Fluorescence Spectroscopy Quiz

Fluorescence Spectroscopy: Key Concepts and Principles

• Fluorescence typically occurs at a rate of 10^8 per second, in contrast to phosphorescence, which has a slower emission rate of 1 to 100 per second.

• Fluorescence was first observed in quinine, a compound found in tonic water, back in 1845, leading to the term "fluorophores" for fluorescent substances.

• Fluorescence involves pi electrons in aromatic groups, with emission spectra displaying vibrational energy levels.

• The emission and absorption spectra in fluorescence are mirror images of each other, due to the Frank-Condon principle, which states that electronic transitions do not alter nuclear geometry.

• The Stokes shift in fluorescence, observed by Sir G.G. Stokes, explains the energy loss from absorption to emission, attributed to internal conversion and other factors.

• Kasha's rule in fluorescence spectroscopy states that the same fluorescence emission spectrum is observed irrespective of the excitation wavelength, except for certain exemptions.

• The emission spectrum of quinine is not an exact mirror image of its absorption due to transitions from different energy levels, which is explained by the internal conversion process.

• The symmetry of the absorbance and emission spectra is a result of the same transitions being involved in both processes, and similar vibrational energy levels of S0 and S1.

• Quantum yield and lifetime are essential parameters in fluorescence spectroscopy, with the former measuring the efficiency of the fluorescence process, and the latter determining the time available for the fluorophore to interact with its environment.

• Quantum yields close to one indicate bright emissions, while lifetime affects the information available from a fluorophore's emission.

• Fluorescence spectroscopy involves a wavelength scan, where the same sample is exposed to various light wavelengths to observe the fluorescence pattern, as per Kasha's rule.

• The emission spectrum of a fluorophore displays the energy difference between its vibrational energy levels, allowing for the determination of the energy gap between specific peaks.Fluorescence Spectroscopy and Quantum Yield

• Quantum yield (Q) is the ratio of emitted photons to the absorbed photons, determined by the radiative decay constant (γ) and the non-radiative decay rate constant (KNR).

• Quantum yield (Q) can approach unity when the non-radiative decay rate (KNR) is significantly smaller than the radiative decay rate (γ).

• Energy yield of fluorescence is always less than unity due to Stokes shift, where emitted energy is lower than absorbed energy.

• Fluorescence lifetime (τ) is the average time a molecule spends in the excited state before returning to the ground state, typically around 10 nanoseconds.

• Fluorescence is a random process, not all molecules emit photons at the same time, explaining the persistent observation of fluorescence.

• Differences in quantum yield and lifetime of similar molecules, such as aocene and erythrosine B, are attributed to variations in non-radiative decay rates (KNR).

• Heavy atoms like iodine result in shorter lifetimes and lower quantum yields due to increased non-radiative decay rates.

• The quantum yield of a fluorophore can be inferred from its emission spectrum intensity, but not its lifetime.

• The natural lifetime of a fluorophore, in the absence of non-radiative decay, is calculated from the intrinsic radiative decay rate (γ).

• The radiative decay rate (γ) can be calculated using an equation involving the emission and absorption spectra and the refractive index of the medium.

• Comparison of natural lifetime, measured lifetime, and quantum yield can provide valuable insights into the behavior of fluorophores.

• For instance, the measured lifetime of the widely used membrane probe dph is much longer than the calculated natural lifetime, near 10 nanoseconds.