Application of UV-visible spectrophotometer Key words organic analysis absorption spectrum ultraviolet-visible spectrophotometry 1 Overview In practice, people have already concluded that different colored substances have different physical and chemical properties. According to these characteristics of the material, it can be effectively analyzed and judged. Since color is already noticeable, the material content is estimated based on the color depth of the material, which can be traced back to ancient and medieval times. In 1852, Beer, referring to articles published by Bouguer in 1729 and Lambert in 1760, proposed the basic law of spectrophotometry, that is, the intensity and the intensity of the color when the liquid thickness is equal. The concentration of the color solution is proportional, which lays the theoretical basis for spectrophotometry, which is the famous Bill lambertian law. In 1854, Duboscq and Nessler et al. applied this theory to the field of quantitative analysis chemistry and designed the first colorimeter. By 1918, the US National Bureau of Standards made the first UV-Vis spectrophotometer. Since then, the UV-VIS spectrophotometer has been continuously improved, and there have been various types of instruments such as automatic recording, automatic printing, digital display, and microcomputer control, which have increased the sensitivity and accuracy of the photometric method, and its application range has been continuously expanded. Since the advent of UV-Vis spectrophotometry, great progress has been made in application, especially on the basis of the development of related disciplines, to promote the constant innovation of the spectrophotometer instrument, more complete functions, making the application of the photometric method more widely range. At present, spectrophotometry has been widely adopted by various departments of industry and agriculture and various fields of scientific research, and has become a powerful test method for people to engage in production and scientific research. China has a solid foundation in the field of analytical chemistry, and has reached a certain level internationally in the production of spectrophotometric analysis methods and instruments [1][2] 2. Principle Absorption spectroscopy of matter essentially means that the molecules and atoms in the material absorb some specific wavelengths of light energy in the incident light, and correspondingly, the vibrational energy level transition and the transition of the electron energy level occur. Since various substances have their own different molecules, atoms and different molecular space structures, their absorption of light energy will not be the same. Therefore, each substance has its own unique, fixed absorption spectrum curve, which can be based on absorption. The level of absorbance at certain characteristic wavelengths in the spectrum is used to determine or determine the content of this substance, which is the basis for qualitative and quantitative analysis of spectrophotometric. Spectrophotometric analysis is based on the absorption spectrum of the substance to study the composition, structure and interaction of substances in the effective means. The basis of quantitative analysis by UV-Vis spectrophotometry is the Lambert-Beer law. That is, the absorbance of a substance at a certain concentration is directly proportional to the thickness of its absorbing medium. Its mathematical expression is as follows: A= 錬c In the formula: A—absorbance (also called optical density, extinction value), The molar absorption coefficient (its physical meaning is: when the concentration of the light-absorbing material is 1 mol/L, the absorption cell thickness is 1 cm, and the light absorption value caused by the passage of a certain wavelength of primary light), b—the thickness of the absorption medium (cm), c—the concentration of the light-absorbing substance (mol/l). The color of a material is closely related to its electronic structure. When radiation (photon) causes an electronic transition and the molecule (or ion) rises from the ground state to the excited state, the molecule (or ion) absorbs light in the visible region or ultraviolet. The occurrence or change of color is associated with the deformation of the normal electronic structure of the molecule. When the molecule contains one or more chromogenic genes (ie, atom groups with unsaturated bonds), radiation can cause changes in the energy of the electrons in the molecule. Common chromophores are: CO, -N=N-, -N=O,-CN,CS If two chromophores are separated by one carbon atom, a conjugated group is formed, which shifts the absorption band toward a longer wavelength (ie, red shift), and the intensity of the absorption band increases significantly. When the molecule contains a color-promoting group (a group that does not share an electron pair), a redshift effect also occurs. Common color forming groups are: -OH -NH2, -SH, -Cl, -Br, -I 3. Features Spectrophotometry is one of the most useful tools for analysts. Almost every analytical laboratory is inseparable from the UV-Vis spectrophotometer. The main features of spectrophotometry are: (1) Wide range of applications Since various inorganic and organic substances absorb in the ultraviolet visible region, they can be determined by this method. So far, almost all elements on the periodic table of chemical elements (except for a few radioactive and inert elements) can be used this method. Of the total number of papers published in the international analysis, photometry accounted for about 28%, and China accounted for about 33% of the total number of published papers. (2) High sensitivity Due to the large amount of new reagents synthesized and promising progress in applied research, the sensitivity of elemental assays has been advanced, especially with regard to the application of multi-complexes and various surfactants. The molar absorptivity of the element increases from the original tens of thousands to hundreds of thousands. (3) good selectivity At present, there are some elements that can be directly determined by the use of appropriate chromogenic conditions, such as the determination of cobalt, uranium, nickel, copper, silver, iron, etc., and there are already satisfactory methods. (4) High accuracy For general spectrophotometry, the relative error of its concentration measurement is in the range of 1 to 3%. If differential photometry is used for measurement, the error can be reduced to 0.X%. (5) Wide range of applicable concentrations From constant (1% to 50%) (especially using differential methods) to trace amounts (10-8 to 10-6%) (after pre-enrichment). (6) Low cost, simple and fast analysis Because spectrophotometry has the above advantages, it is still widely used in chemical, metallurgy, geology, medicine, food, pharmaceutical and other departments and environmental monitoring systems. The application in water quality analysis is very extensive. At present, there are over 70 metallic and non-metallic elements that can be measured by direct and indirect methods. 4 Application 4.1 Verification of substances Absorption according to some characteristics of the absorption spectrum, especially the maximum absorption wavelength ax and molar absorption coefficient æ¾¹ are common physical parameters for the verification of substances. This has a wide range of applications in drug analysis. In the pharmacopoeia at home and abroad, the maximum absorption wavelength and absorption coefficient of many UV absorption spectra of drugs have been loaded into them, providing a good means for drug analysis. 4.2 Comparison with Standards and Standards The analytical sample and the standard sample are prepared in the same solvent at the same concentration, and the ultraviolet-visible absorption spectrum is measured under the same conditions. If the two are the same material, the spectra of the two should be exactly the same. If there is no standard, it can also be compared with a ready-made standard spectrum. This method requires accurate instrumentation, high precision, and the same assay conditions. 4.3 Comparison of Absorption Coefficients of Maximum Absorption Wavelength Since the UV absorption spectrum contains only 2 to 3 broad absorption bands, the UV spectrum is mainly the absorption of the chromophore within the molecule in the UV region, and has little to do with the molecules and other parts. Different molecular structures with the same chromophore do not affect the UV absorption spectrum of the chromophore in larger molecules. Different molecular structures may have the same UV absorption spectrum, but their absorption coefficients are different. If the analysis sample and the standard sample have the same absorption wavelength and the same absorption coefficient, the analysis sample and the standard sample may be considered to be the same substance. Example 1 Although the maximum absorption wavelength of hexadiene-1,5 (CH2=CHCH2CH2=CH2) is 178 nm (molar absorption coefficient is 26000), the maximum absorption wavelength of hexene-1 (CH2=CHCH2CH2CH2CH3) is 177 nm (molar absorption coefficient 逦11800). The two substances have the same chromophore, although the value of ax is basically the same, but the value is different. The diene 逯 when 鹊ãƒâ”‘ thumb 蟆 U 馑 饔 饔 Xing å—¤îƒ oyster î®èˆœ î®èˆœ 还 还 ( ( ( ( (5) î‚Š   îƒ¡æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• æ¹®î• 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 (5) Like butadiene-1,3 (CH2=CHCH=CH2) and hexadiene-1,5 (CH2=CHCH2CH2CH=CH2), there are two double bonds, but in butadiene-1,3 In the conjugate system, the maximum absorption length is 210 nm, while the molar absorption coefficient 逯翟蛴爰 î’– î’– -1, 5 is basically the same. 4.4 Purity test Example 2 Ultraviolet Absorption Spectroscopy can determine trace amounts of UV-absorbing impurities in compounds. If there is no obvious absorption peak in the ultraviolet visible region of the compound, and its impurity has a strong absorption peak in the ultraviolet region, impurities in the compound can be detected. 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