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 Fluorescence spectroscopy. : 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 flourescent substances Phosphorescence spectroscopy: 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. Substances showing phosphorescence are phosphorescent substances. Chemiluminescence (also chemoluminescence) is the emission of light (luminescence) as the result of a chemical reaction. There may also be limited emission of heat Fluorescence Phosphorescence Radiation less processes Vibration relaxation Internal conversion External conversion Intersystem crossing Jablonski diagram is a graphical representation of the various transitions(electronic states, vibrational levels) that can occur after a molecule has been excited photochemically. When a molecule is raised from its ground state to a higher state using light, photochemistry occurs. The molecule in the excited state has a shorter lifetime and significantly more energy than the ground state from which it was formed. As a result, molecules in the excited state are much more reactive. A photochemical or photophysical process deactivates an excited state. Therefore, the fate of the excited molecules is described by using the Jablonski diagram, which only focuses on the photophysical process occurring during the excitation and deactivation process. Radiative transitions involve the absorption of a photon, if the transition occurs to a higher energy level, or the emission of a photon, for a transition to a lower level. Nonradiative transitions arise through several different mechanisms, all differently labeled in the diagram. Relaxation of the excited state to its lowest vibrational level is called vibrational relaxation. This process involves the dissipation of energy from the molecule to its surroundings, and thus it cannot occur for isolated molecules. A second type of nonradiative transition is internal conversion (IC), which occurs when a vibrational state of an electronically excited state can couple to a vibrational state of a lower electronic state. A third type is intersystem crossing (ISC); this is a transition to a state with a different spin multiplicity. In molecules with large spin-orbit coupling, intersystem crossing is much more important than in molecules that exhibit only small spin-orbit coupling. ISC can

1. FLUOROMETRY.
Prepared by: Proff. Priyanka shah
M.PHARM (QA)
KPGU

2. INTRODUCTION:
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
Fluorescence spectroscopy. : 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 flourescent
substances
Phosphorescence spectroscopy: 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.
Substances showing phosphorescence are phosphorescent substances.
Chemiluminescence (also chemoluminescence) is the emission of light (luminescence) as the result of a chemical
reaction. There may also be limited emission of heat

3. Theory of Fluorescence and
Phosphorescence
• A molecular electronic state in which all of the electrons are paired are called singlet state.
• In a singlet state molecules are diamagnetic.
• Most of the molecules in their ground state are paired.
• When such a molecule absorbs uv/visible radiation, one or more of the paired electron
raised to an excited singlet state /excited triplet state.

4. From the excited singlet state one of the following
phenomenon occurs
 Fluorescence
 Phosphorescence
 Radiation less processes
Vibration relaxation
Internal conversion
External conversion
Intersystem crossing

6. Jablonski diagram
 Jablonski diagram is a graphical representation of the various transitions(electronic states,
vibrational levels) that can occur after a molecule has been excited photochemically.
 When a molecule is raised from its ground state to a higher state using light,
photochemistry occurs.
 The molecule in the excited state has a shorter lifetime and significantly more energy than
the ground state from which it was formed.
 As a result, molecules in the excited state are much more reactive.
 A photochemical or photophysical process deactivates an excited state.
 Therefore, the fate of the excited molecules is described by using the Jablonski diagram,
which only focuses on the photophysical process occurring during the excitation and
deactivation process.

7. Radiative transitions involve the absorption of a photon, if
the transition occurs to a higher energy level, or the
emission of a photon, for a transition to a lower level.
Nonradiative transitions arise through several different
mechanisms, all differently labeled in the diagram.
Relaxation of the excited state to its lowest vibrational
level is called vibrational relaxation. This process involves
the dissipation of energy from the molecule to its
surroundings, and thus it cannot occur for isolated
molecules. A second type of nonradiative transition
is internal conversion (IC), which occurs when a vibrational
state of an electronically excited state can couple to a
vibrational state of a lower electronic state.
A third type is intersystem crossing (ISC); this is a
transition to a state with a different spin multiplicity. In
molecules with large spin-orbit coupling, intersystem
crossing is much more important than in molecules that
exhibit only small spin-orbit coupling. ISC can be followed
by phosphorescence.

8. Instrumentation fluorimetry
Parts of Instrumentation
• SOURCE OF LIGHT
•FILTERS AND
MONOCHROMATORS
• SAMPLE CELLS
• DETECTORS

9. 1)SOURCE OF LIGHT:-
Mercury vapor lamp:
• Mercury vapour at high pressure give intense lines on continuous background above
350nm.
• low pressure mercury vapour gives an additional line at 254nm.
• it is used in filter fluorimeter.
Xenon arc lamp:
• It give more intense radiation than mercury vapour lamp.
• it is used in spectrofluorimeter.
Tungsten lamp:
• If excitation has to be done in visible region this can be used.
• It is used in low cost instruments.

10. 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
secondary filter
Primary filter:-absorbs visible radiation and transmit UV radiation.
Secondary filter:-absorbs UV radiation and transmit visible
radiation.

11. 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
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
1. Barrier layer cell/Photo voltaic cells
2. Photomultiplier cells

12. 1. Barrier layer /photovoltaic cell:
• It is employed in inexpensive instruments Filter Fluorimeter.
• It consists of a copper plate coated with a thin layer of cuprous oxide (Cu2O).
• A semi transparent film of silver is laid on this plate to provide good contact.
• When external light falls on the oxide layer, the electrons emitted from the oxide layer move into the copper plate.
Then oxide layer becomes positive and copper plate becomes negative.
2. Photomultiplier tubes:
• These are incorporated in expensive instruments like spectrofluorimeter.
• 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- 100Vhigher than the preceding one.
• 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.

13. Photomultiplier tubes

14. INSTRUMENTS:
The most common three types are:
1. Single beam (filter) fluorimeter
2. Double beam (filter )fluorimeter
3. Spectrofluorimeter (double beam) INSTRUMENTS

15. Single beam (filter) fluorimeter
• 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 (filter)
fluorimeter

16. Advantage:
• Simple in construction
• Easy to use.
• Economical
Disadvantages
• It is not possible to use reference solution & sample solution at a time.
• Rapid scanning to obtain Exitation & emission spectrum of the compound is not possible.
Double beam instrument:
• 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.

18. Application:
1. Determination of inorganic substances
• Determination of ruthenium ions in presence of other platinum metals.
• Determination of aluminum (III) in alloys.
• Determination of boron in steel by complex formed with benzoin.
• Estimation of cadmium with 2-(2 hydroxyphenyl) benzoxazole in presence of tartarate.
2. Neuclear research: Field determination of uranium salts.
3. Flurometric reagents:

19. FACTORS AFFECTING FLUORIMETRY
• 1. Nature of molecules.
• 2. Effect of substituent.
• 3. Effect of concentration.
• 4. Adsorption, Light.
• 5. Photodecomposition.
• 6. Oxygen, PH.
• 7. Temperature and viscosity.
• 8. Quantum yield of fluorescence.
• 9. Intensity of incident light.
• 10.Path length

20. NATURE OF MOLECULES :
Only the molecules absorbs UV/Visible radiation can show the fluorescence.
• Greater the absorbency of the molecules more intense its fluorescence
• Unsaturated molecules with 𝜋 bonds and good resonance stability can exhibit fluorescence.
Eg : Alkenes with conjugate double bond.
• Saturated molecules with sigma bond do not exhibit fluorescence.
Eg : Aliphatic unsaturated cyclic organic compounds
NATURE OF SUBSTITUENTS
•Electron donating groups enhances fluorescence. Eg: NH2 , OH will increase degree of fluorescence.
• While electron withdrawing groups like halogens COOH, NO2 etc diminishes the fluorescence.
• Thus it may noted that cyclohexane is non fluorescent , benzene shows weak fluorescence ,
while compounds like Anthracene, Riboflavin are strongly fluorescent.

21. FLUORESCENCE AND CONCENTRATION. •
Beers law states that in a solution of an absorbing substance the absorbance is directly proportional to the
concentration.
• Thus the fluorescence intensity will be proportional to the concentration of molecules in the excited state and
therefore the intensity of radiation.
• The light absorbed by the sample and intensity of the exciting light does not remain constant but decreases as it
travels through the sample
Thus the fluorescence intensity will be proportional to the amount of light absorbed which can be expressed as
fluorescence intensity = 𝑄Ia
𝑄is fluorescence intensity
Fluorescence efficiency = Fluorescence quanta emitted /EMR quanta absorbed
Ia is intensity of light absorbed
Since emission is proportional to absorption,
Ia = Io-It
Io is intensity of incident light
It is intensity of transmitted light •
According to beer lamberts law It= Io -act
• Substituting it in above equation
Ia = Io- Ioe –act
Ia =Io(I- e-act)
Ia =Io(1-(1-act)

22. Ia = Io(1-1+act)
Ia=Io*act
Fluorescence intensity 𝑄 *Ia =Q𝐼𝑜𝑎𝑐𝑡
ie, F=Q𝐼𝑜𝑎𝑐𝑡
• Q = constant of particular system
• Io constant for an instrument
• a= molecular extinction co-efficient which is constant for a substance.
• t= path length
• C = concentration of substance
F= fluorescence
Fluorescence intensity is directly proportional to concentration.
• But in high concentration it does not obey linearity.
Calibration curve

23. ADSORPTION :
• The extreme sensitivity of the methods requires very dilute solutions.
• Adsorption of fluorescent substance on the container will may therefore present a serious problem
• Stock solution must be kept diluted as required.
• Eg: Quinine is a example of a substance which is adsorbed into the cell wall
LIGHT :
Monochromatic light is essential for the excitation of fluorescence because the intensity will vary with wave length.
PHOTODECOMPOSITION:
• Excitation of a weakly fluorescing or dilute solution with intense light sources will cause photochemical
decomposition of analyte.
• To minimize decomposition
• Use of longest feasible wavelength for excitation.
• Remove dissolved oxygen.
• Protect the unstable solutions from ambient light by storing them in dark bottle

24. OXYGEN
The presence of oxygen may interfere in 2 ways
• 1. By direct oxidation of the fluorescent substance to non florescent substance.
• 2. By quenching of fluorescence.
• The paramagnetic substance like dissolved oxygen and many transition metals with unpaired electron will dramatically decrease
fluorescence.
• Anthracene is well known to be susceptible to the presence of oxygen
PH: Depend on chemical structure of molecule
TEMPERATURE AND VISCOSITY
Temperature: ↑ ↑collision ↓ fluorescence
Viscosity ↑ ↓ collision ↑fluorescence
QUANTUM YEILD OF FLUORESCENCE
• Quantum yield of fluorescence = Number of photons emitted /Number of photons absorbed
• Since the absorbed energy is lost by pathways, the quantum efficiency is less than 1. • Highly fluorescent
substance have ∅ value near 1 which shows that most of the absorbed energy is re- emitted as fluorescenc

25. QUANTUM YEILD OF FLUORESCENCE
• Quantum yield of fluorescence(ΦF) = Number of photons
emitted /Number of photons absorbed
• Since the absorbed energy is lost by pathways, the quantum
efficiency is less than 1.
• Highly fluorescent substance have ∅ value near 1 which shows
that most of the absorbed energy is re- emitted as fluorescence.
Fluorescence quantum yield standards
Compound Solvent λex(nm) Φ
Quinine 0.1 M HClO4 347.5 0.60 ± 0.02
Fluorescein 0.1 M NaOH 496 0.95 ± 0.03
Tryptophan Water 280 0.13 ± 0.01
Rhodamine 6G Ethanol 488 0.94

26. Quenching of fluorescence
• Quenching refers to any process that reduces the fluorescence intensity of a given substance.
This may occur due to various factors like
• pH
• Concentration
• Temperature
• Viscosity
• Presence of oxygen,
• Heavy metals or,
• specific chemical substances etc.
Fig: Quenching of quinine fluorescence in presence of chloride ion

27. Example of quenching agents

28. Types of quenching process:
Quenching
Chemical quenching
Collisional quenching
Concentration
quenching
Static quenching

29. Collisional quenching
• Collisional quenching occurs by the interaction of a quencher molecule (Q) with an
excited molecule of the fluorescing substance (F*).
• A simplified mechanism can be written to describe this process
Here, the interaction results in the dissipation of excitation energy by a non radiative energy
transfer from F* to Q without or, less fluorescence.

30. Simple mechanism of collisional quenching
• Halides ions such as chlorides or, iodides are well known collisional quenchers.
• For example, quenching of quinine drug by chloride ion or, quenching of tryptophan by
iodide ion follow collisional quenching process.
• Weak coupling Light Energy transfer Quenching of light F* Q Distance

31. Static quenching
• Static quenching occurs at the ground state of fluorescing molecule. It
can be simplified by following mechanism.
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.

32. Static quenching
Caffeine and related xanthines and purines reduce intensity of riboflavin by static mechanism.
Quenching that occurs due to oxygen also follows this mechanism.

33. Concentration 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
highly concentrated solution
( >50 μg/ml ).

34. Chemical quenching
• Chemical quenching is due to various factors like change in
• .pH,
• presence of oxygen,
• halides and
• electron withdrawing groups, heavy metals etc.
• Change in pH : Aniline at pH (5-13) gives fluorescence when excited at 290 nm.
But pH <5 or, pH >13 it does not show any fluorescence.
• Oxygen : Oxygen leads to the oxidation of fluorescent substance to non
fluorescent substance and thus, causes quenching

35. Halides and electron withdrawing groups : Halides like chloride ions, iodide ions and electron
withdrawing groups like -NO , -COOH , -CHO groups lead to quenching.
Heavy metals : presence of heavy metals also lead to quenching because of collision
and complex formation.
Conclusion :
In the usual case, quenching is an undesirable effect and the possibility of
encountering this type of interference should always be evaluated in developing a
fluorometric assay.
However, this phenomenon can be used as an analytical means for determining the
concentration of the compounds known to quench fluorescence.
Quenching study can also be used to reveal the localization of fluorophores in
proteins or, membranes and their permeability to the quenchers.