Fluorescence Spectrometry
435
where, S o is the ground singlet electronic state, S 1 and S n represent the first and nth
higher energy singlet electronic states, respectively. The hν (ex) and hν (em) are the
excitation and emission energies, respectively.
According to the thermodynamical prediction, a molecule predominantly resides
in the ground state until its thermal energy is lower than that of the energy gap between
the excited and ground electronic states. When a ground state molecule absorbs hν ex
energy of a photon, the transition from the ground state (S o ) to any suitable singlet
state (S n ) takes place. The molecule then quickly relaxes to the lowest vibrational
level of the first electronic excited state by way of internal conversion. Subsequently,
the emission of a lower energy photon, hν em , occurs before relaxing back to the
ground state S o . If the ending state is other than the ground state, the molecule loses
its remaining energy through further fluorescent emission and/or non-radiative relaxation in which the energy is dissipated in the form of heat or phonons. The different
energy levels that participate in the absorption and emission of light photons by a
fluorophore have been classified in an energy diagram which is known as ‘Jablonski
Energy Level Diagram’ (Fig. 5), in honor of the Polish physicist Professor Alexander
Jablonski. The Jablonski energy diagram simply explains how electrons present in
fluorophores are excited from the ground state to higher energy states by absorbing a
particular photonic energy and various processes occur when the excited molecules
relax back to their ground state by emitting a particular radiation. Fluorescence rarely
renders due to the absorption of UV light of wavelengths shorter than 250 nm because
such radiations are sufficiently energetic to cause the deactivation of the excited state
by dissociation or predissociation. Most organic molecules have at least a few bonds
that can be disparted by this amount of energy. Consequently, fluorescence due to
σ
∗
−σ transitions is rarely observed.
The Jablonski energy level diagram, as depicted in Fig. 5, illustrates a singlet
ground state (S o ), along with the first (S 1 ), and second (S 2 ), excited singlet states.
The thicker lines describe electronic energy levels, whereas the thinner lines render
the vibrational energy states. Here, we have ignored the representation of rotational
energy states for the sake of simplicity. On the right-hand side, excited triplet state
(T 1 ) is presented. After absorbing the photonic energy that occurs approximately in
less than femtosecond, electrons transit from the singlet ground electronic level, S o ,
to an excited state S n (n > l). An excited molecule relaxes back to the ground state
S o into the following steps:
Step 1: Higher excited state (S n ) to the lowest excited state S 1 : In this process, the
excited molecule present at S n returns back to S 1 by dissipating some part of its
energy to the surrounding. This transit process is known as “Internal Conversion”
having the fluorescence lifetimes of 10
−14 to 10
−11 s.
Step 2: Transition of excited molecule form S 1 to S o through different processes,
listed below:
• Fluorescence: Emission of photons having a radiative rate constant k r and lifetime
scale of 10
−9 to 10
−7 s.
• Intersystem crossing: The excited molecules can also go through a transition to
an excited triplet state T 1 , which has lower energy than S 1 . This transient passage
435
where, S o is the ground singlet electronic state, S 1 and S n represent the first and nth
higher energy singlet electronic states, respectively. The hν (ex) and hν (em) are the
excitation and emission energies, respectively.
According to the thermodynamical prediction, a molecule predominantly resides
in the ground state until its thermal energy is lower than that of the energy gap between
the excited and ground electronic states. When a ground state molecule absorbs hν ex
energy of a photon, the transition from the ground state (S o ) to any suitable singlet
state (S n ) takes place. The molecule then quickly relaxes to the lowest vibrational
level of the first electronic excited state by way of internal conversion. Subsequently,
the emission of a lower energy photon, hν em , occurs before relaxing back to the
ground state S o . If the ending state is other than the ground state, the molecule loses
its remaining energy through further fluorescent emission and/or non-radiative relaxation in which the energy is dissipated in the form of heat or phonons. The different
energy levels that participate in the absorption and emission of light photons by a
fluorophore have been classified in an energy diagram which is known as ‘Jablonski
Energy Level Diagram’ (Fig. 5), in honor of the Polish physicist Professor Alexander
Jablonski. The Jablonski energy diagram simply explains how electrons present in
fluorophores are excited from the ground state to higher energy states by absorbing a
particular photonic energy and various processes occur when the excited molecules
relax back to their ground state by emitting a particular radiation. Fluorescence rarely
renders due to the absorption of UV light of wavelengths shorter than 250 nm because
such radiations are sufficiently energetic to cause the deactivation of the excited state
by dissociation or predissociation. Most organic molecules have at least a few bonds
that can be disparted by this amount of energy. Consequently, fluorescence due to
σ
∗
−σ transitions is rarely observed.
The Jablonski energy level diagram, as depicted in Fig. 5, illustrates a singlet
ground state (S o ), along with the first (S 1 ), and second (S 2 ), excited singlet states.
The thicker lines describe electronic energy levels, whereas the thinner lines render
the vibrational energy states. Here, we have ignored the representation of rotational
energy states for the sake of simplicity. On the right-hand side, excited triplet state
(T 1 ) is presented. After absorbing the photonic energy that occurs approximately in
less than femtosecond, electrons transit from the singlet ground electronic level, S o ,
to an excited state S n (n > l). An excited molecule relaxes back to the ground state
S o into the following steps:
Step 1: Higher excited state (S n ) to the lowest excited state S 1 : In this process, the
excited molecule present at S n returns back to S 1 by dissipating some part of its
energy to the surrounding. This transit process is known as “Internal Conversion”
having the fluorescence lifetimes of 10
−14 to 10
−11 s.
Step 2: Transition of excited molecule form S 1 to S o through different processes,
listed below:
• Fluorescence: Emission of photons having a radiative rate constant k r and lifetime
scale of 10
−9 to 10
−7 s.
• Intersystem crossing: The excited molecules can also go through a transition to
an excited triplet state T 1 , which has lower energy than S 1 . This transient passage