2. FLOURESCENCE
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• Molecule absorbs energy and immediately (10-6 to 10-
8
sec) emits energy at a higher wavelength (lower
energy) Phosphorescence is similar but involves a
slower emission step (> 10-4 sec).
• Excitation and emission wavelengths specific to the
compound
• Emission measured at 90° to the excitation light path
• Emission proportional to drug concentration
3. LUMINESCENCE
SPECTROSCOPY
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• The emission of radiation from a species
after that species has absorbed radiation.
FLUORESCENCE
LUMINESCENCE PHOSPHORESCENCE
SPECTROSCOPY
CHEMILUMINESCENCE
5. LUMINESCENCE
SPECTROSCOPY
Faculty of Science
• In favorable cases, luminescence methods
are amongst some of the most sensitive
and selective of analytical methods
available.
• Detection Limits are as a general rule at
ppm levels for absorption
spectrophotometry and ppb levels for
luminescence methods.
6. LUMINESCENCE
SPECTROSCOPY
Faculty of Science
• Collectively, fluorescence and
phosphorescence are known as
photoluminescence.
• A third type of luminescence -
Chemiluminescence - is based upon
emission of light from an excited species
formed as a result of a chemical reaction.
7. LUMINESCENCE
SPECTROSCOPY
Faculty of Science
• Most chemical species are not naturally
luminescent.
• Derivatisation reactions are often
available to form luminescent derivatives
of non-luminescent compounds.
• However, this extra step lessens the
attractiveness of luminescence methods.
8. Energy Level Diagram
SINGLET STATES ↑↓ ↑ TRIPLET STATES ↑↑ ↑
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s2 VIBRATIONAL
RELAXATION
T2
s1
T1
INTERSYSTEM
CROSSING
FLUORESCENCE PHOSPHORESCENCE
INTERNAL INTERNAL
CONVERSION CONVERSION
Ground
State
9. Fluorescence and
Phosphorescence - 1
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• Following absorption of radiation, the
molecule can lose the absorbed energy by
several pathways. The particular
pathway followed is governed by the
kinetics of several competing reactions.
10. Fluorescence and
Phosphorescence - 2
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• One competing process is vibrational
relaxation which involves transfer of
energy to neighbouring molecules which
is very rapid in solution (10-13 sec).
– In the gas phase, molecules suffer fewer
collisions and it is more common to see the
emission of a photon equal in energy to that
absorbed in a process known as resonance
fluorescence.
11. Fluorescence and
Phosphorescence - 3
Faculty of Science
• In solution, the molecule rapidly relaxes
to the lowest vibrational energy level of
the electronic state to which it is excited
(in this case S2). The kinetically favoured
reaction in solution is then internal
conversion which shifts the molecule
from S2 to an excited vibrational energy
level in S1.
12. Fluorescence and
Phosphorescence - 4
Faculty of Science
• Following internal conversion, the
molecule loses further energy by
vibrational relaxation. Because of
internal conversion and vibrational
relaxation, most molecules in solution
will decay to the lowest vibrational
energy level of the lowest singlet
electronic state before any radiation is
emitted.
13. Fluorescence and
Phosphorescence - 5
Faculty of Science
• When the molecule has reached the
lowest vibrational energy level of the
lowest singlet electronic energy level
then a number of events can take place:
14. Fluorescence and
Phosphorescence - 6
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• the molecule can lose energy by internal
conversion without loss of a photon of
radiation, however, this is the least likely
event;
15. Fluorescence and
Phosphorescence - 7
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• the molecule can emit a photon of
radiation equal in energy to the difference
in energy between the singlet electronic
level and the ground-state, this is termed
fluorescence;
16. Fluorescence and
Phosphorescence - 8
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• the molecule can undergo intersystem
crossing which involves and electron spin
flip from the singlet state into a triplet
state. Following this the molecule decays
to the lowest vibrational energy level of
the triplet state by vibrational relaxation;
17. Fluorescence and
Phosphorescence - 9
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• the molecule can then emit a photon of
radiation equal to the energy difference
between the lowest triplet energy level
and the ground-state in a process known
as phosphorescence.
18. Fluorescence and
Phosphorescence - 10
Faculty of Science
• In fluorescence, the lifetime of the
molecule in the excited singlet state is
10-9 to 10-7 sec.
• In phosphorescence, the lifetime in the
excited singlet state is 10-6 to 10 sec
(because a transition from T1 to the
ground state is spin forbidden).
19. Quantum Efficiency
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• Fluorescence, phosphorescence and
internal conversion are competing
processes. The fluorescence quantum
efficiency and the phosphorescence
quantum efficiency are defined as the
fraction of molecules which undergo
fluorescence and phosphorescence
respectively.
21. Factors affecting Flourescence
1. CONJUGATION
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Molecule must have unsaturation for uv/vis
absorption
2. NATURE OF SUBSTITUENT GROUP
Electron donating gp-NH2, OH, Increse the FI
Electron withdrawing gp-NO2,COOH, Reduce the
FI
22. 3. STRUCTURAL RIGIDITY
Rigid structure- More FI
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-Flourene
Flexile structure-Less FI
-Bipheny
23. Faculty of Science
4.TEMPERATURE
High temp reduce the FI due to increase
in collision of molecules & vice versa
5.OXYGEN
Decrease the FI by oxidation of
substance
24. 6.CONCENTRATION AND
FLUORESCENCE INTENSITY
Faculty of Science
• The power of fluorescent radiation, F, is
proportional to the radiant power of the
excitation beam absorbed by the species able to
undergo fluorescence:
F = K'(P0 - P)
where P0 is the power incident on the sample, P
is the power after it traverses a length b of the
solution and K' is a constant which depends
upon experimental factors and the quantum
efficiency of fluorescence.
25. CONCENTRATION AND
FLUORESCENCE INTENSITY
Faculty of Science
• Beer's law can be rearranged to give:
P/P0 = 10-εbc
where A = εbc is the absorbance.
Substitution gives:
F = K'P0(1 - 10- εbc)
• This is the fluorescence law
• Unlike Beer’s Law fluorescence isn’t in
general linear with concentration.
26. QUENCHING OF FLOURESCENCE
• It is decrease in FI due to specific effects of
constituents of the solution itself .
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• It may be due to
con.,PH,Temp,viscosity,presence of specific
chemical substances
27. CONCENTRATION AND
FLUORESCENCE INTENSITY
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which demonstrates two important points:
• that at low concentrations fluorescence
intensity is proportional to concentration;
• that fluorescence is proportional to the
incident power in the incident radiation at
the absorption frequency.
30. 1. Source of light
• Mercury vapour lamp –above350 nm at 8 atm p
• Xenon arc lamp-more intense radiation thsn
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MVP
• Tungston lamp-used only for visible region(400-
800nm)
2. Filters & Monochromotors
• Primary filter- absorbs vis radiation & transmits
uv radiation
• Secondory filters- absorbs uv radiation &
transmits vis radiation
31. DETECTORS
• Photovoltaic cell
• Photo multiplier tubes-most common
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32. INSTRUMENTATION
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• The fluorescence is often viewed at 90°
orientation (in order to minimise
interference from radiation used to excite
the fluorescence).
• The exciting wavelength is provided by
an intense source such as a xenon arc
lamp (remember F ∝ P0).
33. INSTRUMENTATION
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• Because An intense monochromatic light
source is required ...
• Lasers are an almost ideal light source for
fluorimetry (laser-induced fluorescence) but are
too expensive and/or impractical for most
routine applications.
• Two wavelength selectors are required filters
(in fluorimeters) and monochromators (in
spectrofluorometers).
34. Types of Fluorescent Molecules
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• Experimentally it is found that fluorescence is
favoured in rigid molecules, eg.,
phenolphthalein and fluorescein are structurally
similar as shown below. However, fluorescein
shows a far greater fluorescence quantum
efficiency because of its rigidity.
•
phenolphthalein
35. Types of Fluorescent Molecules
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• It is thought that the extra rigidity
imparted by the bridging oxygen group in
Fluorescein reduces the rate of
nonradiative relaxation so that emission
by fluorescence has sufficient time to
occur.
Fluorescein
36. APPLICATION
1. Determination of inorganic substances-
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e.g. detection of uranium, ruthenium,
2. Determination of organic substances-
aromaticpolycylichydrocarbon,
indoles, napthols, proteins,pigments,
steroids, etc
38. APPLICATIONS
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B. Fluorimetric Drug
Analysis
• Many drugs possess
high quantum
efficiency for
fluorescence. For
example, quinine can
be detected at levels
below 1 ppb. Quinine
39. APPLICATIONS
Faculty of Science
• In addition to ethical
drugs such as
quinine, many drugs
of abuse fluoresce
directly. For
example lysergic
acid diethylamide
(LSD) whose
structure is:
