Liquid chromatography fluorescence detector (FLD) is a highly sensitive and selective detector suitable for compounds that can produce fluorescence or substances that emit light after derivatization. Its core advantage lies in its sensitivity that can reach many times that of ultraviolet detectors, and strong anti-interference ability.
The working mechanism of fluorescence detector is based on photoluminescence phenomenon, and the core steps are as follows:
The excitation light source (commonly xenon lamp, mercury lamp or laser diode) emits excitation light of a specific wavelength, which is filtered by a monochromator (grating or filter) to select a single wavelength excitation beam and focused on the effluent (detection cell) of the chromatographic column. When the tested component molecule absorbs the excitation light energy, it will transition from the ground state (stable state) to the excited state (high-energy state).
Molecules emitting fluorescence in an excited state are unstable and will lose some energy in a short period of time (10 ⁻⁸~10 ⁻⁴ seconds) through non radiative transitions, returning to the low vibrational level of the excited state, and then releasing photons through radiative transitions, returning to the ground state. The light produced in this process is fluorescence. The wavelength of fluorescence is always greater than the excitation wavelength (Stokes shift), which is a key characteristic of fluorescence detection.
To avoid interference from excitation light, the fluorescence receiving system of the detector (photomultiplier tube PMT or photodiode) is usually placed at a 90 ° angle to the excitation light path for signal acquisition and conversion. After the emitted fluorescence is selected by a monochromator to have characteristic fluorescence wavelengths, it is received by a photodetector and converted into an electrical signal. The electrical signal intensity shows a linear relationship with the concentration of the tested component within a certain range. After amplification and data processing, chromatographic peaks and quantitative results are obtained.
Testing process:
Excitation light sources (such as xenon lamps or lasers) emit a continuous spectrum, which is then separated by a monochromator to select a specific wavelength as the excitation light.
Excitation light illuminates the fluorescent substance in the sample pool, producing fluorescence.
The fluorescent signal is detected by a photosensitive element (such as a photomultiplier tube) through a right angled optical path (to avoid excitation light interference) and converted into an electrical signal.
After amplification, the electrical signal is transmitted to the data processing system to generate a chromatogram.
Liquid chromatography fluorescence detector has become an important tool for trace analysis due to its high sensitivity and selectivity, especially suitable for fields such as biology and environment. However, its non universality and sensitivity to interference limit its application scope. By combining laser light sources, combined technologies, or derivative methods, its analytical capabilities can be further expanded to meet the needs of complex sample detection.