What is Fluorescence Analysis?


Fluorescence Analysis

Fluorescence analysis refers to the use of certain substances that are in an excited state after being irradiated by ultraviolet light, and the excited state molecules undergo a de-excitation process of collision and emission, which can reflect the characteristics of the substance. The method of qualitative or quantitative analysis . Since some substances themselves do not emit fluorescence (or the fluorescence is very weak), it is necessary to convert the substances that do not emit fluorescence into substances that emit fluorescence. For example, some reagents (such as fluorescent dyes) are used to form complexes with substances that do not emit fluorescence, and various complexes can emit fluorescence, and then measure. Therefore, the use of fluorescent reagents opens the door to fluorescent analysis of some inorganic and organic substances that do not fluoresce originally, and expands the scope of analysis.

Basic Information

①Direct fluorescence photometry
②As a detector for HPLC (used more)
According to the energy level transition mechanism of the absorption spectrum and fluorescence spectrum of the material molecules, the material with the ability to absorb photons can instantly emit light with a wavelength longer than the excitation light, that is, fluorescence, when irradiated by light of a specific wavelength (such as ultraviolet light).

direct assay

Determination and analysis are carried out using the fluorescence emitted by the substance itself.

indirect assay

Regardless of whether it is a direct measurement or an indirect measurement, the standard working curve method is generally used. Various known amounts of fluorescent substances are taken and prepared into a series of standard solutions. The fluorescence intensity of these standard solutions is measured, and then the fluorescence intensity is given. The working curve of the concentration of the standard solution. Under the same instrument conditions, measure the fluorescence intensity of the unknown sample, and then find out the concentration (ie content) of the unknown sample from the standard working curve.
The commonly used fluorescence analysis instruments are: visual fluorescence meter (fluorescence analysis lamp), fluorescence photometer and fluorescence spectrophotometer.

Production of fluorescence

According to the Boltzmann distribution, molecules are basically in the ground state of the electronic energy level at room temperature. After absorbing ultraviolet-visible light, the electrons in the ground state molecule can only transition to different vibration-rotation energy levels of the excited singlet state, and cannot directly transition to the vibration-rotation energy levels of the excited triplet state according to the spin-forbidden selection law. energy level.
Molecules in the excited state are unstable, and usually release excess energy through radiative transitions and nonradiative transitions to return to the ground state, and fluorescence emission is one of the ways.

Vibrational relaxation

It is a process in which the molecules in each vibrational energy level of the excited state transfer part of the vibrational energy to the solvent molecules by colliding with the solvent molecules, and the electrons return to the lowest vibrational energy level of the same electronic excited state. Since energy is not released in the form of optical radiation, vibrational relaxation is a non-radiative transition. Vibrational relaxation can only take place within the same electron energy level.

Internal conversion

Referred to as internal conversion, when the energy difference between two electronic excited states is so small that their vibrational energy levels overlap, the excited molecule is often transferred from a high electronic energy level to a low electronic energy level in a non-radiative manner.

fluorescence emission

No matter which excited singlet state the molecule is initially in, through internal conversion and vibrational relaxation, it can return to the lowest vibrational energy level of the first excited singlet state, and then emit photons in the form of radiation to return to any vibrational state of the ground state At the energy level, the light quantum emitted at this time is called fluorescence. The wavelength of fluorescence is always longer than that of the excitation light due to the loss of some energy from vibrational relaxation and internal conversion. Since electrons can stay at any vibrational level of the ground state when they return to the ground state, the resulting fluorescence lines sometimes exhibit several very close peaks. Through further vibrational relaxation, these electrons all quickly return to the lowest vibrational energy level of the ground state.

External energy conversion

Abbreviated as exoconversion, it is a process in which excited state molecules in a solution collide with solvent molecules or other solute molecules to lose energy and release energy in the form of thermal energy. External transitions often occur during the transition from the lowest vibrational energy level of the first excited singlet state or excited triplet state to the ground state. Exoconversion reduces fluorescence intensity.

Interaystem crossing

It is a process in which electrons in an excited state molecule undergo spin inversion and change the multiplicity of the molecule. After the molecule crosses from the excited singlet state to the excited triplet state, the fluorescence intensity is weakened or even extinguished. In molecules containing heavy atoms such as iodine, bromine, etc., intersystem crossing is the most common, because in atoms with high atomic numbers, the interaction between electron spin and orbital motion is large, which is conducive to electron spin inversion. happened. In addition, the presence of paramagnetic substances such as oxygen molecules in the solution is also prone to crossover between systems, thereby weakening the fluorescence.

Phosphorescence emission

Molecules that have crossed between systems are then reduced to the lowest vibrational energy level of the excited triplet state through vibrational relaxation. The molecules can survive for a period of time at the lowest vibrational energy level of the excited triplet state, and then return to the vibrational energy levels of the ground state to emit light radiation. , this light emission is called phosphorescence. Because the energy level of the excited triplet state is lower than the lowest vibrational energy M of the excited singlet state, the energy of phosphorescence radiation is smaller than that of fluorescence, that is, the wavemin of phosphorescence is longer than that of fluorescence. Phosphorescence emits later than fluorescence because the molecule has a longer lifetime in the excited triplet state. Due to the influence of factors such as the collision between fluorescent substance molecules and solvent molecules, the molecules in the excited triplet state are often deactivated back to the ground state by the X-radiation process, so they rarely exhibit phosphorescence at room temperature, and they can only be reduced by freezing or immobilization. Phosphorescence cannot be detected until conversion, so phosphorescence is not as common as fluorescence analysis.


Fluorescence analysis is an advanced analysis method, which is more widely used and popular than electron probe method, mass spectrometry, spectroscopy, polarography, etc., which has many advantages inseparable from fluorescence analysis. The equipment used for fluorescence analysis is relatively simple, such as visual fluorometer and fluorescence photometer, which are very simple and can be manufactured by themselves. Compared with mass spectrometers, polarographs and electron probes, it is many times cheaper in cost, and the biggest features of fluorescence analysis are: high analytical sensitivity, strong selectivity and easy use. There are not many instruments that have these three characteristics at the same time.

high sensitivity

The biggest feature of fluorescence analysis is its high sensitivity. The sensitivity of fluorescence analysis is 2 to 3 orders of magnitude higher than that of spectrophotometry. This is because the fluorescence of fluorescence analysis and the incident light form a right angle, not in a straight line, so it is Fluorescence was detected on a black background. In spectrophotometry, the receiver is in line with the incident light, so it is detected against a bright background. Therefore, the fluorescence analysis method is more sensitive than the spectroscopic method. The sensitivity of spectroscopic spectroscopy can generally only detect
grams, a difference of three orders of magnitude. Of course, the fluorescence analysis method is less sensitive than the electron probe method with an electron microscope, but the electron probe instrument is expensive and inconvenient to use.

Strong selectivity

The second characteristic of fluorescence analysis is its strong selectivity, especially for organic compounds. Because the fluorescence spectrum includes both the excitation spectrum and the emission spectrum, any substance that can emit fluorescence must first absorb ultraviolet rays of a certain wavelength, and after absorbing ultraviolet rays, it may not necessarily emit fluorescence. Substances that emit fluorescence have different fluorescence wavelengths. If even the fluorescence spectrum is the same, its laser spectrum is not necessarily the same. On the contrary, if their excitation spectra are the same, they can be distinguished by emission spectra, so there are more choices. Therefore, fluorescence analysis is highly selective. For example, there are two substances whose fluorescence spectra are very similar, and it is not easy to separate them. But their laser spectra will not be the same, so they can be separated by scanning the laser spectrum. It is difficult to do this by spectroscopic spectroscopy, because spectroscopic spectroscopy can only obtain the characteristic absorption spectrum of the substance to be tested. Therefore, the selectivity of spectroscopic spectroscopy is not as strong as that of fluorescence analysis.


Fluorescence Analysis of Organic Matter

The fluorescence analysis of organic compounds is widely used, and there are hundreds of organic substances that can be determined, such as the fluorescence analysis of enzymes and coenzymes, the fluorescence analysis of pesticides and poisons, the fluorescence analysis of amino acids and proteins, and the fluorescence analysis of nucleic acids. These constitute the main content of fluorescence analysis technology. Many organic compounds do not fluoresce strongly or do not fluoresce under ultraviolet irradiation, so certain organic reagents must be used so that the resulting product can emit strong fluorescence under ultraviolet irradiation. For example, aliphatic organic compounds are determined by indirect methods.

Fluorescence Analysis of Inorganic Elements

There are not many chemical elements that can directly emit fluorescence under ultraviolet irradiation, so most of the fluorescence analysis of some elements adopts indirect determination method, which is to use organic reagents to form complexes with the elements to be determined. These complexes can emit different wavelengths of fluorescein under ultraviolet irradiation, and then the content of this element is determined from the fluorescence intensity. Due to the wide variety of organic fluorescent reagents, there are more than 60 elements that can be determined by fluorescence analysis.
For example, the fluorescence analysis of lead: lead ions Pb and Cl ions form a lead-chloride complex, which emits blue fluorescence when excited by short-wave ultraviolet light at 270 nm, and the fluorescence peak wavelength is at 480 nm. The content of Pb was determined on the standard working curve according to the fluorescence intensity. This method can determine 0.1~0.6 μg lead/ml.