be absorbed. At low levels of analyte, doubling the concentration
doubles the amount of light absorbed, say from 25% to 50%. If 99% of the light has already
been absorbed, doubling the concentration still doubles the amount of remaining light
absorbed, but the change is only from 99% to 99.5%. This results in the curve becoming flat at
high absorbance.
It can be seen that A = log(I0/I). If I0 = 100 and A = 1.0, then I = 10. Only 10% of the initial
radiation intensity is transmitted. The other 90% of the intensity is absorbed by the sample. If
A = 2.0, I = 1.0, indicating that 99% of the incident light is absorbed by the sample. If A = 3.0,
99.9% of the incident light intensity is absorbed. As we shall see, the error in the measurement
of A increases as A increases (or as I decrease). In practice, Beer’s Law is obeyed for
absorbance values less than or equal to 1.0.

Factors that influence the absorption spectra of molecules

We studied that UV/Visible radiation is absorbed by the molecules through transition of
electrons in the chromophore from low energy molecular orbitals to higher energy molecular
orbitals. We are interested in the transitions that lie in the far UV, near UV, and visible regions
of the electromagnetic spectrum. The molecules that absorb in these regions invariably have
unsaturated bonds. Plants are green due to unsaturated organic compounds, called chlorophylls.
A highly unsaturated alkene, lycopene, imparts red color to the tomatoes (Figure 11).

Figure 11 Structure of lycopene, the pigment that imparts red color to the tomatoes

As can be seen from its structure, lycopene is a highly conjugated alkene. As compared to the
simple non-conjugated alkenes that typically absorb in vacuum UV region, absorption
spectrum of lycopene is hugely shifted towards higher wavelengths (or lower energy). There
can be factors that could shift the absorption spectra to smaller wavelengths or can

18
increase/decrease the absorption intensity. Before understanding how conjugation causes shift
in the absorption spectra, let us look at some important terms that are used to refer to the shifts
in absorption spectra (Figure 12):
Bathochromic shift: Shift of the absorption spectrum towards longer wavelength
Hypsochromic shift: Shift of the absorption spectrum towards smaller wavelength
Hyperchromic shift: An increase in the absorption intensity
Hypochromic shift: A decrease in the absorption intensity

Figure 12 Terminology for shifts in absorption spectra

Conjugation: Conjugation brings about a bathochromic shift in the absorption bands. The
higher the extent of conjugation, the more is the bathochromic shift. Such shift in absorption
spectra can easily be explained using molecular orbital theory. Figure 13 shows the molecular
orbitals drawn for ethylene; 1,3-butadiene; and 1,3,5-hexatriene on a qualitatively same energy
scale for comparing their energies. As is clear from the figure, the energy differences between
the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital
(LUMO) decreases as the conjugation increases. This provides an explanation as to why an
electronic transition is possible at lower energy (higher wavelength) as the conjugation
increases.
Auxochrome: Auxochromes are the chemical groups that result in a bathochromic shift when
attached to a chromophore. The strongest auxochromes like –OH, –NH2, – OR, etc. possess
nonbonding electrons. They exhibit bathochromism by extending conjugation through
resonance.

19
The auxochrome modified chromophore is a new chromophore in real sense. The term
auxochrome is therefore rarely used these days, and the entire group (basic chromophore +
auxochrome) can be considered as a chromophore different from the basic chromophore. Alkyl
groups also result in the bathochromic shifts in the absorption spectra of alkenes. Alkyl groups
do not have non-bonded electrons, and the effect is brought about by another type of interaction
called hyperconjugation.

Figure 13 Molecular orbitals of ethylene; 1,3-butadiene; and 1,3,5-hexatriene. Notice the

decrease in the energy gap of HOMO and LUMO as the conjugation increases.

Solvents: The solvents used in any spectroscopic method should ideally be transparent (non-
absorbing) to the electromagnetic radiation being used. Table 5 shows the wavelength cutoffs
(the lowest working wavelength) of some of the solvents used in UV/visible spectroscopy.

Table 5 Solvents commonly used in UV/visible spectroscopy

Solvent Wavelength cutoff
Water 190 nm
Acetonitrile 190 nm
Cyclohexane 195 nm
Methanol 205 nm
95% ethanol 205 nm

20
Water, the solvent of biological systems, thankfully is transparent to the UV/visible region of
interest i.e. the regions above λ > 190 nm. Solvents also play important role on the absorption
spectra of molecules. Spectrum of a compound recorded in one solvent can look significantly
different in intensity, wavelength of absorption, or both from that recorded in another. This is
not something unexpected because energies of different electronic states will depend on their
interaction with solvents. Polarity of solvents is an important factor in causing shifts in the
absorption spectra. Conjugated dienes and aromatic hydrocarbons are little affected by the
changes in solvent polarity. α,β-unsaturated carbonyl compounds are fairly sensitive to the
solvent polarity. The two electronic transitions π → π* and n → π* respond differently to the
changes in polarity. Polar solvents stabilize all the three molecular orbitals (n, π, and π*), albeit
to different extents (Figure 5.4). The non-bonding orbitals are stabilized most, followed by π*.
This results in a bathochromic shift in the π → π* absorption band while a hypsochromic shift
in n → π* absorption band. Shift to different extents of the two bands will result in the different
shape of the overall absorption spectrum.

Figure 14 Differential stabilization of molecular orbitals in polar solvents

Biological chromophores

Amino acids and proteins: Among the 20 amino acids that constitute the proteins, tryptophan,
tyrosine, and phenylalanine absorb in the near UV region. All the three amino acids show
structured absorption spectra. The absorption by phenylalanine is weak with an εmax of ~200
M-1cm-1 at ~250 nm. Molar absorption coefficients of ~1400 M-1cm-1 at 274 nm and ~5700 M-
1
cm-1 at 280 nm are observed for tyrosine and tryptophan, respectively. Disulfide linkages,

21
formed through oxidation of cysteine resides, also contribute to the absorption of proteins in
near UV region with a weak εmax of ~300 M-1cm-1 around 250-270 nm. The absorption spectra
of proteins are therefore largely dominated by Tyr and Trp in the near UV region. In the far
UV region, peptide bond emerges as the most important chromophore in the proteins. The
peptide bond displays a weak n → π* transition (εmax ≈ 100 M-1cm-1) between 210-230 nm, the
exact band position determined by the H-bonding interactions the peptide backbone is involved
in. A strong π → π* transition (εmax ≈ 7000 M-1cm-1) is observed around 190 nm. Side chains
of Asp, Glu, Asn, Gln, Arg, His also contribute to the absorbance in the far UV region. Figure
15 shows an absorption spectrum of a peptide

Figure 15 Absorption spectrum of a peptide. The absorption band ~280 nm is due to

aromatic residues. Absorption band in the far UV region arises due to peptide bond
electronic transitions.

Nucleic acids: Nucleic acids absorb very strongly in the far and near UV region of the
electromagnetic spectrum. The absorption is largely due to the nitrogenous bases. The
transitions in the nucleic acid bases are quite complex and many π → π* and n → π* transitions
are expected to contribute to their absorption spectra. A 260 nm wavelength radiation is
routinely used to estimate the concentration of nucleic acids. Though the molar absorption
coefficients vary for the nucleotides at 260 nm, the average εmax can be taken as ~104 M-1cm-
1
. It is important to mention that nucleotides show hyperchromicity when exposed to aqueous
environment. The absorbance of the free nucleotides is higher than that of single stranded
nucleic acid which is higher than that of the double stranded nucleic acid (assuming equal
amount of the nucleotides present in all three).

22
Other chromophores: Nucleotides like NADH, NADPH, FMN, and FAD; porphyrins such as
heme, chlorophylls and other plant pigments; retinal (light sensing molecule); vitamins; and a
variety of unsaturated compounds constitute chromophores in the UV and visible region.
Having studied the principles of the UV/visible absorption spectroscopy and various factors
that influence the electronic transitions, we can now have a look at its applications, especially
the applications for analyzing the biological samples.

Applications:

i. Determination of molar absorption coefficient: From Beer-Lambert law, A = εcl. It is

therefore straightforward to calculate the molar absorption coefficient of a compound if the
concentration of compound is accurately determined.
ii. Quality Control: A given organic compounds such as a drug can be studied for

absorbances at 260 and 280 nm. A typical nucleic acid containing all four bases shows an
absorption band centered ~260 nm while a protein having aromatic amino acids shows
absorption band centered ~280 nm. It is possible to determine the purity of protein preparations
by recording absorbances at both 260 and 280 nm. A ratio of the absorbance at 260 nm to that
at 280 nm.

iii. Quantification of compounds: This is perhaps the most common application of a

UV/visible spectrophotometer in a bioanalytical laboratory. If the molar absorption coefficient
at a wavelength is known for the compound, the concentration can easily be estimated using

23
Beer-Lambert law. The compounds can still be quantified if their molar absorption coefficients
are not known. Estimation of total protein concentration in a given solution is an important
example of this. As the given solution is a mixture of many different proteins, the ε is not
available. There are, however, dyes that specifically bind to the proteins producing colored
complex. The color produced will be proportional to the amount of the protein present in the
solution. Performing the experiment under identical conditions using known concentrations of
a protein gives a standard graph between absorbance of the dye and the amount of protein. This
standard graph is then used to estimate the concentration of the given protein sample.
iv. Chemical kinetics: UV/visible spectroscopy can be used to monitor the rate of chemical
reactions if one of the reactants or products absorbs in a region where no other reactant or
product absorbs significantly.
v. Detectors in liquid chromatography instruments: UV/visible detectors are perhaps the
most common detectors present in liquid chromatography systems. Modern instruments use
photodiode array detectors that can detect the molecules absorbing in different spectral regions
(Figure 16).

Figure 16 Diagram of a photodiode array detector

vi. Determination of melting temperature of DNA: A double stranded DNA molecule can
be denatured into the single strands by heating it. Melting temperature, Tm is the temperature
at which 50% of the DNA gets denatured into single strands. Denaturation of DNA is
accompanied by hyperchromic shift in the absorption spectra in the near UV region. A melting
curve (plot between temperature and absorbance at 260 nm) is plotted and Tm is determined
(Figure 17).

24
Figure 17 Thermal denaturation of a DNA sample; a plot of absorbance at 260 nm against
the temperature allows determination of the melting temperature (Tm).

vii. Microbial growth kinetics: A UV/visible spectrophotometer is routinely used to

monitor the growth of microorganisms. The underlying principle behind this, however, is not
absorbance but scattering. As the number of microbial cells increase in a culture, they cause
more scattering in light. The detector therefore receives less amount of radiation, recording this
as absorbance. To distinguish this from actual absorbance, the observed value is referred to as
the optical density.

25
26
Fluorescence Spectroscopy-I
This lecture is a very concise review of the phenomenon of fluorescence and the associated
processes. Let us move a step forward from the absorption of the UV/visible radiation. What
happens to the electrons that absorb UV/visible light and occupy the high energy molecular
orbitals? In a UV/visible absorption experiment, the samples continue absorbing light. This
means that the higher energy molecular orbitals never get saturated. This further implies that
after excitation, the molecules somehow get rid of the excess energy and return back to the
ground state. The electrons can return back to the ground state in different ways such as
releasing the excess energy through collisions or through emitting a photon. In fluorescence,
the molecules return back to the ground state by emitting a photon. The molecules that show
fluorescence are usually referred to as the fluorophores. Various electronic and molecular
processes that occur following excitation are usually represented on a Jablonski diagram as
shown in Figure 18.

Figure 18 Jablonski diagram showing various processes following absorption of light by

the fluorophore

S0, S1, and S2 represent the singlet electronic states while the numbers 0, 1, 2 represent the
vibrational energy levels associated with the electronic states. T1 depicts the first triplet
electronic state. Let us go through the processes shown in Figure 18: Absorbance: S0 state with
0th vibrational level is the state of lowest energy and therefore, the highest populated state.

27
Absorption of a photon of resonant frequency usually results in the population of S1 or S2
electronic states; but usually a higher vibrational state. Transition of electrons from low energy
molecular orbital to a high energy molecular orbital through absorption of light is a
femtosecond (10-15 s) phenomenon. The electronic transition, therefore, is too quick to allow
any significant displacement of the nuclei during transition.
Internal conversion: Apart from few exceptions, the excited fluorophores rapidly relax to the
lowest vibrational state of S1 through non-radiative processes. Non-radiative electronic
transition from higher energy singlet states to S1 is termed as internal conversion while
relaxation of a fluorophore from a higher vibrational level of S1 to the lowest vibration state is
termed as vibrational relaxation. The terms ‘internal conversion’ and ‘vibrational relaxation’,
however, are often interchangeably used. The timescale of internal conversion/vibrational
relaxation is of the order of 10-12seconds.
Fluorescence: Fluorescence lifetimes are of the order of 10-8 seconds, implying that the internal
conversion is mostly complete before fluorescence is observed. Therefore, fluorescence
emission is the outcome of fluorophore returning back to the S0 state through S1 → S0 transition
emitting a photon. This also explains why emission spectra are usually independent of the
excitation wavelength, also known as Kasha’s rule (However, there are exceptions wherein
fluorescence is observed from S2 → S1 transition). The S1 → S0 transition, like S0 → S1
transition, typically results in the population of higher energy vibrational states. The molecules
then return back to the lowest vibrational state through vibrational relaxation.
Intersystem crossing: Intersystem crossing refers to an isoenergetic non-radiative transition
between electronic states of different multiplicities. It is possible that a molecule in a
vibrational state of S1 can move to the isoenergetic vibrational state of T1. The molecule then
relaxes back to the lowest vibrational state of the triplet state.
Phosphorescence: The molecule in the triplet state, T1, can return back to the S0 state emitting
a photon. This process is known as phosphorescence and has time scales of several orders of
magnitudes higher than that of fluorescence (10-3 – 10 s).

Characteristics of fluorescence:
Figure 19 shows absorption and fluorescence emission spectrum of a hypothetical fluorophore.
The important characteristics of the fluorescence emission can be briefly summarized as
follows:

28
Figure 19 Absorption and fluorescence emission spectrum of a hypothetical fluorophore

Stokes shift: A fluorescence emission spectrum is always shifted towards longer wavelengths
with respect to the absorption spectrum. This shift is known as Stokes shift and is expected as
excited molecules lose energy through processes like internal conversion and vibrational
relaxation. The emitted radiation is therefore expected to be of lower energy i.e. higher
wavelength

.
Figure 20 Potential energy diagrams showing the Franck-Condon principle
Kasha’s rule: As fluorescence emission is observed from S1 → S0 transtions (except a few
exceptions), fluorescence absorption spectrum is independent of the excitation wavelength.

29
Franck-Condon principle: The Franck-Condon principle states that the positions of the nuclei
do not change during electronic transitions. The transitions are said to be vertical. This implies
that if the probability of 0th → 2nd vibrational transition during S0 → S1 transition is highest,
the 2nd → 0th transition will be most probable in the reciprocal transition (Figure 20).
This results in an emission spectrum that is a mirror image of the S0 → S1 transition in terms
of the shape. There are several exceptions to the mirror image rule that arise largely due to the
excited state reactions of the molecule.

Fluorescence quenching, resonance energy transfer and anisotropy

Fluorescence spectroscopy comprises of experiments exploiting various different phenomena
related to it. Discussion of all these experiments is beyond the scope of this course, but we shall
have a quick look at a few important phenomena related to fluorescence.

Fluorescence quenching: A decrease in fluorescence intensity is referred to as quenching. A

molecule that quenches the fluorescence of a fluorophore is called a quencher. A quencher can
be either a collisional quencher or a static quencher. A collisional quencher brings about
decrease in fluorescence intensity by de-exciting the excited fluorophore through collisions.

30
Addition of another non-radiative process to the system leads to lower quantum yield. A static
quencher forms a non-fluorescent complex with the fluorophore. It effectively leads to a
decrease in the concentration of the fluorophore thereby decreasing the fluorescence emission
intensity.
Resonance energy transfer: Resonance energy transfer (RET), also known as fluorescence
resonance energy transfer (FRET) is an excited state phenomeneon wherein energy is
transferred from a donor molecule (D) to an acceptor molecule (A). The prerequisite for the
energy transfer is that there should be an overlap between the emission spectrum of the D and
the absorption spectrum of the A (Figure 21).

Figure 21 Diagrammatic representation of spectral overlap between donor’s emission and

acceptor’s absorption spectrum.
The efficiency of energy transfer depends upon
i. the distance between D and A
ii. the relative orientation of the transition dipoles of D and A
iii. the extent of the overlap between D’s emission spectrum and A’s absorption spectrum.

where,
r is the distance between D and A.
R0 (also called the Förster distance) is the distance (r) between D and A at which the efficiency
of energy transfer is 50% and is characteristic of a D-A FRET pair.

31
Resonance energy transfer can be used to determine the distances between D and A and is
therefore also termed as molecular ruler.
Fluorescence anisotropy: The radiation emitted by a sample following excitation with
polarized light can be polarized. Polarization is measured in terms of anisotropy. Zero
anisotropy implies isotropic/non-polarized radiation while non-zero anisotropy implies some
degree of polarization. Figure 22 shows how fluorescence anisotropic measurements are made.

Figure 22 A schematic diagram showing the measurement of fluorescence anisotropy

The sample is excited with the linearly polarized light and emission is recorded at 90°. A
polarizer is placed before the detector that allows intensity measurement of the light polarized
parallel ( I ∥) and perpendicular ( I⊥) to the direction of excitation radiation. The anisotropy (r)
is given by:

Molecular tumbling before emission changes the orientation of the transition dipole moment,
resulting in the loss of polarization (Figure 23). As rotational diffusion of the molecules
depends on their sizes, fluorescence anisotropy can be used to measure the diffusion coefficient
and therefore the sizes of the molecules.

32
Figure 23 Depolarization of radiation as a result of molecular tumbling

33
Biological fluorophores

Amino acids: Aromatic amino acids tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe)
are perhaps the most important intrinsic biological fluorophores. Proteins harboring these
amino acids become intrinsically fluorescent.
Proteins: Proteins are fluorescent due to the presence of aromatic amino acids that fluoresce in
the near UV region. Certain proteins, however, do fluoresce in the visible region. Green
fluorescent protein (GFP), for example, fluoresces in the green region of the electromagnetic
spectrum. The discovery of green fluorescent protein has revolutionized the area of cell biology
research. It is therefore important to see what green fluorescent protein is and why it fluoresces
in the visible region.
Green Fluorescent Protein (GFP)
Green fluorescent protein, abbreviated as GFP was discovered by Shimomura and coworkers
in 1962. The protein was isolated from the jellyfish, Aequorea victoria, that glows in the dark.
GFP is a 238 amino acid long protein that folds into an 11-stranded β-barrel structure wherein
an α-helix passes through the barrel.
The fluorophore of the GFP, p-hydroxy benylideneimidazolinone is formed by the residues 65-
67 (Ser-Tyr-Gly) and is present in the α-helix passing through the barrel.
The excitation spectrum of GFP exhibits a strong absorption band at 395 nm and a weak band
at 475 nm. Emission is observed at ~504 nm i.e. in the green region. GFP is an excellent
fluorophore with a molar absorption coefficient of ~30000 M-1cm-1 at 395 nm and fluorescence
quantum yield of 0.79. GFP has been engineered through extensive mutations to remove the
undesirable properties that could affect its use as a potential fluorophore. For example, a Ser65
→ Thr65 mutant has improved quantum yield and its major excitation band shifted to 490 nm.
GFP has the tendency to form oligomers, seriously questioning its use as a fluorescent probe.
The aggregation tendency has also been removed through extensive mutations. GFP can easily
be tagged to a protein by expressing the fusing gene (GFP gene fused with the gene expressing
the desired protein). The GFP then acts as a reporter for all the processes the linked protein is
involved in. Several color variants of GFP have been generated through modifications in the
residues that constitute the fluorophore. Development of the GFP variants with varying
excitation and emission characteristics has made it possible to label the proteins differentially.

Nucleotides: Nicotinamide adenine dinucleotide in its reduced form, NADH and the flavin
adenine dinucleotide in its oxidized form, FAD are fluorescent in the visible region of the

34
electromagnetic spectrum. It is not necessary for all the biomolecules to have an intrinsic
fluorophore to perform fluorescence experiments. Fluorescent groups can be covalently
incorporated into the molecules making them fluorescent with desirable fluorophore. Such
externally incorporated fluorophores are called extrinsic fluorophores.

Applications of fluorescence
Protein folding: High sensitivity of tryptophan fluorescence to the polarity of solvent makes it
an interesting intrinsic fluorescent probe for studying protein folding. In the proteins having
Phe and Tyr, Trp can be selectively excited at 295 nm. In water and other aqueous solutions,
tryptophan fluoresces with an emission maximum, λmax around 350 nm. A tryptophan present
in the hydrophobic environment usually displays a blue shift in the emission spectrum and an
increase in quantum yield. Due to the hydrophobic nature of the indole side chain, tryptophans
are usually buried inside the core of the proteins. The folding can therefore be studied by
monitoring the Trp fluorescence as protein folds burying the water-exposed Trp residues inside
the protein.
Peptide-lipid interactions: Interaction of the peptides having Trp residues with lipid bilayers
can easily be studied using fluorescence spectroscopy. Interaction of the peptide with lipids
brings the tryptophan in relatively hydrophobic environment causing a blue shift in emission
spectrum (Figure 24).

Figure 24 Spectral changes in tryptophan fluorescence upon binding to lipid bilayers

Binding studies: Binding of small fluorescent molecules to the biomacromolecules can be

studied using fluorescence anisotropy. Binding of the fluorophore to a macromolecule will

35
reduce its tumbling (increase its rotational correlation time) thereby resulting in higher
fluorescence anisotropy.

FRET:
i. The distance between two sites in a biomacromolecule such as a protein can be
calculated by labeling these sites with suitable donor-acceptor FRET pair. FRET can also be
used to study the intermolecular interaction if the interacting molecules comprise of the
fluorophores making a FRET pair.
ii. Interactions of peptides and other molecules with lipid bilayers comprising fluorophore
labeled lipids. If the interacting molecule makes a FRET pair with the fluorescent lipid, the
distance between them can be calculated providing information about the insertion of the
molecule in the lipid bilayer.
iii. FRET has been utilized to study the kinetics of enzymatic reactions. For example, a
DNA molecule, tagged with the fluorescence donor at one end and an acceptor at the other end
can be used as a substrate to study the restriction endonuclease activity and cleavage reaction
kinetics (Figure 25). A similar assay can be used to study the proteases using peptides as the
substrates.

Figure 25 Decrease in fluorescence intensity of the acceptor following cleavage of DNA

molecules.

Fluorescence quenching:
i. Interaction of a fluorophore with other molecule(s) may provide it protection against a
collisional quencher. For example, interaction of a Trp containing peptide with lipid bilayers
can be studied using iodide (I−) as the collisional quencher. The peptide sample in the presence
of lipid vesicle is titrated with the potassium iodide (KI) and fluorescence spectra recorded at
each quencher concentration. The collisional fluorescence quenching is described by a plot of

36
‘the ratio of quantum yield in the absence of quencher to that in the presence of quencher’
against ‘the quencher concentration’. Such a plot is known as the Stern-Volmer plot. The Stern-
Volmer equation is given by

Where,
F0 = Fluorescence intensity in the absence of quencher
F = Fluorescence intensity in the presence of quencher
kq = Bimolecular quenching constant.
τ0 = Fluorescence lifetime in the absence of quencher.
[Q] = Quencher concentration
[R] Ksv = Stern-Volmer constant
A normalized accessibility factor (NAF) is defined as the ratio of ‘the Ksv in the presence of
the binding partner of the fluorophore’ to ‘that without the binding partner’.

ii. The fluorescence intensity of a sample increases with an increase in the fluorophore
concentration. Beyond certain concentration, however, the fluorescence intensity decreases due
to self-collisional quenching. This property is often used to study the membranolytic activities
of a compound. A fluorescent dye at self-quenching concentrations is trapped inside a lipid
vesicle. A membranolytic compound results in the release of the fluorescent dye causing
increase in fluorescence emission intensity (Figure 26).

Figure 26 Membranolytic activity of a compound monitored through dye release assay. Release
of dye from the lipid vesicle diminishes the self-quenching resulting in enhanced fluorescence
emission.

iii. Fusion of lipid vesicles can also be studied using the same approach. Vesicles that
contain self-quenching concentrations of the fluorescent dye are titrated with the vesicles

37
without fluorophores. A fusion will result in the dilution of fluorophores; the consequent
decrease in self-quenching is exhibited as an increase in the fluorescence intensity (Figure 27).

Figure 27 Fusion of fluorescent dye-containing lipid vesicles with vesicles without dye
results in dilution of dye. The dilution results in lesser self-quenching thereby increasing
the fluorescence intensity.

38
39