The document discusses the principles of fluorescence and phosphorescence. Fluorescence occurs when electrons in an excited singlet state fall back down to the ground singlet state, emitting a photon. This process is fast, taking 10-12 seconds. Phosphorescence occurs when electrons in an excited triplet state fall to the ground singlet state. This is a spin-forbidden process and is therefore slower, taking up to hours. Applications of fluorescence include drug analysis and studying drug-protein binding. Phosphorescence has applications in signs, toys, and molecular spectroscopy.

1. Principle And Application Of Fluorescence And Phosphorescence
Submit to,
Nishu Singla Ma’am
Submit by,
Harsh Kumar Pandey
21MPI1003 (1st sem)
M. Pharma (Industrial Pharmacy)
Chandigarh University (UIPS)
MODERN PHARMACEUTICAL
ANALYTICAL TECHNIQUES
21PHT-641

2. CONTENT
• Introduction
• Flouroscence
• Phosphorescence
• Reference

3. INTRODUCTION
Luminescence is the emission of light by a substance. It occurs when an electron returns to the
electronic ground state from an excited state and loses its excess energy as a photon.
It is of 3 types.
• Fluorescence spectroscopy
• Phosphorescence spectroscopy
• Chemiluminescence spectroscopy

4. Principle of fluorescence
The electronic states of most organic molecules can be divided into singlet states and triplet.
• Singlet ground state : All electrons in the molecule are spin-paired
• Singlet excited state : Unpaired electrons of opposite spin present
• Triplet state : Unpaired electrons of same spin present

5. Principle of fluorescence
Energy of emitted radiation is less than that of absorbed radiation because a part of energy is lost due to
vibrational or collisional processes. Hence the emitted radiation has longer wavelength (less energy) than
the absorbed radiation.
Vibrational deactivation takes place through intermolecular collisions at a time scale of 10 -12 s (faster
than that of fluorescence process).

6. Internal Conversion
As electronic energy increases, the energy levels grow more closely spaced.It is more likely that there will be overlap between
the high vibrational energy levels of S n-1 and low vibrational energy levels of S n. This overlap makes transition between
states highly probable.
Internal conversion is a transition occurring between states of the same multiplicity and it takes place at a time scale of 10 -¹²
(faster than that of fluorescence process).
The energy gap between S ₁ and S ₀ is significantly larger than that between other adjacent states → S lifetime is longer →
radiative emission can compete effectively with non-radiative emission.

7. Stokes shift
The difference btw the max wavelength of the excitation light and the max wavelength of the emitted fluorescence lights is a
constant.
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

8. • Mirror-image rule typically applies when only S₀ → S₁ excitation takes place.
• Deviations from the mirror-image rule are observed when S₀ → S₂ or transitions to even higher excited states also take
place.

9. Applications
• Determination of fluorescent drugs in low-dose formulations in the presence of non-fluorescent
excipients.
• In carrying out the limit tests where the impurity is fluorescent.
• Useful for studying the binding of drugs to component in complex formulations.
• Widely used in bioanalysis for measuring small amounts of drug and for studying drug-protein binding.

10. PHOSPHORESCENCE
Definition:
• It is Delayed and long lived emission of light energy in the form of a photon after an electron has been excited due to
radiation.
• It is type of photoluminescence.
• It is spin-forbidden process.

11. PRINCIPAL OF PHOSPHORESCENCE
• Phosphorescence occurs when electrons from the excited triplet state return to the ground singlet state.
• Excitation of electrons to a higher state is accompanied with the change of a spin state . Once in a different spin state,
electrons cannot relax into the ground state quickly because the re- emission involves quantum mechanically forbidden
energy state transitions. As these transitions occur very slowly in certain materials, absorbed radiation may be re-emitted at
a lower intensity for up to several hours after the original excitation.

12. Principle of Phosphorescence
• The indirect process of conversion from the excited state produced by absorption of energy with an even number of
electrons, to an excited trinergy, the singlet state, to a triplet state, is known as intersystem crossing.
• Direct transition from the ground state, usually a singlet state, for a molecule triplet state is theoretically forbidden, which
means that the reverse transition from triplet to ground state will be difficult.
• Thus, while the transition from an excited singlet state, for example, S1, to the ground state with the emission of
fluorescence can take place easily and within 109 - 106 seconds, the transition from an excited triplet state to the ground
state with the emission of phosphorescence requires at least 104 seconds and may take as long as 102seconds.
• The triplet state of a molecule has a lower energy than its associated singlet state so that transitions back to the ground
state are accompanied with the emission of light of lower energy than from the singlet state. Therefore, we would typically
expect phosphorescence to occur at longer wavelengths than fluorescence.

13. Applications
• Currently, phosphorescent materials have a variety of uses, and molecular phosphorescence
spectrometry is applicable across many industries.
• Phosphorescent materials find use in radar screens, glow-in-the-dark toys, and in pigments, some of
which are used to make highway signs visible to drivers.
• Molecular phosphorescence spectroscopy is currently in use in the pharmaceutical industry, where its
high selectivity and lack of need for extensive separation or purification steps make it useful.
• It also shows potential in forensic analysis because of the low sample volume requirement.

14. Reference
• https://www.vedantu.com/physics/fluorescence-and-phosphorescence
• https://en.wikipedia.org/wiki/Fluorescenc
• https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supple
mental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Electronic_Spectroscopy/Fluo
rescence_and_Phosphorescence
• https://www.britannica.com/science/spectroscopy/Fluorescence-and-phosphorescence
• https://microbenotes.com/fluorescence-microscope-principle-instrumentation-applications-
advantages-limitations/