Molecular fluorescence spectroscopy
Molecular fluorescence spectra and development history
1. principle
In the absorption of ultraviolet and visible electromagnetic radiation in the process of molecular excitations excited excited state, most of the molecules will collide with other molecules in the heat way to dissipate this part of the energy, some molecules in the form of light radiation out of this part Energy, the wavelength of the radiated light is different from the wavelength of the absorbed radiation. The latter process is called photoluminescence. Molecular luminescence includes fluorescence, phosphorescence, chemiluminescence, bioluminescence and scattering spectroscopy. The analytical method that is based on the fluorescence measurement of a compound is called molecular fluorescence spectroscopy.
The light emitted by the light source is turned into intermittent light by the light cutting device and becomes monochromatic light by the excitation light monochromator, which is the excitation light of the fluorescent material. Fluorescence emitted by the measured fluorescent substance under excitation light is changed into monochromatic fluorescent light by a monochromator and then irradiated on the photomultiplier tube. The photocurrent generated by the measured fluorescent substance is amplified by an amplifier to be input to the recorder. One excitation, one emission, using a dual monochromator system, can measure the excitation spectrum and the fluorescence spectrum, respectively.
2. classification
Fluorescence spectrometer is the basic equipment to determine the luminescent properties of materials. General fluorescence spectrometer can be divided into three kinds:
(1) Basic: Steady-state spectrometer at 200-800 nm in the UV-visible band.
(2) Expanded: Ultraviolet visible-near-infrared steady-state spectrometer covering 200-1700 nm band.
(3) Integrated: a spectrometer that covers the above two bands while measuring transient spectra.
3. The main purpose
(1) fluorescence excitation and fluorescence emission spectra;
(2) Synchronous fluorescence (wavelength and energy) scanning spectra;
(3) 3D (Ex Em Intensity);
(4) Time Base and CWA (fixed wavelength single point measurement);
(5) Fluorescence lifetime measurement, including life-time resolution and time resolution;
(6) Computer to collect spectral data and process data (Datamax and Gram32).
4. Development History
For the first time a fluorescent phenomenon was recorded by Spanish physician and botanist N. Monardes in the 16th century, who in 1575 referred to the extremely lovely sky blue in an aqueous solution containing a slice of wood called "Lignum Nephriticum" . In the 17th century, famous scientists such as Boyle (1626-1691) and Newton (1624-1727) once again observed the phenomenon of fluorescence. Fluorescence then aroused the interest of many scientists, fluorescence analysis methods are also more and more applied to biological and chemical analysis.
Of course, the development of fluorescence analysis method is inseparable from the development of instrument application. In general, fluorescence spectrometer has been through three stages of development since its inception:
(1) manual;
(2) automatic scanning
(3) Computerized.
Before the 19th century, the observation of fluorescence was carried out by the naked eye. It was not until 1928 that Jette and West proposed the first photoelectric fluorometer. The sensitivity of the photoelectric fluorometer is limited. After the invention of a photomultiplier tube by Zworykin and Rajchman in 1939, it was a very important phase in terms of increasing sensitivity and allowing the use of monochromators with higher resolution. In 1943 Dutton and Bailey proposed a manual correction step of fluorescence spectroscopy. The first automatic spectral correction device was introduced by Studer in 1948 and only appeared in 1952 as commercial calibrated spectrometers.
The main components and functions
Fluorescence spectrometer mainly includes light source, excitation monochromator, sample cell, fluorescent monochromator and detector and other major components.
1. light source
Early fluorescent spectrophotometers equipped with low pressure mercury lamps that produced very narrow mercury lines. With high pressure mercury lamps, the spectrum is broadened and there is also a continuous band of high strength. However, a complete excitation spectrum measurement requires a lamp capable of emitting higher intensity light radiation from the visible to the ultraviolet range. Xenon arc lamps are suitable for this condition and as such are the most widely used light sources in fluorescence spectrophotometers.
2. Monochromator
The function of the monochromator is to decompose the continuous spectrum emitted by the light source into monochromatic light and to accurately and conveniently "remove" the light of a certain wavelength that is required, which is the heart of the spectrometer. Monochromators are mainly composed of slits, dispersive elements and lens systems, of which dispersive elements are the key components. Dispersive elements are prisms and reflective gratings or a combination of the two that disperses continuous spectrum light into monochromatic light.
(1) prism monochromator
Prism monochromator is the use of different wavelengths of light in the prism of different refractive index composite light will be dispersed as monochromatic light. Prismatic dispersion of the size of the prism material and the geometry of the production. Common prism made of glass or quartz. Visible spectrophotometer can be used glass, it is suitable for UV, the entire visible spectrum.
(2) grating monochromator
Gratings have many unique advantages as dispersive components. Raster can be defined as a series of equal width, equidistant parallel slit. The principle of dispersion of a grating is based on the phenomenon of diffraction and interference of light. Commonly used grating monochromator for the reflective grating monochromator, which is divided into two kinds of planar reflective grating and concave reflective grating, one of the most commonly used planar reflective grating. Grating monochromators have a higher resolution (up to ± 0.2 nm) than prism monochromators, and their usable wavelength range is also wider than prism monochromators, with 80% of the energy of the incident light being in the primary spectrum. In recent years, the engraved copy technology of grating is also constantly improving, and its quality is constantly improving, so its application is more and more widely.
(3) slit
Slit is an important part of the monochromator, a direct impact on the resolution. The smaller the slit width, the better the monochromaticity, but the light intensity also decreases.
3. Sample cell
Fluorescent sample cells need to use low fluorescence materials, usually glass and quartz materials, the shape of a square and rectangular is appropriate.
4. Detector
Main selenium photocell, photocell and photomultiplier tube. At present, due to the weak fluorescence, photomultiplier tubes are generally used as detectors. Select a photomultiplier tube to consider the response wavelength, sensitivity and noise levels. Blue tube for protein, nucleic acid measurement applicable, while the red tube is suitable for fluorescent dye detection.
application
At present, fluorescence analysis has become an important and effective spectral chemical analysis. In China, only a few analytical chemists in the early 1950s engaged in the research of fluorescence analysis. However, by the late 1970s, the fluorescence analysis method has drawn widespread attention in the domestic analytical community. Among the many analytical chemists in the country, , Has gradually formed a team engaged in the field of work.
Fluorescence analysis features
(1) The main features of fluorescence analysis are high sensitivity and good selectivity. The sensitivity of fluorescence analysis is 2-3 orders of magnitude higher than that of absorption spectroscopy. Spectrophotometry is usually in the 10-7 level, and fluorescence sensitivity of 10-9.
(2) strong selectivity, the fluorescent material has two characteristic spectra: the excitation spectrum and the absorption spectrum, compared with the spectrophotometric single absorption spectrum, the fluorescence spectrum can be identified according to the excitation spectrum and the emission spectrum.
(3) informative, can provide a variety of fluorescent material parameters.
(4) However, fluorescence analysis methods also have their shortcomings: ① many substances do not fluoresce; ② the relationship between fluorescence and compound structure is not clear; ③ many interference factors, photolysis, oxygen quenching, easy to pollution.
2. The main application
(1) Application in the field of biology
The field is mainly used for the clinical determination of the content of certain components in biological samples, biotechnology and immunological technology analysis, such as deoxyribose and deoxyribonucleic acid content determination, DNA, antibodies, antigens and other aspects of research. Mainly in this area using a variety of fluorescent probes for analysis and detection, mainly divided into bio-nano-fluorescent probe and biological non-nano-fluorescent probe.
The rise of nanotechnology, which opened up a new area of fluorescence analysis. Due to their excellent fluorescence properties, wide excitation and narrow emission, nanomaterials have become an important research object in fluorescence analysis and have aroused the interest of researchers.
(2) in the field of food applications
The field is mainly used for the analysis and detection of minerals and metal elements, amino acids, vitamins, fungi contamination, additives, preservatives, harmful substances in food packaging and pesticide residues in food. Especially with the combination of HPLC, TLC, FIA and other technologies can better achieve the detection of various substances in food. At present, the internationalization of food standards in our country is increasingly becoming more and more sensitive and micro-quantified for the requirements of food analysis. Fluorescence analysis is to meet the analytical requirements in this area.
(3) in the application of drug analysis
The field of drug analysis can use fluorescence analysis for the identification of the active ingredients of drugs, pharmacokinetic studies, clinical pharmacology and efficacy analysis. Drug fluorescence analysis can be divided into three categories: direct fluorescence analysis, indirect fluorescence analysis and nano-fluorescence analysis. Conventional fluorescence analysis was first applied to the analysis of anti-malaria drug quinine. With the development of fluorescence analysis, its application has been expanding. It is widely used in the analysis of antibiotics, analgesics, sedatives and hemostatic agents.
(4) Application in environmental analysis
The field mainly uses fluorescence analysis to detect the content of substances in the environment, mainly for the detection of water bodies, ores and soils. With the development of organic chemicals, petrochemicals, pharmaceutical industry and the extensive use of pesticides (pesticides, herbicides, etc.), the environmental hazards and pollution of organic compounds have become increasingly serious. Currently included in the national standard organic pollutants monitoring methods of fluorescence analysis; Determination of organic mercury by cold atomic fluorescence spectrometry; Acetylation filter paper chromatography fluorescence spectrophotometry of atmospheric fly ash and water in the benzo (a) pyrene Determination; phenols, lignosulfonates, polycyclic aromatic hydrocarbons (pyrene, firefly, anthracene) [PASH] Determination of fluorescence analysis.
Instrument development
In recent years, the development of a variety of new fluorescence analysis techniques, such as laser induced fluorescence method, synchronous fluorescence method, derivative fluorescence method, fluorescence probe method, photochemical fluorescence method, time-resolved fluorescence method, three-dimensional fluorescence method, polarization fluorescence method, Assay, Fluorescence Imaging, Fluorescent Fiber Optic Sensors and more. The application of these technologies has accelerated the development of a variety of new fluorescence analytical instruments, enabling fluorescence analysis to continue to evolve in high efficiency, trace, micro and automation.
With the development of science and technology, the introduction of new technologies such as laser, microcomputer, electronics and so on, the fluorescence spectrometer has been rapidly developed both in theory and in practice. The main development directions are as follows:
(1) resolution increase;
(2) scanning speed;
(3) multi-function, can be the same plane fluorescence, phosphorescence and chemiluminescence measurement; using fiber-optic flatbed scanner accessories can also be thin-layer chromatographic separation spot scanning determination;
(4) to promote the expansion of application technology:
① time resolution (phase resolution)
② fluorescence polarization (fluorescence immunity)
③ synchronous fluorescence
④ three-dimensional fluorescence
⑤ fluorescence fiber chemical sensor analysis of new methods have emerged, 10-6S level fluorescence life determination.
(5) compact, convenient type.
1. principle
In the absorption of ultraviolet and visible electromagnetic radiation in the process of molecular excitations excited excited state, most of the molecules will collide with other molecules in the heat way to dissipate this part of the energy, some molecules in the form of light radiation out of this part Energy, the wavelength of the radiated light is different from the wavelength of the absorbed radiation. The latter process is called photoluminescence. Molecular luminescence includes fluorescence, phosphorescence, chemiluminescence, bioluminescence and scattering spectroscopy. The analytical method that is based on the fluorescence measurement of a compound is called molecular fluorescence spectroscopy.
The light emitted by the light source is turned into intermittent light by the light cutting device and becomes monochromatic light by the excitation light monochromator, which is the excitation light of the fluorescent material. Fluorescence emitted by the measured fluorescent substance under excitation light is changed into monochromatic fluorescent light by a monochromator and then irradiated on the photomultiplier tube. The photocurrent generated by the measured fluorescent substance is amplified by an amplifier to be input to the recorder. One excitation, one emission, using a dual monochromator system, can measure the excitation spectrum and the fluorescence spectrum, respectively.
2. classification
Fluorescence spectrometer is the basic equipment to determine the luminescent properties of materials. General fluorescence spectrometer can be divided into three kinds:
(1) Basic: Steady-state spectrometer at 200-800 nm in the UV-visible band.
(2) Expanded: Ultraviolet visible-near-infrared steady-state spectrometer covering 200-1700 nm band.
(3) Integrated: a spectrometer that covers the above two bands while measuring transient spectra.
3. The main purpose
(1) fluorescence excitation and fluorescence emission spectra;
(2) Synchronous fluorescence (wavelength and energy) scanning spectra;
(3) 3D (Ex Em Intensity);
(4) Time Base and CWA (fixed wavelength single point measurement);
(5) Fluorescence lifetime measurement, including life-time resolution and time resolution;
(6) Computer to collect spectral data and process data (Datamax and Gram32).
4. Development History
For the first time a fluorescent phenomenon was recorded by Spanish physician and botanist N. Monardes in the 16th century, who in 1575 referred to the extremely lovely sky blue in an aqueous solution containing a slice of wood called "Lignum Nephriticum" . In the 17th century, famous scientists such as Boyle (1626-1691) and Newton (1624-1727) once again observed the phenomenon of fluorescence. Fluorescence then aroused the interest of many scientists, fluorescence analysis methods are also more and more applied to biological and chemical analysis.
Of course, the development of fluorescence analysis method is inseparable from the development of instrument application. In general, fluorescence spectrometer has been through three stages of development since its inception:
(1) manual;
(2) automatic scanning
(3) Computerized.
Before the 19th century, the observation of fluorescence was carried out by the naked eye. It was not until 1928 that Jette and West proposed the first photoelectric fluorometer. The sensitivity of the photoelectric fluorometer is limited. After the invention of a photomultiplier tube by Zworykin and Rajchman in 1939, it was a very important phase in terms of increasing sensitivity and allowing the use of monochromators with higher resolution. In 1943 Dutton and Bailey proposed a manual correction step of fluorescence spectroscopy. The first automatic spectral correction device was introduced by Studer in 1948 and only appeared in 1952 as commercial calibrated spectrometers.
The main components and functions
Fluorescence spectrometer mainly includes light source, excitation monochromator, sample cell, fluorescent monochromator and detector and other major components.
1. light source
Early fluorescent spectrophotometers equipped with low pressure mercury lamps that produced very narrow mercury lines. With high pressure mercury lamps, the spectrum is broadened and there is also a continuous band of high strength. However, a complete excitation spectrum measurement requires a lamp capable of emitting higher intensity light radiation from the visible to the ultraviolet range. Xenon arc lamps are suitable for this condition and as such are the most widely used light sources in fluorescence spectrophotometers.
2. Monochromator
The function of the monochromator is to decompose the continuous spectrum emitted by the light source into monochromatic light and to accurately and conveniently "remove" the light of a certain wavelength that is required, which is the heart of the spectrometer. Monochromators are mainly composed of slits, dispersive elements and lens systems, of which dispersive elements are the key components. Dispersive elements are prisms and reflective gratings or a combination of the two that disperses continuous spectrum light into monochromatic light.
(1) prism monochromator
Prism monochromator is the use of different wavelengths of light in the prism of different refractive index composite light will be dispersed as monochromatic light. Prismatic dispersion of the size of the prism material and the geometry of the production. Common prism made of glass or quartz. Visible spectrophotometer can be used glass, it is suitable for UV, the entire visible spectrum.
(2) grating monochromator
Gratings have many unique advantages as dispersive components. Raster can be defined as a series of equal width, equidistant parallel slit. The principle of dispersion of a grating is based on the phenomenon of diffraction and interference of light. Commonly used grating monochromator for the reflective grating monochromator, which is divided into two kinds of planar reflective grating and concave reflective grating, one of the most commonly used planar reflective grating. Grating monochromators have a higher resolution (up to ± 0.2 nm) than prism monochromators, and their usable wavelength range is also wider than prism monochromators, with 80% of the energy of the incident light being in the primary spectrum. In recent years, the engraved copy technology of grating is also constantly improving, and its quality is constantly improving, so its application is more and more widely.
(3) slit
Slit is an important part of the monochromator, a direct impact on the resolution. The smaller the slit width, the better the monochromaticity, but the light intensity also decreases.
3. Sample cell
Fluorescent sample cells need to use low fluorescence materials, usually glass and quartz materials, the shape of a square and rectangular is appropriate.
4. Detector
Main selenium photocell, photocell and photomultiplier tube. At present, due to the weak fluorescence, photomultiplier tubes are generally used as detectors. Select a photomultiplier tube to consider the response wavelength, sensitivity and noise levels. Blue tube for protein, nucleic acid measurement applicable, while the red tube is suitable for fluorescent dye detection.
application
At present, fluorescence analysis has become an important and effective spectral chemical analysis. In China, only a few analytical chemists in the early 1950s engaged in the research of fluorescence analysis. However, by the late 1970s, the fluorescence analysis method has drawn widespread attention in the domestic analytical community. Among the many analytical chemists in the country, , Has gradually formed a team engaged in the field of work.
Fluorescence analysis features
(1) The main features of fluorescence analysis are high sensitivity and good selectivity. The sensitivity of fluorescence analysis is 2-3 orders of magnitude higher than that of absorption spectroscopy. Spectrophotometry is usually in the 10-7 level, and fluorescence sensitivity of 10-9.
(2) strong selectivity, the fluorescent material has two characteristic spectra: the excitation spectrum and the absorption spectrum, compared with the spectrophotometric single absorption spectrum, the fluorescence spectrum can be identified according to the excitation spectrum and the emission spectrum.
(3) informative, can provide a variety of fluorescent material parameters.
(4) However, fluorescence analysis methods also have their shortcomings: ① many substances do not fluoresce; ② the relationship between fluorescence and compound structure is not clear; ③ many interference factors, photolysis, oxygen quenching, easy to pollution.
2. The main application
(1) Application in the field of biology
The field is mainly used for the clinical determination of the content of certain components in biological samples, biotechnology and immunological technology analysis, such as deoxyribose and deoxyribonucleic acid content determination, DNA, antibodies, antigens and other aspects of research. Mainly in this area using a variety of fluorescent probes for analysis and detection, mainly divided into bio-nano-fluorescent probe and biological non-nano-fluorescent probe.
The rise of nanotechnology, which opened up a new area of fluorescence analysis. Due to their excellent fluorescence properties, wide excitation and narrow emission, nanomaterials have become an important research object in fluorescence analysis and have aroused the interest of researchers.
(2) in the field of food applications
The field is mainly used for the analysis and detection of minerals and metal elements, amino acids, vitamins, fungi contamination, additives, preservatives, harmful substances in food packaging and pesticide residues in food. Especially with the combination of HPLC, TLC, FIA and other technologies can better achieve the detection of various substances in food. At present, the internationalization of food standards in our country is increasingly becoming more and more sensitive and micro-quantified for the requirements of food analysis. Fluorescence analysis is to meet the analytical requirements in this area.
(3) in the application of drug analysis
The field of drug analysis can use fluorescence analysis for the identification of the active ingredients of drugs, pharmacokinetic studies, clinical pharmacology and efficacy analysis. Drug fluorescence analysis can be divided into three categories: direct fluorescence analysis, indirect fluorescence analysis and nano-fluorescence analysis. Conventional fluorescence analysis was first applied to the analysis of anti-malaria drug quinine. With the development of fluorescence analysis, its application has been expanding. It is widely used in the analysis of antibiotics, analgesics, sedatives and hemostatic agents.
(4) Application in environmental analysis
The field mainly uses fluorescence analysis to detect the content of substances in the environment, mainly for the detection of water bodies, ores and soils. With the development of organic chemicals, petrochemicals, pharmaceutical industry and the extensive use of pesticides (pesticides, herbicides, etc.), the environmental hazards and pollution of organic compounds have become increasingly serious. Currently included in the national standard organic pollutants monitoring methods of fluorescence analysis; Determination of organic mercury by cold atomic fluorescence spectrometry; Acetylation filter paper chromatography fluorescence spectrophotometry of atmospheric fly ash and water in the benzo (a) pyrene Determination; phenols, lignosulfonates, polycyclic aromatic hydrocarbons (pyrene, firefly, anthracene) [PASH] Determination of fluorescence analysis.
Instrument development
In recent years, the development of a variety of new fluorescence analysis techniques, such as laser induced fluorescence method, synchronous fluorescence method, derivative fluorescence method, fluorescence probe method, photochemical fluorescence method, time-resolved fluorescence method, three-dimensional fluorescence method, polarization fluorescence method, Assay, Fluorescence Imaging, Fluorescent Fiber Optic Sensors and more. The application of these technologies has accelerated the development of a variety of new fluorescence analytical instruments, enabling fluorescence analysis to continue to evolve in high efficiency, trace, micro and automation.
With the development of science and technology, the introduction of new technologies such as laser, microcomputer, electronics and so on, the fluorescence spectrometer has been rapidly developed both in theory and in practice. The main development directions are as follows:
(1) resolution increase;
(2) scanning speed;
(3) multi-function, can be the same plane fluorescence, phosphorescence and chemiluminescence measurement; using fiber-optic flatbed scanner accessories can also be thin-layer chromatographic separation spot scanning determination;
(4) to promote the expansion of application technology:
① time resolution (phase resolution)
② fluorescence polarization (fluorescence immunity)
③ synchronous fluorescence
④ three-dimensional fluorescence
⑤ fluorescence fiber chemical sensor analysis of new methods have emerged, 10-6S level fluorescence life determination.
(5) compact, convenient type.
