Fluoroscence Spectroscopy

1. Fluorescence Spectroscopy
Principle and application
NIPER Hyderabad
National Institute of Pharmaceutical Education and Research

2. LIST OF
CONTENTS
FACTORS AFFECTIG FLUORESCENCE
6
1 INTRODUCTION
2 FLUORESCENCE AND ITS PRNCIPLE
3 JABOLONSKI DIAGRAM
4 TYPES OF FLUORESCENCE
5 GENERAL INSTRUMENTATION
8 APPLICATION AND CASE STUDY
ADVANTAGES AND DISADVANTAGES
7

3. What is Fluorescence
• Molecular absorption of light energy at one wavelength
and its instantaneous remission at a higher wavelength
• A Proton emission process involved in molecular
relaxation from an excited state to ground state.
• The molecules showing Fluorescence are called
Fluorophores and it mostly includes the likes of
aromatic compounds

4. Fluorophores
General Principle of Fluorescence
• A molecule will absorb radiation and go to higher
energy state
• Then, it relaxes to lowest energy vibrational
transition of respective electronic state
• Fluorescence Emission- The Fluorophores decay
from the excited singlet and relax to allowable
electronic ground state singlet.

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
Reemission 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 the following-
—An Ozone Free Light Source
— A Monochromator
—A Sample cuvette
—A second Monochromator
— A photo-multiplier tube

12. FACTORS
Factors affecting Fluoroscence
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
• Involves High Precision (up
to 1 percent)
• 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 crytals(FDA)

15. Case Study 1