Solvent Effects on Fluorescence Emission

Introduction

• Environmental factors significantly influence fluorescence emission.
• Key factors include:
  • Interactions between fluorophore and solvent molecules.
  • Solvent polarity, dissolved inorganic and organic compounds.
  • Temperature, pH, and localized concentration of fluorescent species.
• Absorption and emission spectra, and quantum yields, are sensitive to these factors.
• Fluorescence sensitivity is linked to local environment interactions during the excited state lifetime.
• Tutorial explores relaxation effects and spectral shifts as a function of solvent polarity.

Jablonski Energy Diagram

• Tutorial uses a Jablonski energy diagram.
• Displays a fluorophore with solvent molecules above a spectral plot.
• Indicates dipole moments of fluorophore and solvent molecules with arrows.

Solvent Polarity Effects

• Fluorophore excitation:
  • Molecule excited to Franck-Condon excited state.
  • Solvent relaxation follows, shifting emission spectrum to longer wavelengths.
• Solvent polarity changes with a slider.
  • Higher polarity lowers solvent-relaxed S(1) energy level.
  • Increases solvent re-orientation with respect to fluorophore dipole.

Solvent-Fluorophore Interaction

• Solvent molecules interact with ground state fluorophore dipole moment.
• Energy level differences cause rearrangement of surrounding molecules.
• Franck-Condon principle:
  • Excitation to a higher electronic energy level occurs faster than molecular re-orientation.
• Time delay occurs between excitation and solvent molecule re-ordering.

Solvent Relaxation and Energy Levels

• Post-excitation:
  • Fluorophore loses excess vibrational energy rapidly.
  • Solvent molecules re-orient around the fluorophore.
  • Solvent relaxation reduces energy separation between states.
• Results in a red shift in fluorescence emission.
• Increased solvent polarity notably reduces excited state energy level.
• Polarity of fluorophore affects sensitivity to solvent effects.
  • Polar/charged fluorophores exhibit stronger effects.

Applications and Observations

• Solvent effects can significantly impact Stokes shifts.
• Example: Tryptophan in protein denaturation:
  • Shift from non-polar to polar environment increases emission wavelength.
• Utilization:
  • Probing solvent polarity effects, molecular associations, complex formation.

Considerations for Quantitative Fluorescence

• Monitor for potential emission profile shifts.
• Homogeneous systems show progressive emission intensity increase with concentration.
• Complex systems may exhibit intensity fluctuations or spectral shifts due to:
  • pH changes, calcium ion concentrations, energy transfer, quenching agents.
• Consider unexpected solvent or environmental effects in experimental evaluations.

Contributing Authors

• Brian Herman - Department of Cellular and Structural Biology, University of Texas Health Science Center.
• Joseph R. Lakowicz - Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland.
• Ian D. Johnson and Michael W. Davidson - National High Magnetic Field Laboratory, Florida State University.