ASSIGNMENT –II
TOPIC:
FACTORS AFFECTING FLUORESCENCE
SUBJECT: MODERN PHARMACEUTICAL ANALYTICAL TECHNIQUES
SUBMITTED BY,
RAKHY REGY
CLASS NO: 07
[Link] 1ST SEMESTER
NAZARETH COLLEGE OF PHARMACY
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FLUORESCENCE
Fluorescence and phosphorescence are photon emission
processes that occur during molecular relaxation from electronic
excited states.
• These photonic processes involve transitions between
electronic and vibrational states of polyatomic fluorescent
molecules (fluorophores). The Jablonski diagram (Figure 1)
offers a convenient representation of the excited state
structure and the relevant transitions.
• Each electronic state is split into multiple sublevels
representing the vibrational modes of the molecule.
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FACTORS AFFECTING THE FLUORESCENCE
Fluorescence is affected by number of factors. The factors are
broadly divided as those, which are related to intrinsic structure
of the molecule, called as intrinsic factors and the environmental
of the molecule called as extrinsic factors.
1. INTRINSIC FACTORS
1.1. Transition type in Fluorescence:
• Fluorescence produced mainly arises due to transition of
electrons from the first excited electronic state to one of the
ground vibrational states.
• The radiation is produced by a transition involving π-π* excited
state rather n- π* because for π-π* transition the molar
absorptivity is 100 - 1000 folds greater than for an n- π*
transition.
• The inherent lifetime associate with π-π* is shorter 10-7 to 10-9
sec as compared to the n-π* (10-5 to 10-7) and fluorescence is
larger because less time for deactivation is available.
1.2. Quantum efficiency or yield:
• Quantum yield is the ratio of molecule that fluoresces to the
total number of molecules that are excited.
• For highly fluorescent molecule the quantum efficiency
approach unity under the same condition e.g. fluorene.
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• Molecules which do not show fluorescence have quantum
efficiency that approach to zero. It is denoted by φ and is
determined by rate constant for the processes by which lowest
excited singlet state is deactivated.
• e.g. fluorescent (Kf), internal conversion (Kic), external
conversion (Kec), intersystem crossing (Kisc), predissociation
(Kpd) and dissociation (Kd)
Φ= Kf
Kf + Kic+ Kec + Kisc + Kpd + Kd
• The equation permits the quantitative interpretation of
structural and environmental factor which effect the
fluorescence e.g. Kf, Kpd, Kd is mainly dependent on the
structure of the molecule, whereas the other on the
environment.
1.3. Structure
• Aromatic compounds or compounds having aromatic
functional groups with low energy transition level (π-π*) show
the most intense and most useful fluorescent behavior.
• Aliphatic, alicyclic or highly conjugate systems may also show
fluorescence.
• The quantum yield/efficiency increases with increase in
number of rings and their degree of condensation.
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• Heterocyclic compounds have lowest energy electronic
transition level of n-π* that rapidly converts to the triplet state
and thus prevents fluorescence .
• e.g. pyridines, furans, thiophene etc. Enhance
phosphorescence.
• Fused benzene rings also show fluorescence.
• Fluorescence decreases with halogen substitution,
because with increase in atom number of halogens cause
(heavy atom effect which enhances inter system crossing).
• Substitution by carboxylic or carbonyl group also decreases
fluorescence due to n-π* transition.
• The nature of groups attached to the fluorophore also affects
the intensity of fluorescence, for example, electron donating
groups- fluorescence intensity increases. That is because
the substituents delocalize the pi-electrons and they tend to
increase the transition probability between the lowest excited
singlet state and the ground state: –OH, –NH2, –OEt, –OMe.
• Electron Withdrawing Groups- fluorescence intensity
Decreases –
–COOH, –COR, –CHO
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– COOR, –F, –Cl, –Br, –I, –SH,
• Groups having no effect on fluorescence –
–NH4+, –SO3H, –alkyl group.
1.4. Structure rigidity:
• Fluorescence from a molecule has been observed to increases
with increase in structure rigidity. The shape and wavelength
position of the fluorescence spectra and fluorescence
parameters of aromatic molecules are largely determined by the
molecular structure of the fluorescence species.
• For example, structure less absorption and fluorescence
spectra are seen with non-planar molecules where in case of
rigid molecules of the high-symmetry group show well resolved
absorption and fluorescence spectra with well-resolved
vibrational bands are observed.
Sometimes the absorption and fluorescence spectra of a planar
and rigid compound show a similar structural pattern and display
mirror symmetry.
• Transition from a nonplanar molecule to a similar but more
planar and rigid molecule is accompanied by an increase in
quantum yield of fluorescence.
For instance, biphenyl in solution is nonplanar and has a very
wide structureless absorption band and a structural
fluorescence spectrum.
They do not show any mirror symmetry between them, but
when phenyl rings of biphenyl are forced into a planar position
by bridging with the introduction of a methylene group
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(fluorene), both the absorption and fluorescence spectra
become very sharp.
They also display mirror similarity, and the quantum yield
values increase from 0.18 to 0.80.
• Complexation also results in increase in fluorescence.
e.g. 8-hydroxy quinolone has less fluorescence than its Zn
complex.
2. Extrinsic factors
2.1. The light source
• The fluorescence photons that pass through the emission
monochromator (EmM) are only a small fraction of the total
fluorescence intensity. It is necessary therefore to use a light
source that has a relatively high intensity light output.
• In very simple fluorescence spectrometers used for routine
dedicated analyses, a Mercury arc lamp may be satisfactory,
but it only produces light at a limited number of wavelengths in
the ultraviolet and visible spectral range (e.g. 253.7, 302.2,
313.2, 435.8, 546.1 nm).
• The continuum lamps used in absorption spectrometry,
deuterium and quartz/ iodine sources have too low an intensity
and hence the fluorescence intensity is also very low.
• Most fluorescence spectrometers use a high-pressure xenon
arc lamp as an excitation source, since it produces a useful
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high intensity continuum light spectrum from 200 nm to 1000
nm.
2.2. Concentration:
The power of fluorescence radiation (F) is proportional to the
radiant power of the excitation beam that is absorbed by the system.
F= Kc
The above equation shows F is directly related to
concentration .i.e. with increase in concentration of the
fluorescence species the fluorescence will increase linearly but
this holds true for dilute solutions only.
2.3 Temperature
Quantum efficiency decreases with increase in temperature, as
frequency of collision increase which increases probability for
deactivation by external conversion.
Decrease in viscosity of solvent also leads to external conversion
and it has same effect as with increase in temperature.
2.4 Solvent Effect
• Solvent effect A change of solvent is accompanied by a change
in polarity, dielectric constant and change in polarizability of
the surrounding medium.
• Thus,the change of solvent affects the ground state and the
excited state differently. The absorption as well as emission
spectra are affected by the properties of the solvent.
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• With the increasing solvent polarity, both absorption and
emission bands undergo a bathchromic shift, the latter being
more pronounced than the former.
• Polarity of the solvents causes increase in n-π* transition while
π-π* suffers opposite.
2.4. Effect of pH on fluorescence
• The fluorescence of the acidic and basic compound is usually
pH dependent. Both wavelength and intensity are different for
the ionized and unionized forms of the compound.
e.g aniline and anilinium ion
• The change in the emission results from the differencing
number of resonance species that are associated with the
acidic and basic forms of the molecule.
• The additional resonance forms lead to a more stable first
excited state fluorescence.
• Fluorescence of phenolic form of 1- napthol-4-sulphonic acid
is not detectable by eye but when it is converted to phenolate
ion by addition of base.
• The emission shifts to visible wavelength.
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2.5 Effect of dissolved oxygen:
• Dissolved oxygen causes decrease in emission intensity of
fluorescent solution.
• It may be due to photochemical oxidation of the fluorescence
species. The paramagnetic properties of molecular oxygen are
responsible for loss of fluorescence.
• It also promotes intersystem crossing and conversion of
excited singlet state molecule to the triplet state molecule.
REFERENCE:
1) Rouessac F, Annick Rouessac. Chemical Analysis. John Wiley
& Sons; 2013. Page no : 243-245.
2) Kemp W. Organic spectroscopy. Palgrave Macmillan;
[Link] edition, page no 271-274.
3) Albani, J., 2007. Principles and applications of fluorescence
spectroscopy. Oxford; Ames, Iowa: Blackwell Science.
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