This document discusses fluorescence spectroscopy. It begins by defining fluorescence as the emission of light by a substance when an electron returns to the ground state from an excited state. Factors that affect fluorescence include temperature, viscosity, oxygen concentration, pH, and molecular structure. Applications of fluorescence in pharmacy include determining inorganic substances, in nuclear research, as fluorescent indicators, in organic analysis, in liquid chromatography, and for determining vitamins B1 and B2. Instrumentation for fluorescence spectroscopy includes various light sources, filters, sample cells, and detectors such as photomultiplier tubes.

1. Presented By-
ROHIT
M.Pharmacy
(Pharmaceutics)

2. 
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INTRODUCTION
DEFINITION
THEORY
FACTORS AFFECTING FLOURESCENCE
APPLICATIONS IN PHARMACY
INSTRUMENTATION

3. 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

4. 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

5. 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 arephosphorescent
substances.

6.  A molecular electronic state in which all of the
electrons are paired are called singletstate.
In a singlet state molecules are diamagnetic.
Mostof the molecules in their ground state are paired.
When such a mole cule absorbs uv/visible radiation,
one or more of the paired electron raised to anexcited
singlet state /excited tripletstate.

7. tripletstate
spins unpaired
Ground
singlet
states
excited singlet
state
spin paired
no net mag.field net mag.field

8. Fluorescence
Phosphorescence
Radiation less
processes
Vibration
relaxation
Internal

9. LIGHT EMITING AT ONCE SOURCE STARTS & STOPS
WHEM SOURCE STOPS

10. JABLONSKI ENERGY
DIAGRAM

11. FLUORESCENCE & THEIR
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
no: of rings and degree of
condensation.

12. CONTD…
Simple heterocyclic do not
exhibit fluorescence.

13. Fusion of heterocyclic nucleus to
benzene ring increases fluorescence.

14. Fluorescence is favored in
molecules with structural
rigidity.
organic chelating agents complexed
with metal ion increases
fluorescence.(A chelating agent is a
substance whose molecules can form

15. Nature of molecule
Nature of substituent
Effect of concentration
Adsorption
 Light
Oxygen
pH
Temp . &viscosity
Intensity of incident light
Path length

16. 1.Temperature:it is inversely proportional to
luminescence.
Increasein temp. causes increase
in collision
of
molecules and finally increase in F & P.
Decrease in temp. Causes decrease
in collision of molecules and
finally decrease in F & P.
2.Viscosity : It is directly proportional
to luminescence.
Increase in viscosity causes
decrease in molecule collision
and finally increase in F & P.

17. 3. Oxygen: It decrease the fluorescence in 2
ways;
Oxidises the fluorescent substance to
non fluorescent substance.
Decreasefluorescence because of
paramagnetic property as it has
triplet ground state.
4. pH: its effects depends on chemical
structure of molecules. E.g-
Aniline in neutral & alkaline medium gives
visible flourescence & in acidic medium
gives fluorescence in uv region only.

18. 5. Rigidity in structure:
Fluorinehasrigid structureand it shows
more fluorescence.
Biphenylhas flexible structureand shows
less fluorescence.
6. Nature of group:
Electrondonatinggroups like amino,
hydroxyl etc causes increase in
fluorescence.
Electronwithdrawing groups like NO2,
COOH etc causes decrease in

19. Decrease in fluorescence intensity due to
specific effects of constituents of the
solution.
Due to concentration, pH, pressure of
chemical substances, temperature,
viscosity, etc.
Types of quenching
Self quenching
Chemical quenching
Static quenching
Collision quenching

20. Fluorescenc
e
Concentration of
fluorescing species
Deviations at higher concentrations can be
attributed to self- quenching or self-
absorption.
Fluorescenc
e
Concentration of
fluorescing species
Calibration curve
(Low con)
calibration curve
(High con)

21. 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 atpH
<5 &>13 it does not exhibitfluorescence.
 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 groundstate.

22. of riboflavin by
This occurs due to complexformation.
e.g- caffeine reduces flourescence
complex formation.
COLLISIONAL QUENCHING
It reduces fluorescence by collision. where no. of
collisions increased hence quenching takes place.

23. Types of flourescence
(when excited by
1. Chemiluminescence (when excited by chemicals)
2. Electrochemiluminescence
electrochemical reaction)
3. Photoluminescence (when excited by
electromagnetic radiation)
 Based on the wavelength of emitted radiation
1. Stroke’s flourescence (wavelength of the emitted
radiation is longer than the absorbedradiation)

24. Contd.
2. Anti-stroke’s Flourescence (wavelength of
emitted radiation is shorter than absorbedradiation)
3. Resonance Flourescence (wavelength of emitted
radiation = absorbed radiation)
 Based on Phenomena:
1. Sensitised flourescence (when elements like Th, Zn,
Cd or an alkali metal are added to mercury vapour
these elements are sensitised & thus gives
fluorescence.
2. Thermally assisted Flourescence (the excitation is
partly by electromagnetic radiation and partly by
thermal energy)

25. INSTRUMENTATION

26. SOURCE OF LIGHT
FILTERS AND MONOCHROMATORS
SAMPLE CELLS
DETECTORS

27. MERCURY ARCLAMP.
XENON ARCLAMP.
TUNGSTEN LAMP.
TUNABLE DYELASERS.

28. 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.

29.  Intense radiation by passage of current
through
an atmosphere of xenon.
Spectrumis continuous over the range between
over 250- 600nm,peak intensity about 470nm.

30. Intensity of the lamp is low.
If excitation is done in the
visible region this lamp is
used.
It does not offer UV radiation.

31. Pulsed nitrogen laser as
the primary source.
Radiation in the range
between 360 and 650 nm is
produced.

32. FILTERS
Primary filter-absorbs visible light & transmits uv light.
Secondary filter-absorbs uv radiations & transmits
visible light.
MONOCHROMATORS
Exitation monochromaters-isolates only the radiation
which is absorbed by the molecule.
Emission monochromaters-isolates only the radiation
emitted by the molecule.

33. The majority of fluorescence assays are
carried out in solution.
Cylindrical or rectangular cells fabricated of silica
or glass used.
Path length is usually 10mm or 1cm.
All the surfaces of the sample holder are
polished in fluorimetry.

34. PHOTOVOLTAIC CELL
PHOTO TUBE
PHOTOMULTIPLIER TUBES –
Best
and accurate.

35. SINGLE BEAM FLUORIMETER
DOUBLE BEAM FLUORIMETER
SPECTROFLUORIMETER(DOUBL
E BEAM)

36. Tungsten lamp as source of light.
The primary filter absorbs visible
radiation and transmits uv radiation.
Emitted radiation measured at
90oby secondary filter.
Secondary filter absorbs uv
radiation and transmits visible
radiation.

37. 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.

38. Power
supply
Source primary filter
Sample cell
Slit
secondary filter
Detector
Data processor

39.  The primary filter in double beam
fluorimeter is replaced by excitation
monochromaters.
 The secondary filter is replaced by
emission monochromaters.
 The incident beam is split into sample and
reference beam using a beam splitter.
 The detector is photomultiplier tube.

40. Power
supply
Source Excitation
monochromator
Emission
monochromator
Detector
Sample
cell
Data processor

42. 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.

43. 2]Nuclear research
Field determination of uranium salts.
3]Fluorescent indicators
Mainly used in acid-base titration.
e.g.:
eosin- colorless-green.
Fluorescein:colourless-green.
Quinine sulphate: blue-violet.
Acridine: green-violet

44. 5] organic analysis
Qualitative and quantitative analysis of
organic aromatic compounds present in
cigarette smoke, air pollutants, automobile
exhausts etc.
6] Liquid chromatography
Fluorescence is an imp method of
determining compounds as they appear at
the end of chromatogram or capillary
electrophoresis column.
7] determination of vitamin B1 &B2.