Introduction, theoretical principle, quantum efficiency of fluorescence, molecular structure of fluorescence, instrumentation, factors influencing the intensity of fluorescence, comparison of fluorometry with spectrophotometry, application of fluorometry in pharmaceutical analysis

2. Luminescence: Luminescence is the phenomenon of a
chemical species to absorb radiation of UV or visible
region and emit a radiation of longer wavelength. Loss of
energy and concomitant transition of molecules from
excited states to ground states with emission of radiation is
called luminescence.
Luminescence can be divided into two types depending
on the lifespan of the excited state –
1. Fluorescence
2. Phosphorescence

3. Singlet and triplet states
• Ground state – two electrons per orbital; electrons have opposite spin and are paired
Singlet excited state
Electron in higher energy orbital has the opposite spin orientation relative to
electron in the lower orbital
Triplet excited state
The excited valence electron may spontaneously reverse its spin (spin flip).
This process is called intersystem crossing. Electrons in both orbitals now have
same spin orientation.
Types of emission
Fluorescence – return from excited singlet state to ground state; does not require change in
spin orientation (more common of relaxation)
An atom or molecule that fluoresces is termed a Fluorophore
Fluorometry is defined as the measurement of the emitted fluorescence light
Phosphoresence – return from a triplet excited state to a ground state; electron requires change
in spin orientation

4. Vibrational energy level: Whether the molecule is in ground state or
excited state, the molecule contains many energy levels which are called
vibrational energy levels.
Vibrational relaxation: Vibrational relaxation is the transition of molecule
from any of the vibrational energy levels to the lowest vibrational energy
level of the excitatory state.
Resonance fluorescence: Resonance fluorescence is the phenomenon
where the molecule absorbs and emits equal amount of energy. Practically
resonance fluorescence doesn’t occur or occur rarely as vibrational
relaxation occurs.
Internal conversion: The phenomenon of excited molecule to return to the
ground state by losing energy by means other than photo radiation is
termed internal conversion.
Intersystem crossing: The transfer of a molecule present in the lowest
vibrational energy level of the excited singlet state to an excited triplet state
is called intersystem crossing.

5. Property Fluorescence Phosphorescence
Transition Molecule transits from excited singlet state
to ground state.
Molecule transits from excited triplet
state to ground state.
Lifespan Fluorescence is continued for only 10-8 to 10-
4 seconds.
Phosphorescence continues for 10-4
seconds to 10 seconds.
Afterglow Not present. Occurs and luminescence slowly fades.
Analytical application Yes. No.
Quantum efficiency: Quantum efficiency is defined as the ratio of number of light quanta emitted and the
number of light quanta absorbed.
Its significance is that, it is an indicator of how fluorescent a molecule is. If Q is near 1, the molecule is highly
fluorescent molecule and if Q is near 0, the molecule is a very low fluorescent molecule.
absorbedlightofEnergy
emittedlightofEnergy
absorbedquantalightofNo.
emittedquantalightofNo.
orQ

6. The method of analysing a sample by measuring its fluorescence i.e.
intensity and composition of light emitted by it, is called fluorometry.
Fluorescence spectroscopy aka fluorometry or spectrofluorometry is an
analytical technique for identifying and characterizing minute amounts of a
fluorescent substance by excitation of the substance with a beam of
ultraviolet light and detection & measurement of the characteristic
wavelength of the fluorescent light emitted.
It is a spectrochemical method.

7. When energy is applied to certain molecules in the form of UV or visible
electromagnetic radiation, the molecules temporally transit to an
excited singlet state where the excited electron is in paired condition
with the ground electron. In the excited state, the molecules lose energy
in radiationless manner to descend to the lowest vibrational energy
level of the excited state. The excited state lasts only 10-8 to 10-4 seconds
and then the excited molecule will return to ground state by losing
energy through emitting radiation. This is termed fluorescence and the
emitted radiation is of longer wavelength.
By measuring the emitted wavelength we can determine the presence
and amount of a compound in a sample.

9. Relationship between fluorescence and
chemical structure
Definite correlations between chemical structure and fluorescence can’t be made.
Degree of conjugation
Delocalization of electron
Electron donating groups
Exception

10. Relationship between fluorescence and
chemical structure
• Electron withdrawing
groups
• Exception

11. Relationship between fluorescence and
chemical structureMolecular geometry
Rigidity and planarity
cis-trans isomerism
Heterocylic compounds
Ionization
Complexation

13. ISS PC1 (ISS Inc., Champaign, IL, USA)
Fluorolog-3 (Jobin Yvon Inc, Edison, NJ, USA )
QuantaMaster (OBB Sales, London, Ontario N6E 2S8)

14. Concentration of fluorescing species
Presence of other solutes/impurities
Chemical quenching
pH of the sample solution
Stability of the sample compound (Degradation of Sample)
Solvent effect
Temperature

15. Features Absorption spectroscopy Fluoroscence spectroscopy
Theoretical consideration
Measurement of amount of light
absorbed.
Measurement of intensity of fluorescence.
Wavelength of light used Which gives maximum absorption. Which gives maximum fluorescence.
Instruments Determines only the absorption of light.
Determines absorption of light as well as
emission of radiation.
Light source Tungsten, H2-discharge lamp. Mercury arc lamp, Xenon arc lamp.
Cell used Silica cell. Glass and metal cells.
Detector
Phototube or photo multiplier is used to
detect the radiation absorbed
Emission filter is used to separate the
emitted light from the transmitted light.
Concentration
Concentration depends on the molar
absorptivity.
Concentration depends on the
characteristics of the instrument.
Electrical transition
Applicable for both ππ* & nπ*
transition.
Not applicable for the compound
containing nπ* transition.
Experimental variables
temperature & Extraneous
solution
Not so restricted. Highly restricted.
Sensitivity and selectivity Less sensitive and less specific. More sensitive and highly specific.

16. Application in chemistry
Determination of metal ions
Separation and identification
Application in biopharmaceutics
Pharmaceutical applications

17. Sensitivity
Specificity
Wide Concentration Range
Simplicity and Speed
Low Cost
Limitations of Fluorometry

18. Excitation spectrum and emission spectrum
The excitation spectrum is a measure of the ability of the impinging radiation to raise a
molecule to various excited states at different wavelengths. An excitation spectrum is recording
of fluorescence versus the wavelength of the exciting or incident radiation and it is obtained by
setting the emission monochromator to a wavelength where fluorescence occurs and scanning the
excitation monochromator. An excitation spectrum looks very much like an absorption spectrum,
because the greater the absorbance at the excitation wavelength, the more molecules are promoted to
the excited state and the more emission will be observed.
The emission (fluorescence) spectrum is a measure of the relative intensity of radiation given
off at various wavelength as the molecule returns from the excited states to the ground state.
The emission spectrum is recording of fluorescence versus the wavelength of the fluorescence
radiation, and it is obtained by setting the excitation monochromator to a wavelength that the sample
absorbs and scanning the emission monochromator.
Since some of the absorbed energy is usually lost as heat, the emission spectrum occurs at longer
wavelengths (lower energy) than does the corresponding excitation spectrum. If an emission spectrum
occurs at shorter wavelengths than the excitation spectrum, the presence of a second fluorescing
species is confirmed.
The absorption and emission spectra will have an approximate mirror image relationship if the
spacings between vibrational levels are roughly equal and if the transition probabilities are similar.

19. Energy level diagram showing why structure is seen in the absorption and emission spectra, and why the spectra seem
roughly mirror images of each other.

20. Mirror image rule
• Vibrational levels in the excited states and ground states are
similar
• An absorption spectrum reflects the vibrational levels of the
electronically excited state
• An emission spectrum reflects the vibrational levels of the
electronic ground state
• Fluorescence emission spectrum is mirror image of
absorption spectrum
S0
S1
v=0
v=1
v=2
v=3
v=4
v=5
v’=0
v’=1
v’=2
v’=3
v’=4
v’=5

21. References
• Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York.
• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York. 3 Id., p. 7.
• Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.
• G. K. Turner, "Measurement of Light From Chemical or Biochemical Reactions," in Bioluminescence and Chemiluminescence: Instruments and
Applications, Vol. I, K. Van Dyke, Ed. (CRC Press, Boca Raton, FL, 1985), pp. 45-47.
• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 51-57.
• Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, chap. 2.
• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 67-69.
• Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, pp. 23-26.
• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 57-58.
• Stotlar, S. C. 1997. The Photonics Design and Applications Handbook, 43rd Edition, Laurin Publishing Co., Inc., Pittsfield, MA, p. 119.
• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 63.
• Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.
• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 30.
• Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular Biology, School of Medicine.
• Iain Johnson, Product Manager, and Ian Clements, Technical Assistant Specialist (May 1998 communication from Molecular Probes, Eugene,
Oregon).
• Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), pp. 14-15.
• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 172.
• Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), p. 21.
• Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York., p. 28.
• Teitz Textbook of Clinical Chemistry and Molecular diagnosis (5th Edition)
• Dr.B.K.Sharma, Instrumental methods of chemical analysis.
• Gurdeep R Chatwal, Instrumental methods of chemical analysis
• http://en.wikipedia.org/wiki/Fluorescence
• http://images.google.co.in/imghp?oe=UTF-8&hl=en&tab=wi&q=fluorescence
• http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm