FLOURIMETRY

Dr. Shital Patel

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Introduction
 A large number of substance absorb ultraviolet and visible light energy. But, there are
some substances which lose excess energy as heat through collisions with neighboring
atoms or molecules. But fluorophores lose excess energy in the form of photons.
 The energy emitted by these substances has a wavelength larger than that of
absorbed. This process of emitting radiation with larger wavelength than that of
absorbed is known as luminescence.
 Fluorescence spectroscopy provides two types of spectrum (1) Excitation or absorption
spectrum (2) Emission spectrum

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Luminescence
 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 3 types.
• Fluorescence spectroscopy.
• Phosphorescence spectroscopy.
• Chemiluminescence spectroscopy

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 Fluorescence:
• When a beam of light is incident on certain substances they emit visible
light or radiations. This is known as fluorescence.
• Fluorescence starts immediately after the absorption of light and stops as
soon as the incident light is cut off. The substances showing this
phenomenon are known as fluorescent substances.
• Time: less than 10-4 s
 Phosphorescence:
• When 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.
• Time: more than 10-4 s
• Substances showing phosphorescence are phosphorescent substances.
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• At the ground state, the molecular orbitals are occupied by two
electrons. The spins of the two electrons in the same orbital must be
antiparallel. This implies that the total spin, S, of the molecule in the
ground state is zero.
• This energy state is called “singlet state” and is labeled as S0.
• The electron spins in the excited state achieved by absorption of
radiation may either be parallel or antiparallel. Accordingly, this may
be a triplet (parallel) or a singlet (antiparallel) state.

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From exited singlet one of the
following phenomenon occurs
• Fluorescence
• Phosphorescence
• Radiation less processes
• Vibration relaxation
• Internal conversion
• External conversion
• Intersystem crossing

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Fluorimetry

• Fluorimetry is an analytical method for the measurement of

fluorescence, based upon emission of absorbed radiation by the
molecules. When a molecule absorbs incident electromagnetic
radiations, it is excited to higher energy level, where it is
unstable.
• Therefore, it returns to the ground state by emitting the
absorbed radiations. When the emission occurs directly from
singlet excited state to singlet ground state without any
transitions and without any change in spin orientation, it is
referred as fluorescence.

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 And when the emission occurs within a fraction of second to
few days of absorption of radiations due to transition from
singlet excited state to triplet state and then to singlet ground
state, it is referred as phosphorescence. It involves the change
in spin orientation during the transition from excited singlet to
triplet and from triplet to singlet ground state.
 The movement of electrons from excited singlet state to triplet
state, ie from unpaired electrons with opposite spin to
unpaired electrons with same spin is termed as inter system
crossing.
 Fluorescence and phosphorescence are combinedly called
photoluminescence.
 The energy emitted by these mechanism has a wavelength
larger than that of absorbed. 10
Fluorescence and Chemical
Structure
 Fluorescence is most commonly observed in compounds containing
aromatic functional groups with low energy.
 Most unsubstituted aromatic hydrocarbons show fluorescence -
quantum efficiency increases with the number of of rings and
degree of condensation.
 The presence of the benzene ring and the nature of substituents on
it seem to favor the fluorescent behaviour of the molecule.
 The halogen substituents tend to decrease the fluorescence and
shift the fluorescence bands to longer wavelengths; the effects
increase with increase in the atomic mass of the substituted
halogen.
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Factors affecting flourescence

• Concentration
• Temperature& viscosity
• pH
• Oxygen
• Quantum yield of fluorescence
• Intensity of incident light
• Adsorption
• Photodecomposition
• Quenchers
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Concentration

 Fluorescence intensity is proportional to concentration of substance only when the

absorbance is less than 0.02
 A=log Io/It or A= abc
 Io=intensity of incident light
 a= absorptivity of constant
 b= Pathlength
 c= concentration

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Temperature

• A rise in temperature is almost always accompanied by a

decrease in fluorescence.
• The change in temperature causes the viscosity of the medium
to change which in turn changes the number of collisions of the
molecules of the fluorophore with solvent molecules.
• The increase in the number of collisions between molecules in
turn increases the probability for deactivation by internal
conversion and vibrational relaxation.

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pH
• Relatively small changes in pH can sometimes cause substantial
changes in the fluorescence intensity and spectral characteristics of
fluorescence.
• For example, serotonin shows a shift in fluorescence emission
maximum from 330 nm at neutral pH to 550 nm in strong acid without
any change in the absorption spectrum.
• In the molecules containing acidic or basic functional groups, the
changes in pH of the medium change the degree of ionisation of the
functional groups. This in turn may affect the extent of conjugation or
the aromaticity of the molecule which affects its fluorescence.
• For example, aniline shows fluorescence while in acid solution it does
not show fluorescence due to the formation of anilinium ion.
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• Therefore, pH control is essential while working with such molecules
and suitable buffers should be employed for the purpose.
Dissolved Oxygen

 •The paramagnetic substances like dissolved oxygen and

many transition metals with unpaired electrons dramatically
decrease fluorescence and cause interference in fluorimetric
determinations.
 •The paramagnetic nature of molecular oxygen promotes
intersystem crossing from singlet to triplet states in other
molecules.
 •The longer lifetimes of the triplet states increases the
opportunity for radiationless deactivation to occur.

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 •Presence of dissolved oxygen influences phosphorescence too
and causes a large decrease in the phosphorescence intensity.
 •It is due to the fact that oxygen which is in triplet state at the
ground state gets the energy from an electron in the triplet
state and gets excited.
 •This is actually the oxygen emission and not the
phosphorescence. Therefore, it is advisable to make
phosphorescence measurement in the absence of dissolved
oxygen.

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• Quantum yield
 ø=Number of photons emitted/number of photons absorbed
 It is always less than 1.0 since some energy is lost by radiation less pathways
(Collisional, Intersystem Crossing, Vibrational Relaxation)
• Nature of molecule:
All the molecules cannot show the phenomenon of fluorescence. Only the molecules
absorb uv/visible radiation can show this phenomenon. Greater the absorbency of the
molecule the more intense its fluorescence.
• Nature of substituent:
Electron donating group enhances fluorescence. e.g.: NH2, OH etc.
Electron withdrawing groups decrease or destroy fluorescence. e.g.: COOH, NO2, N=N
etc.
High atomic no. atom introduced into  electron system decreases fluorescence.
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• Intensity of incident light
Increase in the intensity of incident light on the sample
fluorescence intensity also increases.
• Adsorption
 Adsorption of sample solution in the container may leads to A
serious problem
• Photochemical decomposition:-
Absorption of intense radiation leads to photochemical
decomposition of a fluorescent substance to less fluorescent or
non fluorescent substance.
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Quenchers:-

• Quenching is the reduction of fluorescence intensity by the presence

of substance in the sample other than the fluorescent analyte.
• Decrease in fluorescence intensity due to specific effects of
constituents of the solution.
• Due to concentration, pH, pressure of chemical substances,
temperature, viscosity, etc.
• Quenching is following types:-
 Self quenching
 Chemical quenching
 Static quenching
 Collision quenching 23
 Self quenching:
 Deviations at higher concentrations can be attributed to self-
quenching or self-absorption.

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 Chemical quenching:
• Here decrease in fluorescence intensity due to the factors like
change in pH, presence of oxygen, halides &heavy metals.
• pH- aniline at pH 5-13 gives fluorescence but at pH <5 &>13 it
does not exhibit fluorescence.
• Halides like chloride, bromide, iodide & electron withdrawing
groups like NO2,COOH etc. leads to quenching.
• Heavy metals leads to quenching, because of collisions of
triplet ground state.

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 Static quenching:
 This occurs due to complex formation. e.g. caffeine reduces
the fluorescence of riboflavin by complex formation.
 Collisional quenching
 It reduces fluorescence by collision. Where no. of collisions
increased hence quenching takes place.

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Instrumentation

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Sources of light

• Mercury Arc Lamp.

• Xenon Arc Lamp.
• Tungsten Lamp.
• Tunable Dye Lasers.
 Mercury Arc Lamp:
• Produce intense line spectrum above 350nm.
• High pressure lamps give lines at 366,405, 436,
546,577,691,734nm.
• Low pressure lamps give additional radiation at 254nm.
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 Xenon arc lamp:
• Intense radiation by passage of current through an atmosphere
of xenon.
• Spectrum is continuous over the range between over 250-
600nm,peak intensity about 470nm.
 Tungsten lamp:
• Intensity of the lamp is low.
• If excitation is done in the visible region this lamp is used.
• It does not offer UV radiation
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 Tunable Dye Lasers:
 Pulsed nitrogen laser as the primary source.
 Radiation in the range between 360 and 650 nm is produced.

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Filters and monochromators:

 Filters:
 These are nothing but optical filters works on the principle of
absorption of unwanted light and transmitting the required
wavelength of light.
 In inexpensive fluorimeter primary filter and secondary filter
are present.
 Primary filter:-absorbs visible radiation and transmit UV
radiation.
 Secondary filter:-absorbs UV radiation and transmit visible
radiation.
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 Monochromators:
 they convert polychromatic light into monochromatic light.
They can isolate a specific range of wavelength or a particular
wavelength of radiation from a source.
 Excitation monochromators:-provides suitable radiation for
excitation of molecule.
 Emission monochromators:-isolate only the radiation emitted
by the fluorescent molecules.

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Sample cells:

• These are meant for holding liquid samples. These are made up
of quartz and can have various shapes ex: cylindrical or
rectangular etc.
• Detectors:
Photometric detectors are used they are
• Barrier layer cell/Photo voltaic cells
• Photomultiplier cells

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Single Beam Fluorimeter

• Tungsten lamp as source of light.

• The primary filter absorbs visible radiation and transmits UV
radiation.
• Emitted radiation measured at 90o by secondary filter.
• Secondary filter absorbs UV radiation and transmits visible
radiation.
• Advantage:
 Simple in construction
 Easy to use.
 Economical disadvantages
• Disadvantage:
 It is not possible to use reference solution & sample
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solution at
a time.
Double Beam Fluorimeter

 Similar to single beam instrument.

 Two incident beams from light source pass through primary
filters separately and fall on either sample or reference
solution.
 The emitted radiation from sample or reference pass
separately through secondary filter.
 Advantage: Sample & reference solution can be analyzed
simultaneously.
 Disadvantage: Rapid scanning is not possible due to use of
filters.
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Spectrofluorometer

• In this primary filter in double beam fluorimeter is replaced by

excitation monochromator and the secondary filter is replaced
by emission monochromator.
• Incident beam is split into sample and reference beam by
using beam splitter.

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Application:

•Determination of inorganic ions. These ions form fluorescent

chelates with non-fluorescent organic molecules.
 •Determination of ruthenium ion in the presence of other
platinum metals
 • Determination of aluminium (III) in alloys
 • Estimation of traces of boron in steel
 • Estimation of cadmium
 • Determination of calcium

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• Determination of uranium in salts used extensively in the field
of nuclear research.
• Estimation of traces of boron in steel by means of the complex
formed with benzene.
• Estimation of calcium by fluorimetry with a calcium solution.
• Determination of Vitamin B (B1 thiamine and B2 riboflavin) in
the food samples like meat, cereals, etc.

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 Pharmaceuticals applications
 •Compounds which are fluorescent
 •Compounds readily converted to fluorescent products by
chemical reactions
 Liquid chromatography: Fluorescence is an important method
of determining compounds as they appear at the end of
chromatogram or capillary electrophoresis column.
 Fluorescent indicators:
 Eosin, Fluorescein, Quinine sulphate, Acridine, 2-
napthaquinone, 2-hydroxy cinnamic acid
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 Determination of organic substances
 •Fluorimetry has been used to carry out qualitative as well as
quantitative analysis for a great many aromatic compounds
present in a cigarette smoke, air-pollutants, concentrates and
automobile exhausts.eg. Determination of benzopyrene in the
nanogram range
 •Aromatic polycyclic hydrocarbons, indoles, napthols, proteins,
plant pigments, steroids etc. can be determined at low conc.
by fluorimetry.

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