Fluorescence detector

The fluorescence detector is a commonly used detector for high pressure liquid chromatography. Ultraviolet rays are used to irradiate the chromatographic fraction. When the sample components have fluorescent properties, they can be detected. Its characteristics are high selectivity, only respond to fluorescent substances; sensitivity is also high, the minimum detection limit of up to 10-12g/ml, suitable for trace analysis of polycyclic aromatic hydrocarbons and various fluorescent substances. It can also be used to detect substances that do not fluoresce but can fluoresce after chemical reactions. For example, most phenols do not fluoresce in the analysis of phenols. For this purpose, they are first treated to make them fluorescent, and then analyzed. Working Principles Fluorescence Generation From the point of view of electronic transitions, fluorescence refers to the transition of certain electrons in an atom from the lowest vibrational energy level in the ground state to higher levels after certain substances have absorbed light with the same frequency as its own characteristic. Some vibration levels. Electrons collide in homogeneous molecules or other molecules, consuming a considerable amount of energy, and thus dropping to the lowest vibrational level in the first electronically excited state. This transfer of energy is called a radiative transition. From the lowest vibrational energy level to some of the different energy levels in the ground state, a light with a lower frequency and a longer wavelength than originally absorbed is emitted, that is, fluorescence. The light absorbed by the compound is called excitation light, and the resulting fluorescence is called emission light. The wavelength of fluorescence is always longer than the wavelength of ultraviolet light absorbed by the molecule, and is usually in the visible light range. The nature of fluorescence is closely related to the molecular structure. After molecules of different structures are excited, they cannot emit fluorescence.

Quantitative basis In photoluminescence, the emitted radiation is always dependent on the amount of radiation absorbed. Since an excited molecule returns to the ground state, it may generate energy loss in the form of no radiative transition. Therefore, the number of photons emitting radiation is usually less than the number of photons that absorb radiation. It is expressed by the quantum efficiency Q.

Under fixed experimental conditions, the quantum efficiency is a constant, usually Q is less than 1. For substances that can be used for fluorescent detection, the Q value is generally between 0.1 and 0.9. The fluorescence intensity F is proportional to the intensity of the absorbed light.

For dilute solutions, the fluorescence intensity is positively correlated with the concentration of the fluorescent substance solution, the molar absorptivity, the thickness of the absorption cell, the incident light intensity, the quantum efficiency of the fluorescence, and the fluorescence collection efficiency. The fluorescence intensity of a substance is directly proportional to the concentration of the substance under the condition that other factors remain unchanged, which is the quantitative basis of the fluorescence detector. Fluorescence detectors are solute detectors that can be used directly for quantitative analysis.

Excitation spectrum and emission spectrum fluorescence involve the two processes of light absorption and emission. Therefore, any fluorescent compound has two kinds of characteristic spectrum: excitation spectrum and emission spectrum.

Fluorescence belongs to photoluminescence, and it is necessary to select an appropriate excitation wavelength (Ex) to facilitate detection. The excitation wavelength can be determined by the excitation spectrum of the fluorescent compound. The specific detection method of the excitation spectrum is to scan the excitation monochromator so that the incident light of different wavelengths excites the fluorescent compound, and the generated fluorescence is detected by the light detection element through a fixed wavelength emission monochromator. The resulting fluorescence intensity versus excitation wavelength curve is the excitation spectrum. At the maximum wavelength of the excitation spectrum curve, the number of molecules in the excited state is the largest, that is, the absorbed light energy is also the highest, and the strongest fluorescence can be generated. When considering sensitivity, the determination should select the maximum excitation wavelength.

The so-called fluorescence spectrum actually refers only to the fluorescence emission spectrum. It is a curve that the fluorescence intensity of a monochromator for wavelength scanning is changed with the fluorescence wavelength (ie, the emission wavelength, Em) when the monochromator wavelength is fixed. Fluorescence spectroscopy can be used to identify fluorescent substances and serve as a basis for selecting an appropriate measurement wavelength for fluorescence measurement.

In addition, due to the characteristics of the fluorescence measuring instrument, the energy distribution of the light source, the transmittance of the monochromator, and the response of the detector will change with the wavelength, so that the same compound will have different spectra on different instruments, and No analogy with each other, this spectrum is called the apparent spectrum. To make the same compound have fluorescence spectra with the same characteristics on different instruments, the above characteristics of the instrument need to be corrected. The corrected spectrum is called a true fluorescence spectrum.

Excitation wavelength and emission wavelength are necessary parameters for fluorescence detection. Choosing the proper excitation wavelength and emission wavelength is very important for the sensitivity and selectivity of the detection, especially the detection sensitivity can be greatly improved.