Spectroscopy

1. Fluorescence Spectroscopy
Principle and application
NIPER HYDERABAD
National Institute of Pharmaceutical Education and Research
Roll no - PA/2023/107
Name - Vedang Sachin Junagade
Department- Pharmaceutical Analysis
Code- GE510

2. LIST OF
CONTENTS
FACTORS AFFECTIG
FLUORESCENCE
6
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
7

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.

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.
Crowney J, Jameson J Biochemistry and Molecular Biology Education 29 (2001) 60-65, Elsevier.

5. Jablonski diagram

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

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.

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 - two
Photon absorption method, hot band
absorption method and up conversion
method.

10. General Instrumentation
It includes
• An ozone free light source
• A monochromator
• A sample cuvette
• A second monochromator
• A photo-multiplier tube

12. FACTORS
Factors affecting Fluorescence
Conjugation
in the
compound
No. of
rotatable
bonds
The kind of
substituent
attached
Temperature
Viscosity pH Concentration

13. 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.

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).

15. Major manufacturers
Hitachi PerkinElmer Agilent

16. Case Study 1

17. Case Study 2

18. Case Study 3