This document discusses fluorimetry and phosphorimetry. It defines them as measurement techniques, with fluorimetry measuring fluorescence intensity at a particular wavelength, and phosphorimetry measuring phosphorescence in conjunction with pulsed radiation. It describes the principles behind photoluminescence, including fluorescence and phosphorescence. Factors affecting these processes and instrumentation used are summarized, including light sources, filters, monochromators, and detectors. Applications in pharmaceutical, clinical, environmental, and entertainment fields are also briefly outlined.

1. FLUORIMETRY
and
Phosphorimetry
g. Enosh
2016672003

2. FLUORIMETRY
It is measurement of fluorescence
intensity at a particular wavelength with
the help of a filter fluorimeter or a
spectrofluorimeter.

3. Phosphorimetry
A form of fluorimetry in which
phosphorescence of a sample is measured
in a conjunction with a pulsed sourse of
radiation.

4. Photoluminesence
“Photoluminescence (PL) is the spontaneous
emission of light from a material under optical
excitation.”
It is futher subdivided into two types
a. Flourescence
b. Phosphorescence

5. Fluorescence
When a beam of light is incident on certain substances they
emit visible light or radiation .This phenomenon is called
fluorescence.
Substances showing this phenomenon are fluorescent
substances.
This phenomenon is Fluorescence instantaneous i.e start
emitting radiation immediately after the absorption and stops
when incident light is cut off.
Fluorescent substances emit maximum radiation with in 10-
10 to 10-8sec of absorption. This is also called delayed
fluorescence

6. Phosphorescence
When light is incident on certain substances ,they
emit light continuously even after the light is cut off .
This is called phosphorescence and such substances
are called as phosphorescent substances.
In the fluorescence process, the electron did not
change its spin direction But under the appropriate
conditions, a spin-flip can occur
The light emission process must wait until electron
undergoes a spin-flip to revert back to it original state
Phosphorescent substances emit maximum radiation
within 10-4 to 20 sec or longer.

7. Difference between Phosphorescence and fluorescence
Phosphorescence Fluorescence
The emission could proceed
either from a singlet or triplet
state.
The emission could proceed
only from a singlet state.
short-live electrons (<10-5 s) in
the excited state of
fluorescence
longer lifetime of the excited
state (second to minutes)

8. CONTENTS
•Principle
•Factors effecting
Fluorescence,Phosphorescence
•Intensity
•Instrumentation
•Applications
•Conclusion

9. PRINCIPLE
•Molecule contains electrons, electrons and non
bonding (n) electron.
•The electrons may be present in bonding molecular
orbital.
•It is called as highest occupied molecular orbital
(HOMO).
•It has lest energy and more stable.
•When the molecules absorbs radiant energy from a
light source, the bonding electrons may be promoted
to anti bonding molecular orbital. It has more energy
and hence less stable.

10. The process of promotion of electrons from
HOMO to LOMO with absorption of energy is
called as excitation.
Singlet state:- a state in which all the electrons in a
molecule are paired  
Doublet state:- a state in which un paired electrons is
present  or 
Triplet state:- a state in which unpaired electrons of
same spin present  
Singlet excited state:- a state in which electrons are
unpaired but of opposite spin like   (un paired
and opposite spin)

11. When light of appropriate wavelength is absorbed by
a molecule the electrons are promoted from singlet
ground state to singlet excited state. Once he
molecule is in this excited state relaxation can occur
via several process like emission of radiation.
The process can be the following:-
1) Collision deactivation
2)Fluorescence
3)Phosphorescence.

12. Collisional de activation :-
In which entire energy lost due to collision de activation and
no radiation emitted.
Fluorescence:-
Excited singlet state is highly unstable. Relaxation of electrons
from excited singlet to singlet ground state with emission of
light.
Phosphorescence:-
At favorable condition like low temperature and absence of
oxygen there is transition from excited singlet state to triplet
state which is called as intersystem crossing . The emission of
radiation when electrons undergo transition from triplet state
to singlet ground state.

13. 1. Concentration
2. Quantum yield of fluorescence
3. Intensity of incident light
4. Oxygen
5. Ph
6. Temperature& viscosity
7. Photodecomposition
8. Quenchers
9. Scatter
10. Solvent

14. Concentration:-
•Fluorescence intensity is proportional to concentration of
substance only when the absorbance is less than 0.02
Beer-Lambert Law
A = log(I /I 0) = €.c.l
I = transmitted light intensity
Io = incident light intensity
c = concentration (M)
l = path length of cell (cm)
€= molar extinction coefficient (usually in the range 0-105 ,
with <103 considered to be a week absorption

15. NUMBER OF PHOTONS EMITTED
() = ———————————
NUMBER OF PHOTONS ABSORBEDS
It Is Always Less Than 1.0 Since Some Energy Is Lost By
Radiation less Pathways (Collisions, Intersystem Crossing,
Vibrational Relaxation)
Increase In The Intensity Of Incident Light On The
Sample Fluorescence Intensity Also Increases effect.

16. Dissolved oxygen
•Oxygen 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 radiation less deactivation to occur.
PH
•Relatively small changes in pH can sometimes cause
substantial changes in the fluorescence intensity and spectral
characteristics of fluorescence.

17. 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.

18. Photochemical decomposition:-
Absorption of intense radiation leads to
photochemical decomposition of a fluorescent
substance to less fluorescent or non fluorescent
substance.
Quenchers:-
Quenching is the reduction of fluorescence
intensity by the presence of substance in the
sample other than the fluorescent analyte.

19. •Scatter is mainly due to colloidal particles in solution
•Scattering of incident light after passing through the
sample leads to decrease in fluorescence intensity.
•A higher fluorescence is observed when the solvents
do not contain heavy atoms while phosphorescence
increases due to the presence of heavy atoms in the
solvent.

20. Solvent
•The changes in the “polarity” or hydrogen bonding
ability of the solvent may also significantly affect the
fluorescent behaviour of the analyte.
•The difference in the effect of solvent on the
fluorescence is attributed to the difference in their
ability to stabilise the ground and excited states of the
fluorescent molecule.
•Increased viscosity increases fluorescence as the
deactivation due to collisions is lowered.

21. INSTRUMENTATION

22. INSTRUMENTATION
•SOURCE OF LIGHT
•FILTERS AND MONOCHROMATORS
•SAMPLE CELLS
•DETECTORS

23. 1)SOURCE OF LIGHT:-
Mercury vapour 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.

24. mercury vapour lamp
xenon arc lamp
tungsten lamp

25. 2) FILTERS ANDMONOCHROMATORS:-
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:- Absorbs visible radiation and transmit
UV radiation.
•Secondary filter:-Absorbs UV radiation and transmit
visible radiation.

26. 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.

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

28. INSTRUMENTS
The most common types are:-
•Single beam (filter) fluorimeter
•Double beam (filter )fluorimeter
•Spectrofluorimeter(double beam

29. •It contains tungsten lamp as a source of light and has an
optical system consists of primary filter.
•The emitted radiations is measured at 900 by using a
secondary filter and detector. Primary filter absorbs visible
radiation and transmit uv radiation which excites the
molecule present in sample cell.

30. Cont.
•In stead of 900 if we use 1800 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.

31. It is 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 combinly on a detector.

32. Spectrofluorimeter:
•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.

33. Single beam (filter) fluorimeter

34. Double beam (filter )fluorimeter

35. Spectrofluorimeter(double beam

36. Phosphorimetry
•Instrumentation
•Instrument
•Applications

37. Instrumentation
• Excitation Source
• Filters/Monochromators for excitation
radiations
• Phosphorscope(sample Cell)

38. Excitation Source
High intensity source of UV light are used
Lasers – A laser makes it possible to have narrow
wavelength intervals that offer very high energy
irradiation. This is useful when a large amount of
energy is needed to produce the Phosphorescence
in the sample.
Photodiodes – Photodiodes are specialized diodes
that can be configured in a manner that allows
electrons to flow towards the sample so that the
excess energy excites the phosphorescent particles.

39. Xenon Arcs – Arcs of Xenon can produce the right
amount of radiation for Phosphorescent materials.
Mercury Vapor – Since mercury vapor can create
ultraviolet radiation when electrical current is
passed through it, it is good for use with materials
that shows Phosphorescence under the ultraviolet
radiation.
note: Care should be taken as Intense UV light is
hazardous for health

40. Filters and monochromators used in a phosphorimeter device.
• Filters
– Absorption
– Interfernce
• Monochromators
It allow wavelength adjustment.
Monochromators make it possible to do so with a
diffraction grating.
 Diffraction grating is an optical component with a periodic structure, which splits
and diffracts light into several beams travelling in different directions.

41. Cont.
-The primary filters that excite the sample
provide the appropriate wavelength
-While the secondary filters monochromate the
emitted light when sent to the detector.
Phosphoresence spectroscopy detectors may
have a
single channel (single wavelength from sample)
multiple channels (multiple wavelengths detection)

42. Instrument
phosphoroscope
A phosphoroscope is piece of experimental
equipment devised to measure how long it takes
a phosphorescent material to stop glowing after it
has been excited.

43. Phosphoroscope
1.The Becquerel or rotating disc
phosphoroscope
• A rotating disk excitation optical chopper, with
three open and three larger opaque areas, is
used to alternately excite the sample and
allow phosphorescence to be measured.
• By measuring the phosphorescence intensity
at several time intervals along the emission
decay curve, a recorder trace of the decay
with respect to time can be produced.

44. Cont.
The analytical precision and accuracy for quantitative
measurements is improved by rotating the sample
tube. This minimizes variation in the signal due to
sample in homogeniety resulting from imperfect
glass formation at low-temperatures .

45. 2.The Rotating-Can Phosphoroscope:
• It consists of hollow cylinder having one or more
slit which are equally spaced in the circumference.
• This is rotated by a variable-speed
motor(>1000rpm)
• when the rotating-can is rotated by a motor the
sample is excited show fluorescence and
phosphorescence
For Fluorescence the emission monochromator is
blocked by the can so that fluorescence and scattered
radiation cannot be detected.

46. Cont.
• As the can rotates, the excitation beam is blocked
and the fluorescence and scattered radiation decay
to a negligible amount.
• Further rotation of the can will bring the window
into alignment with the emission monochromator
entrance slit and phosphorescence radiation will
pass into the emission monochromator and onto
the sample photomultiplier

48. Applications of Phosphorescence Spectroscopy
• Pharmaceutical Applications
• Clinical Applications
• Environmental Applications
• Entertainment Applications

49. Pharmaceutical Applications
• The majority of phosphorescence applications
have been applied in the drug and
pharmaceutical field and in the analysis of
pesticides
• A number of the sulphonamide class of drugs
exhibit phosphorescence as do cocaine,
procaine, chlorpromazin and salicylic acid.

50. Clinical Applications
• The phosphorescence intensity of the rare
earths increases tremendously when they are
covalently bound to certain molecules and this
feature has been used in the analysis of
transferin in blood
• dual-wavelength phosphorimeter used to
measure microvascular PO2 (µPO2) in
different depths in tissue and demonstrates its
use in rat kidney.

51. Environmental Applications
•Phosphorescence has been used in the
detection
•In air and water-borne pollutantsfor the
analysis of impurities in polycyclic aromatic
hydrocarbons and in petroleum products.

52. Entertainment Applications
•Use of phosphor coated stamps and envelopes
has appeared which gives a fascinating insight
into the practical use of phosphorescence.
•They used phosphors in lamps
•TV tubes.

53. Advantages
 SENSTIVITY: This technique can measure
concentration as low as microgram/ml or even ng/ml.
 SPECIFICITY: More specific than absorption
technique.
 PRECISION: Up to 100% can easily achieved in
fluorimetery and Phosphorimetry
 .

54. Limitations
Change in pH affects fluorescent and
Phosphorimetry intensity.
Dissolved oxygen may affect fluorescent
intensity.
Traces of halides, heavy metals, etc can affect
fluorescent intensity.

55. Applications of photoluminescence methods
1) Determination of uranium salt by flourimetry.
2) Determination of inorganic species
-by direct method involve formation of fluorescing
chelates.
eg. aromatic structure with two aromatic donor
function group that permit chelate formation with
metal ion decrease in fluorescing emission because
of quenching

56. Cont.
3) Organic analysis- (adenine, antranilic acid, uric
acid, morphine, proteins etc..)
4) Determination of aspirin in blood serum with high
sensitivity by phosphometric at liq. Nitrogen
temperature.
- low conc. of procaine, cocaine, chlorpromazine can
be determined.
5 Determination of Vit B1(Thiamine).
- determination of Vit B2(riboflavin)

57. Comparison of fluorimetry and phosphorimetry with absorption
methods.
 Sensitivity of luminescence methods is generally 10 to 103
times greater than sensitivity of absorption method.
 Luminescence method have greater specificity and
selectivity than absorption methods.

58. References :
•Phosphorimetry
R. J. Keirs, R. D. Britt, W. E. Wentworth
• Phosphorimetry : theory, instrumentation, and applications
Robert J. Hurtubise.
•Principles of Fluorescence Spectroscopy
Authors: Lakowicz, Joseph R.
• Google Scholar and Google images.