fluorescence spectroscopy

Meaning: A molecule absorbs a photon, and an electron moves to an excited electronic state. The molecule quickly loses some energy as heat through vibrational relaxation, then returns to the ground state and emits a photon. Because energy was lost first, the emitted photon typically has lower energy and a longer wavelength than the absorbed photon. This gap is the Stokes shift. Fluorescence emission follows absorption within a very short time (nanoseconds). Phosphorescence is a slower, related process. The emission intensity can report on concentration, binding or the local environment, which is why fluorescent tags such as GFP are useful.

Equation: E = hf = hc/λ, where E is photon energy (J), h is Planck's constant (6.63 × 10⁻³⁴ J·s), f is frequency (Hz), c is the speed of light (3.00 × 10⁸ m/s), and λ is wavelength (m).

On the MCAT: A passage gives excitation and emission wavelengths or plots a spectrum. You compare which light has the longer wavelength, higher frequency or greater energy. You may also pick a detection wavelength or filter, or predict how emission changes with concentration or after quenching.

Common confusion: Students treat the emitted light as the same color as the excitation light, or as scattered excitation light. Emission is usually lower in energy, so its wavelength is longer and its frequency lower. Absorption spectroscopy measures light removed from a beam. Fluorescence measures new light given off by the sample.