Untitled Quiz

Fluorescence spectroscopy is a technique used to analyze medicinal substances.
The lecture covers the principles of fluorescence, instrumentation, interfering factors, and applications in pharmaceutical analysis.
Key learning outcomes include explaining the principles of fluorescence, describing the fluorescence process, understanding the components of a fluorimeter, identifying fluorescent molecules, describing interfering factors in quantitative analysis, and providing pharmaceutical application examples.

Luminescence is the emission of light by a molecule stimulated by light energy/photons.
Types of luminescence include fluorescence, phosphorescence, and chemiluminescence.

Fluorescence occurs when a photon is absorbed by a molecule, causing an electron to be excited to a higher energy level.
The excited state is unstable, resulting in rapid thermal energy loss via vibrations.
The molecule returns to its ground state, emitting a photon of lower energy (longer wavelength)
The emitted photon allows the molecule to return to its original ground state.

The Jablonski diagram visually represents the energy levels and transitions involved in fluorescence.
It displays the various energy levels (electronic, vibrational) and the corresponding time scales for transitions.
Transitions, including absorption, fluorescence, phosphorescence, internal conversion, vibrational relaxation, and intersystem crossing, are labelled with their respective timeframes.

Fluorescence involves three key events:
- Excitation of a susceptible molecule by an incoming photon (10-15 seconds), causing the molecule to move to an excited state.
- Vibrational relaxation of the excited state electrons to the lowest vibrational level of an excited state (10-12 seconds). This involves the dissipation of excess energy from the vibrational states.
- Emission of a longer wavelength photon (10-9 seconds) with the molecule returning to its original ground state.

Molecular energy levels are depicted with excited singlet states above the ground state.
Excitation by a photon can move an electron to an excited singlet state.

Excitation of a molecule by an incoming photon (10-15 seconds).
Vibrational relaxation of excited state electrons to lower vibrational levels of that excited state (10-12 seconds).
Emission of a longer wavelength photon (10-9 seconds), returning to the ground state.

Transition from a higher vibrational energy level to a lower vibrational level within the same electronic state.
Energy released in the form non-radiative kinetic energy, within one electronic state.
Same electronic level, lower vibrational level.

Excited electron transitions from a vibrational level within one electronic state to another vibrational level in a lower electronic state.
Transition happens due to overlapping of vibrational levels in different electronic levels, involving transition between different electronic states.
Non-radiative process involving energy dissipation in the form of emitted photons within electronic states.
Occurs instantaneously upon photon absorbance.

Both are types of luminescence
Fluorescence is characterized by almost instantaneous (nanoseconds) emission.
Phosphorescence involves a longer lifetime (milliseconds/seconds).
Phosphorescence involves intersystem crossing to a triplet state, which has a different spin multiplicity and a longer lifetime

Transition between excited singlet and excited triplet states.
Involves a change in electronic state and spin multiplicity, resulting in a longer lifetime for the triplet state than for the singlet state.

Emission of light from an excited triplet state to a singlet ground state.
Characterized by long lifetimes (milliseconds to seconds).
Often involves intersystem crossing (a transition from one spin state to another during the relaxation process).

Similar to UV Spec, uses a light source, monochromator, sample holder, and detector to measure fluorescence emission spectra.

The excitation of a fluorescent molecule at a single wavelength provides an emission spectrum, with the Y-axis displaying fluorescence intensity and the X-axis emission wavelength.
Emission spectrum shows peaks with the highest intensity of emitted light.

The distance between excitation and emission wavelengths.
The Stokes shift is a useful tool in distinguishing fluorescence from other types of light-emitting phenomenon because of the large energy difference

Characteristics include a high extinction coefficient, high quantum yield, large Stokes shift, and a long emission wavelength.

Structurally rigid molecules with multiple conjugated double bonds (or cyclic structures).
The rigidity allows for less vibrational relaxation, making energy transfer to the emitted photons more efficient.

Quenching — some molecules can decrease fluorescence intensity by interacting with the excited fluorophore. Include collisional quenching, excited state reactions, energy transfer, and complex formation.
Concentration — high concentration can cause non-linearity between concentration and fluorescence emission.
Temperature — higher temperatures increase the probability of deactivation.
PH — changes in pH can affect wavelength and emission intensity; some molecules lose their fluorescence at higher pH values
Inner filter effects — reabsorption of emitted light by other compounds in solution.

Drug Analysis & Identification — ensuring safe and effective drug preparations, useful in quality control, and providing patients and healthcare providers with confidence in the quality.
Clinical Pharmacy — therapeutic drug monitoring (ADME — Absorption, Distribution, Metabolism, Excretion), detecting drugs in biological fluids (e.g., blood, urine).
Drug Dissolution — determining how quickly drugs dissolve, quantify drug amount.
Drug Target Interactions — understanding how drugs interact, fluorescent probes