Untitled Quiz

Fluorescence Spectroscopy

• 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

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

Fluorescence

• 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.

Jablonski Diagram

• 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 Process

• 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.

Principles of Fluorescence

• 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.

Fluorescence Process (detailed)

• 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.

Vibrational Relaxation

• 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.

Internal Conversion

• 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.

Fluorescence Process (steps)

• Excitation (Absorption) - 10-15 seconds
• Internal Conversion and Vibrational Relaxation - 10-12 seconds
• Fluorescence Emission - 10-9 seconds

Fluorescence vs Phosphorescence

• 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

Intersystem Crossing

• 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.

Phosphorescence

• 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).

Fluorometer

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

Fluorescence Emission Spectrum

• 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.

Stoke's Shift

• 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

Ideal Fluorescent Molecule

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

Fluorophore Structure

• 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.

Qualitative Analysis

• Selectively identifies fluorescent agents.
• Tracks drug-target interactions, especially useful for biopharmaceuticals.

Quantitative Analysis

• Fluorescence intensity proportional to fluorophore concentration to a limit.
• Obeys the Beer-Lambert law.

Interfering Factors

• 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.

Other Factors

• Turbidity (inhomogenous solutions), scattering of light away from detector.
• Bubbles
• Photochemical decay (photobleaching)

Applications

• 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

Learning Outcomes

• Explain principles of fluorescence spectroscopy
• Describe fluorescence process
• Understand components of fluorometer
• Identify molecules exhibiting fluorescence
• Factors interfering with quantitative analysis by fluorescence
• Examples of pharmaceutical applications.

Recommended Reading

• Watson, D.G., "Pharmaceutical Analysis" Chapter 7.
• Available online; resources available via Biomedical and McClay libraries.