Fluorescence was first observed about 550 years ago in the extracts of wood of certain trees. This phenomenon is observed in minerals (i.e., aragonite (CaCO₃), calcite (CaCO₃), rubies, emeralds, diamonds), biological systems (i.e., certain scorpions, fish, parrots, flowers) and organic chemicals (i.e., anthracene in toluene). Note that fluorescence is also found outside the lab in tonic water (blue to the presence of quinine, see below), in highlighters (pyranine), in bank notes (security feature), etc.

Fundamentally speaking, fluorescence is the emission of light by a substance that has previously absorbed electromagnetic radiation. In order to excite an electron from the ground state (S₀) to the excited state (S₁), a certain amount of energy is required (purple arrow below). The excited molecule can subsequently relax by various pathways. In a non-radiative relaxation, the energy is dissipated as heat in form of vibrational energy to the solvent (red arrows). Due to the lower remaining energy of the electron, the emitted light displays a longer wavelength than the absorbed radiation (green arrow). Depending on the intensity of the absorbed light and the quantum yield, the fluorescence can be more or less intense.

In Chem 30BL, the students will look at the fluorescence of hymecromone. The spectrum below on the right (solid red line) is the emission spectrum of hymecromone in methanol for the excitation at λ=365 nm.² Note that the peak maximum is at λ=445 nm, which means that there is a 80 nm Stokes' shift. Going backwards from this peak maximum, one can acquire the excitation spectrum (dashed black line), which shows that the used wavelength of λ =365 nm is very close of the excitation maximum. Note that the correlating maxima in the spectra are very close in intensity, which means that most of the excited photons are emitted during the emission. The quantum yield Φ is very high (=photons emitted/ photons absorbed) in this case. Thus, if the compound is excited using the long wavelength of the UV-lamp, it will appear bluish to the observer. If the student would use different wavelength, the emission would be weaker.

In strongly acidic conditions, the excitation wavelength shifts to λ_ex=320 nm and the emission from λ_em=445 nm to 455 nm because of the protonation of the carbonyl oxygen atom (I). In near neutral pH-values (pH=4-7), the molecular form of the compound (II) is observed, which displays an enhanced emission at λ_em =445 nm. Under weakly basic conditions (pH=9-12), the mono-anion (III) is favored due to the deprotonation of the phenol group. A very strong emission is observed atλ_em =445 nm. In strongly alkaline conditions (pH>12), the lactone undergoes hydrolysis (IV) leading to a decreased fluorescence.¹

(I)                                                                     (II)                                                               (III)                                                           (IV)

References:

1. Zhi, H.; Wang, J.; Wang, S.; Wei, Y. J. Spectrosc. 2013, Article ID 147128

2. Thanks to Dr.Martin Phillips for the acquisition of the spectra