2. Content :
Introduction
Theory
Concepts of singlet, doublet and triplet electronic states
Factors affecting fluorescence
Quenching
Instrumentation and Applications
3. • Absorption of UV Visible causes transitions of electron from singlet ground state to
singlet excited state.
• As this state is not stable , it emits the energy in the form of UV-Visible radiation and
returns to singlet ground state.
• This study or measurement of this emitted radiation (when electron undergoes
transition from singlet excited to singlet ground state) is the principle of fluorimetry.
• Phosphorence is also a related phenomenon , which is the study of emitted radiation
when electron undergoes transition from triplet state to singlet ground state
Introduction:-
4. • A large number of substances are known which absorb UV or Visible light energy .
• But most of these substances loose excess of energy as heat through collisions with
neighboring atoms or molecules(collosional deactivation).
• Some of the substances loose only part of this excess energy as heat and emit remaining
energy as electromagnetic radiation(light). This process of emitting radiation is
collectively called luminescence.
Introduction:-
6. Fluorescence :
• When a beam of light is incident on certain substances they emit
visible light or radiations.
• This phenomenon is called fluorescence and the substances
showing this phenomenon are known as fluorescent substances.
• The phenomenon of fluorescence is instantneous starts
immediately after absorption of light and stops as soon as
incident light is cut off.
• Materials exhibiting fluorescence generally re-emit excess
radiation within 10-6
to 10-4
sec.
7. 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.
• Materials exhibiting phosphorescence generally reemit excess radiation within 10-4
to 20sec or longer.
8. Chemiluminescence:-
• Chemiluminescence occurs when a chemical reaction produces an
electronically excited species which emits a photon in order to reach the
ground state.
• These sorts of reactions can be encountered in biological systems; the effect
is then known as bioluminescence.
• The number of chemical reactions which produce chemiluminescence is
small.
9. Theory of fluorescence
• Fluorescence: It is a phenomenon of emission of radiation when the molecules are excited by
radiation at certain wavelength.
• Fluorimetry: It is measurement of fluorescence intensity at a particular wavelength with the
help of a filter fluorimeter or a spectrofluorimeter.
• Molecule contains σ electrons, π electrons and nonbonding (n) electron.
• The electrons may be present in bonding molecular orbital. It is called as highest occupied
molecular orbital (HOMO). It has least energy and more stable.
• When the molecules absorb radiant energy from a light source, the bonding electrons may be
promoted to anti bonding molecular orbital (LUMO).
• It has more energy and hence less stable. The process of promotion of electrons from
10. • Singlet state: a state in which all the electrons in a molecule are paired.
• Doublet state: a state in which un paired electrons are present. Eg : Free radical or
• Singlet excited state: a state in which electrons are unpaired but of opposite spin like (un
paired and opposite spin).
• Triplet excited state: a state in which unpaired electrons of same spin present. or
• When light of appropriate wavelength is absorbed by a molecule the electrons are promoted
from singlet ground state to singlet excited state.
• Once the molecule is in this excited state, relaxation can occur via several processes by
emission of radiation. The processes can be the following 1)Collisional deactivation 2)
Fluorescence 3) Phosphorescence
12. • Collisional deactivation: In which entire energy lost due to collision de activation
and no radiation emitted and energy is lost in the form of heat.
• Fluorescence: excited singlet state is highly unstable. Relaxation of electrons from
excited singlet excited to singlet ground state with emission of light.
13. • Phosphorescence: At favourable condition like low temperature and absence of oxygen there is
transition from excited singlet state to triplet state which is called as inter system crossing. The
emission of radiation when electrons undergo transition from triplet state to singlet ground state is
called as phosphorescence.
• Internal conversion: Intermolecular process by which a molecule passes to a lower energy
electronic state without emission of light. It involves vibrational relaxation in singlet excited state ,
singlet excited state to triplet excited state and vibrational relaxation in triplet state.
14. • External conversion: It is process by which a molecule brings down its energy
electronic state by emission of light. It involves singlet excited state to singlet ground
state and triplet excited state to a singlet ground state with emission of light
• Intersystem crossing: Process in which spin of an excited electron is reversed and
change in multiplicity results. Most common when vibrational manifold overlap exists
and when the molecule has a heavy atom substituent.
16. 1. Concentration: Fluorescence intensity is proportional to concentration of substance only
when the absorbance is less than 0.02.
• i.e. f = Q Io act
In the above equation,
Q= constant for a particular substance
Io=constant for a instrument
a= molecular extinction coefficient, which is constant for a substance
t= path length (constant for a sample cell)
c= concentration of substance
This is true in low concentrations (μg or ng/ml), but in high concentration(mg/ml) it does not
obey the linearity.
17. 2. Conjugation :
• A molecule must have unsaturation (π electrons i.e conjugation) so that UV Visible
radiation can be absorbed .
• If there is no absorption then there is no fluorescence intensity.
3. Intensity of incident light: Increase in the intensity of incident light on the sample
fluorescence intensity also increases.
4. Oxygen: Oxidation of fluorescent species to a non-fluorescent species, quenches
fluorescent substance.
19. 6. pH: The effect of pH depends on chemical structure of the molecule. Alteration of pH of a
solution will have significant effect on fluorescence.
• Eg : Aniline in neutral or alkaline medium gives visible fluorescence but in acidic conditions gives
fluorescence in UV region.
7. Temperature and viscosity:
Temperature increases can increase the collisional de activation, and reduce fluorescent
intensity.
If viscosity of solution is more the frequency of collisions are reduced and increase in fluorescent
intensity.
8. Photochemical decomposition: Absorption of intense radiation leads to photochemical
decomposition of a fluorescent substance to less fluorescent or non-fluorescent substance.
20. 9. Quenchers:
• Quenching is the reduction of fluorescence intensity by the presence of
substance in the sample other than the fluorescent analyte.
1. Collisional quenching
2. Static quenching
3. Self quenching
4. Chemical quenching
21. • Quenching is following types:
Collisional quenching:
• Collisional quenching occurs when the excited fluorophore experiences contact with an
atom or molecule that can facilitate non-radiative transitions to the ground state.
• Collisions between the fluorescent substance and halide ions leads to reduction in
fluorescence intensity.
• Common quenchers include O2, I-
, Cs+, and acrylamide.
• For example, quenching of quinine drug by chloride ion and quenching of tryptophan by
iodide ion.
22. Static quenching:
• This occurs because of complex formation between the fluorescent molecule and other molecules..
• Here, a complex formation occurs between the fluorescing molecule at the ground state (F) and the quencher
molecule (Q) through a strong coupling.
• Such complex may not undergo excitation or, may be excited to a little extent reducing the fluorescence
intensity of the molecule.
• For example, Caffeine and related xanthines and purines reduce the intensity of riboflavin by the static
mechanism.
23. • Inner fluorescent effect:
• Absorption of Incident (UV) light or emitted (fluorescent) light by primary and secondary filters
leads to decrease in fluorescence intensity.
Self-quenching:
• Concentration quenching is a kind of self-quenching. It occurs when the concentration of the
fluorescing molecule increases in a sample solution. The fluorescence intensity is reduced in a
highly concentrated solution (>50 μg/ml).
Chemical quenching :
• Chemical quenching is due to various factors like change in pH, presence of oxygen, halides, and
electron-withdrawing groups, heavy metals, etc
• Quinine is highly fluorescent in 0.05M H2SO4, but non-fluorescent in 0.1M HCl due to Collisional quenching
by halide ion.
24. • Change in pH :
• Aniline at pH (5-13) gives fluorescence when excited at 290 nm.
• But pH 13 does not show any fluorescence.
• Heavy metals :
• The presence of heavy metals also lead to quenching because of collision and complex formation.
10. Scatter :
• Scatter is mainly due to colloidal particle in solution.
• Scattering of incident light after passing through the sample lead to decrease in fluorescence intensity.
27. Source of light should emit a radiation over a continuous region and be of
adequate stability and intensity.
Factors for selecting light source:
– Lamp intensity
– Wavelength distribution of emitted light
– Stability
Four types:
1.High pressure Xenon arc lamp
2.Xenon flash lamp
3.Mercury vapor lamp
4.Tungsten lamp
SOURCE OF LIGHT:-
28. SOURCE OF LIGHT:-
Mercury vapour lamp: Mercury vapor at high pressure gives intense lines on a continuous background
above 350nm.
Lines are seen at 365, 398,436,546,579,690 and 734nm.
Low pressure mercury vapors give additional lines at 254nm.
It is used as source in filter type of fluorimeter.
29. B} xenon arc lamp:
• It give more intense radiation than mercury vapour lamp.
• It is used in spectrofluorimeter.
• Spectrum is continuous over the range between 250- 600nm,
C} Tungsten lamp:
• If excitation has to be done in visible region this can be used.
• It is used in low cost instruments
• It does not offer UV radiation and Intensity of lamp is low.
• It offers radiation in the range between 350 to 650nm.
30. 2) 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 instruments fluorimeter primary filter
and secondary filter are present.
• Primary filter/ Excitation filter : Isolates the
wavelength that will cause the compounds to
fluorescence. Absorbs visible radiation and transmit
UV radiation.
• Secondary filter / Emission filter : Isolates the
desired emitted wavelength . Absorbs UV radiation
and transmit visible radiation.
31. Monochromators
• A monochromator is an optical device that transmits a mechanically
selectable narrow band of wavelength of light or other radiation
chosen from wide range of wavelengths .
• They convert polychromatic light into monochromatic light.
• They can isolate a specific range of wavelength or a particular
wavelength of radiation from a source.
32. • These are meant for holding liquid samples.
• The sample cells are cylindrical or polyhedral like those used in
Colorimetry .
• The sample cells made up of color corrected fused glass and path length is
normally 10mm or 1cm.
• It need not be made up of quartz, since it required measuring only the
emitted radiation and not the absorbed radiation.
• Hence if there is absorption of radiation by glass, it will not affect the
results
• ex: cylindrical or rectangular etc.
Sample cells:
33. • The emitted radiation is mostly visible radiation and some times UV radiation.
• To measure the intensity of such radiation photo voltaic cell or photo multiplier tubes can
be used.
• Measuring low concentration of substance and the intensity of emitted radiation is weak, so
photo multiplier tubes are best and accurate.
• In inexpensive instruments like photofluorimeters, phototubes can be used.
Photometric detectors are used ,they are
Barrier layer cell/Photo voltaic cells
Photomultiplier cells (Best and accurate)
Detectors:
34. Barrier layer /photovoltaic cell:
• It is employed in inexpensive instruments. For ex: Filter Fluorimeter.
• It consists of a Iron plate coated with a thin layer of Selenium
• A semi transparent film of silver is laid on this plate to provide good contact.
• When external light falls on the silver layer, the electrons emitted from the selenium
layer move into the silver selenium surface.
35. • Then silver layer becomes positive and Iron plate becomes negative.
• Hence an electromotive force(emf) develops between the silver layer and Iron plate and behaves
like a voltaic cell. So it is called photovoltaic cell.
• A galvanometer is connected externally between silver film and Iron plate and the deflection in
the galvanometer shows the current flow through it. The amount of current is found to be
proportional to the intensity of incident light.
36. 2. Photomultiplier tubes:
• These are incorporated in expensive instruments like spectro-fluorimeter.
• Its sensitivity is high due to measuring weak intensity of light.
• The principle employed in this detector is that, multiplication of photoelectrons by secondary
emission of electrons.
• This is achieved by using a photo cathode and a series of anodes (Dyanodes). Up to 10 dyanodes
are used.
• Each dyanode is maintained at 75- 100V higher than the preceding one.
37. • At each stage, the electron emission is multiplied by a factor of 4 to 5 due to secondary
emission of electrons and hence an overall factor of 106 is achieved.
• PMT can detect very weak signals, even 200 times weaker than that could be done using
photovoltaic cell. Hence it is useful in fluorescence measurements.
• PMT should be shielded from stray light in order to have accurate results. .
39. • It contains tungsten lamp as a source of light and has an optical system consists of primary filter.
• The emitted radiations is measured at 90* by using a secondary filter and detector.
• Primary filter absorbs visible radiation and transmit UV radiation which excites the molecule present
in sample cell.
• In stead of 90 if we use 180 geometry as in colorimetry secondary filter has to be highly efficient
other wise both the unabsorbed UV radiation and fluorescent radiation will produce detector
response and give false result.
• Single beam instruments are simple in construction cheaper and easy to operate.
40. DOUBLE BEAM FLUORIMETER
• It similar to single beam except that the two incident beams from a single light source
pass through primary filters separately and fall on the another reference solution.
• Then the emitted radiations from the sample or reference sample pass separately through
secondary filter and produce response comb inly on a detector.
41. In Spectro fluorimeter:-
• The primary filter in double beam fluorimeter is replaced by excitation monochromater.
• The secondary filter is replaced by emission monochromater.
• The incident beam is split into sample and reference beam using a beam splitter.
• The detector is photomultiplier tube.
42. APPLICATIONS :
1. Determination of inorganic substances. Al3+,Li+,ZN2+.
2. Determination of thiamine Hcl.
3. Determination of indoles, phenols, & phenothiazines, phenytoin.
4. Determination of napthols, proteins, plant pigments and steroids.
5. Fluorimetry ,nowadays can be used in detection of impurities in nanogram level better than
absorbance spectrophotometer with special emphasis in determining components of sample at the
end of chromatographic or capillary column.
6. Determination of ruthenium ions in presence of other platinum metals.
7. Estimation of cadmium with 2-(2 hydroxyphenyl) benzoxazole in presence of tartarate.
