1. Fluorescence Spectroscopy
Principle and application
NIPER HYDERABAD
National Institute of Pharmaceutical Education and Research
Name - Vedang Sachin Junagade
PA/2023/107
Department- Pharmaceutical Analysis
Code- GE510
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2. LIST OF
CONTENTS
FACTORS AFFECTING
FLUORESCENCE
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1 INTRODUCTION
2 FLUORESCENCE AND ITS
PRNCIPLE
3 JABLONSKI DIAGRAM
4 TYPES OF FLUORESCENCE
5 GENERAL INSTRUMENTATION
8 APPLICATION AND CASE STUDY
ADVANTAGES AND DISADVANTAGES
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3. What is Luminescence ?
• Luminescence : Emission of light from an excited state of a molecule.
• Characterized by - λemission > λabsorption
• Luminescence comprises of two types -
a) Chemiluminescence- A reaction where a molecule gets produced at
an excited state and it emits light.
b) Photoluminescence- Upon absorption of light, a molecule will go to
a higher energy state and upon relaxation will emit radiation.
• Fluorescence is a type of molecular photoluminescence.
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Mohammad A. Omary, Howard H. Patterson,Luminescence Theory,Encyclopedia of Spectroscopy and Spectrometry,Elsevier,
4. Fluorophores
General Principle of Fluorescence
• An electron in a molecule will absorb radiation and will
move to higher energy state.
• Then, it relaxes to lowest energy vibrational transition of
respective electronic state.
• Fluorescence emission- The fluorophores decay and the
electrons move from the excited singlet and relax to
allowable electronic ground state singlet.
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Crowney J, Jameson J Biochemistry and Molecular Biology Education 29 (2001) 60-65, Elsevier.
7. TYPES OF FLUORESCENCE
The energy of the emitted radiation is
less as compared to the absorbed
radiation. The emitted radiation is of
longer wavelength.
The energy of the emitted radiation is
more as compared to the absorbed
radiation. The emitted radiation is of
shorter wavelength.
STOKES SHIFT ANTI STOKES SHIFT
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Zhu X, Feng W, Royal Society Of Chemistry DOI: 10.1039/c6cs00415f
8. 01
02
Stokes Fluorescence
Re-emission of less
energetic photons, with an
increase wavelength than
the absorbed one.
Shift associated with rapid
decay to lowest vibrational
level.
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Crowney J, Jameson J Biochemistry and Molecular Biology Education 29 (2001) 60-65, Elsevier
9. 01
02
Anti-Stokes Fluorescence
High energy emission at a lower
wavelength in comparison to the
excitation.
Done by utilization of heat,
lanthanide complexes etc.
Involves 3 methods that are –
a)Two Photon absorption method
b)Hot band absorption method
c)Up conversion method.
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Zhu X, Feng W, Royal Society Of Chemistry DOI: 10.1039/c6cs00415f
10. General Instrumentation
It includes
• An ozone free light source
• A monochromator
• A sample cuvette
• A second monochromator
• A photo-multiplier tube
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Thomas I, Abraham E, International Journal of Advances in Pharmaceutical Analysis , DOI - 10.7439/ijapa
12. Advantages Disadvantages
• A sensitive method.
• Specificity present as the excitation
wavelength can be controlled.
• Involves high precision.(up to 1 %).
• Careful buffering is necessary.
• UV light may cause photochemical change and
damage the sample.
• Not suitable for determination of large
constituents- less accuracy for large molecules.
• Florescence is not shown by all molecules and
compounds.
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13. Factors affecting Fluorescence
Conjugation
Rigidity of the molecule
Substituent attached
Temperature
Viscosity
pH
Concentration
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Naresh K, Journal of Chemical and Pharmaceutical Research ISSN: 0974-2115
14. Applications of Fluorescence Spectroscopy
• Laser induced spectroscopy for tumour diagnosis.
• Investigation of chemical structures.
• Glucose determination.
• Determination of inorganic substances like ruthenium, boron in
steel etc.
• Avidin-Biotin fluorescent assays for biomolecules by using
Fluorescein Diacetate crystals(FDA).
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Sin, KK., Chan, C.PY., Pang, TH. et al. A highly sensitive fluorescent immunoassay based on avidin-labeled nanocrystals. Anal Bioanal Chem 384, 638–644 (2006).
