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D. P. Singh et al.
1 Fundamental Principles of Fluorescence Spectrometry
Certain materials, substances and natural species glow when high energy radiation
falls on them. Such materials have been utilized in all possible branches of science
and technology as depicted in Fig. 1. The mechanism that renders the absorption of
electromagnetic radiation and subsequent emission of light of a particular wavelength
is termed as “Luminescence”. Usually, the wavelength of the emitted light is longer
than that of the incident radiation. The light emission abolishes just after stopping the
electromagnetic exposure; however, the phosphorescent materials continue to emit
light for a certain period of time. Fluorescence is a member of the ubiquitary family of
luminescence in which a substance or molecule emits electromagnetic radiation from
excited states that are created either via physical (i.e., absorption of light), chemical
or mechanical (like friction) mechanisms. The evolution of luminescence under the
exposure of ultraviolet or visible light photons is defined as “photoluminescence”
which is commonly divided into fluorescence and phosphorescence. The existence
of fluorescence and phosphorescence depends upon the electronic configuration of
the excited states of the molecules and emission pathway. The mechanism of phosphorescence is alike to fluorescence having a much longer excited state lifetime. In
contrast to fluorescence, phosphorescence is usually not observed in solutions at room
temperature because of the dominative processes like quenching and nonradiative
decay over the emission.
The term fluorescence is taken from the name of a rock. The name ‘fluorspar’ is
also derived from the Latin ‘fluo’ (for flow) and ‘spar’ (a term referred to non-metallic
minerals). The early observations of fluorescence were recorded by Bernardino de
Sahagún and Nicolás Monardes in 1560 and 1565, respectively. They found fluorescence in the infusion named lignum nephriticum (formally known as ‘kidney
wood’) which was derived from the wood of two trees namely Pterocarpus indicus
and Eysenhardtia polystachya. A chemical compound named matlaline (Fig. 2), was
mainly responsible for this fluorescence which could be obtained by the oxidation
of flavonoids. Later on, fluorescence in fluorite was described by Edward Clarke
and René Haüy in 1819 and 1822, respectively. In 1833, Sir David Brewster noticed
the red emission from chlorophyll extracts under UV light illumination. Afterwards,
Sir George Stokes (1819–1903) observed the blue light emission in fluorspar and
uranium glasses upon exposure to ultraviolet light and called this phenomenon “Fluorescence”. He also noted down an offset between the absorption and emission
wavelengths which has been termed as the ‘Stokes shift’ in his honour.
1.1 Absorption, Excitation and Emission Spectra
Every material or substance absorbs light or electromagnetic radiation according to
the Planck-Einstein quantum relation, i.e., E = hν = hc/λ; where, E is the energy of
a photon, h is known as the Planck’s constant and its value is equivalent to 6.62607 ×
D. P. Singh et al.
1 Fundamental Principles of Fluorescence Spectrometry
Certain materials, substances and natural species glow when high energy radiation
falls on them. Such materials have been utilized in all possible branches of science
and technology as depicted in Fig. 1. The mechanism that renders the absorption of
electromagnetic radiation and subsequent emission of light of a particular wavelength
is termed as “Luminescence”. Usually, the wavelength of the emitted light is longer
than that of the incident radiation. The light emission abolishes just after stopping the
electromagnetic exposure; however, the phosphorescent materials continue to emit
light for a certain period of time. Fluorescence is a member of the ubiquitary family of
luminescence in which a substance or molecule emits electromagnetic radiation from
excited states that are created either via physical (i.e., absorption of light), chemical
or mechanical (like friction) mechanisms. The evolution of luminescence under the
exposure of ultraviolet or visible light photons is defined as “photoluminescence”
which is commonly divided into fluorescence and phosphorescence. The existence
of fluorescence and phosphorescence depends upon the electronic configuration of
the excited states of the molecules and emission pathway. The mechanism of phosphorescence is alike to fluorescence having a much longer excited state lifetime. In
contrast to fluorescence, phosphorescence is usually not observed in solutions at room
temperature because of the dominative processes like quenching and nonradiative
decay over the emission.
The term fluorescence is taken from the name of a rock. The name ‘fluorspar’ is
also derived from the Latin ‘fluo’ (for flow) and ‘spar’ (a term referred to non-metallic
minerals). The early observations of fluorescence were recorded by Bernardino de
Sahagún and Nicolás Monardes in 1560 and 1565, respectively. They found fluorescence in the infusion named lignum nephriticum (formally known as ‘kidney
wood’) which was derived from the wood of two trees namely Pterocarpus indicus
and Eysenhardtia polystachya. A chemical compound named matlaline (Fig. 2), was
mainly responsible for this fluorescence which could be obtained by the oxidation
of flavonoids. Later on, fluorescence in fluorite was described by Edward Clarke
and René Haüy in 1819 and 1822, respectively. In 1833, Sir David Brewster noticed
the red emission from chlorophyll extracts under UV light illumination. Afterwards,
Sir George Stokes (1819–1903) observed the blue light emission in fluorspar and
uranium glasses upon exposure to ultraviolet light and called this phenomenon “Fluorescence”. He also noted down an offset between the absorption and emission
wavelengths which has been termed as the ‘Stokes shift’ in his honour.
1.1 Absorption, Excitation and Emission Spectra
Every material or substance absorbs light or electromagnetic radiation according to
the Planck-Einstein quantum relation, i.e., E = hν = hc/λ; where, E is the energy of
a photon, h is known as the Planck’s constant and its value is equivalent to 6.62607 ×