1. Excitation:
A sample is exposed to a beam of light, typically from a UV or visible light source.
2. Absorption:
Molecules in the sample absorb this light and get excited to a higher energy state.
3. Non-Radiative Relaxation:
Before fluorescence, there's usually a loss of some energy through non-radiative processes, leading the molecule to a lower vibrational level within the excited state.
4. Emission:
The excited molecules return to their ground state by releasing the absorbed energy in the form of emitted light (fluorescence).
This emitted light is typically of longer wavelength (lower energy) than the excitation light due to the energy loss in non-radiative relaxation.
5. Detection:
A detector, placed perpendicular to the excitation beam to minimize interference, measures the intensity of the emitted light.
6. Spectral Analysis:
Fluorescence spectra are plotted, with emission intensity versus emission wavelength, providing qualitative and quantitative information about the fluorophores in the sample.
7. Quantification:
By comparing the fluorescence intensity of unknown samples to calibration standards, the concentration of fluorescent species can be determined.
