The ultraviolet visible spectrophotometer is based on the principle of ultraviolet visible spectrophotometry, and uses the material molecules toradiationAn analytical instrument that absorbs for analysis.It is mainly composed of light source, monochromator, absorption cell, detector, signal processor and other components.The function of the light source is to provide a continuous spectrum of sufficient intensity and stability.Hydrogen lamp or deuterium lamp is usually used for ultraviolet light area. Tungsten lamp orTungsten halogen lamp。The function of the monochromator is to decompose the composite light emitted by the light source and separate the monochromatic light of the required wavelength from it.Dispersion elements include prism and grating.Glass absorption cell shall be used for the measurement of visible light area, and quartz absorption cell shall be used for the measurement of ultraviolet light area.The function of the detector is to detect the intensity of the transmitted light through the photoelectric conversion element and convert the optical signal into an electrical signal.Commonly used photoelectric conversion elements include photocell, photomultiplier and photodiode array detector.There are many ways to classify spectrophotometer: according to the light path system, it can be divided into single beam spectrophotometer and double beam spectrophotometer;According to the measurement mode, it can be divided into single wavelength spectrophotometer and dual wavelength spectrophotometer;It can be divided into spectroscopic scanning detection and diode array full spectrum detection according to the detection method of plotting spectrum[1]。
In 1852, Beer referred to the articles published by Bouguer in 1729 and Lambert in 1760, and proposed the basic law of spectrophotometry, that is, when the thickness of the liquid layer is equal, the intensity of color is proportional to the concentration of the chromogenic solution, thus laying the theoretical foundation of spectrophotometry, which is the famous Beer Lambert law.
In 1854, Duboscq, Nessler and others applied Lambertian law to the field of quantitative analytical chemistry and designed the first colorimeter.
In 1918, the United States National Bureau of Standards made the first ultraviolet visible spectrophotometer.Since then, the ultraviolet visible spectrophotometer has been continuously improved, and various types of instruments, such as automatic recording, automatic printing, digital display, microcomputer control, have emerged, which has continuously improved the sensitivity and accuracy of the spectrophotometry and expanded its application range.
working principle
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The UV visible absorption spectrum of a molecule is the absorption spectrum generated by the electronic energy level transition after some groups in the molecule absorb the UV visible radiation light.Since various substances have different molecules, atoms and different molecular spatial structures, their absorption of light energy will not be the same. Therefore, each substance has its own unique and fixed absorption spectrum curve, which can be judged or measured according to the absorbance at some characteristic wavelengths on the absorption spectrum,This is the basis of qualitative and quantitative analysis of spectrophotometry.
Spectrophotometric analysis is an effective means to study the composition, structure and interaction between substances according to the absorption spectrum of substances.It is a band spectrum, reflecting the information of some groups in the molecule.Qualitative analysis can be carried out by standard light spectrum combined with other means.
According to Lambert Beer's law, light absorption is proportional to the thickness of the absorption layer, and Beer's law indicates that light absorption is proportional to the concentration of the solution;If both the thickness of the absorption layer and the concentration of the solution are considered, Lambert Beer law is obtained.That is, A=ε bc, (A is the absorbance, ε is the molar absorbance coefficient, b is the thickness of the liquid pool, and c is the concentration of the solution).
The analytical sample and the standard sample are prepared in the same solvent with the same concentration, and the ultraviolet and visible absorption spectra are measured respectively under the same conditions.If both are the same substance, their spectrograms should be identical.If there is no standard sample, it can also be compared with the existing standard spectrogram.This method requires accurate instrument, high precision and the same determination conditions.
The experiment proved that the strength of hydrogen bond generated by different polar solvents is also different, which can use UV spectrum to judge the strength of hydrogen bond of compounds in different solvents to determine which solvent to choose.
Structure and function
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It consists of light source, monochromator, absorption cell, detector and signal display system.
Light source: It is a device to provide incident light that meets the requirements, including thermal radiation light source and gas discharge light source.The thermal radiation light source is used in the visible light area, generally tungsten lamp and tungsten halogen lamp, and the wavelength range is 350~1000nm;The gas discharge light source is used in the ultraviolet light area, generally hydrogen lamp and deuterium lamp, with a continuous wavelength range of 180~360 nm.
Monochromator: It is used to decompose the composite light generated by the light source into monochromatic light and separate the required monochromatic light beam. It is the heart of the spectrophotometer.
Absorption cell: also called cuvette, used to hold test solution for absorbance measurement. Its bottom and both sides are ground glass, and the other two sides are optical transparent surfaces. In order to reduce light reflection loss, the optical surface of the absorption cell must be completely perpendicular to the beam direction.According to the material, it can be divided into glass cell and quartz cell. The former is used for visible light zone measurement, and the latter is used for ultraviolet light zone.
Detector: a device that converts optical signals into electrical signals. When measuring absorbance, it does not directly measure the light intensity through the absorption cell, but converts the light intensity into current signals for testing. This photoelectric converter is called a detector.
Signal display system: It is a device to amplify and display the signal output by the detector[2]。
characteristic
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1. High sensitivity.
2. Good selectivity.
3. High accuracy.
4. Widely used.
5. Wide range of concentration.
6. Low analysis cost.
7. Easy to operate.
8. Fast analysis speed.
Product application
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In the application of water and wastewater monitoring, the monitoring analysis and comprehensive evaluation of a water system generally include water phase (solution itself), solid phase (suspended solids, sediment), and biological phase (aquatic organisms).In the routine monitoring of water quality, ultraviolet visible spectrophotometry occupies a large proportion.Because the composition of water and waste water is complex and changeable, the concentration of the substance to be measured and the concentration of the interfering substance are very different, so the analysis method must be selected for specific analysis.
The components or ingredients that can be used for detection in agricultural products and food analysis areprotein、Lysine、glucose、vitamin C、Nitrate、nitrite, arsenic, mercury, etc;
It can be used to detect chlorophyll, total nitrogen and enzyme activity in plant biochemical analysis;
It can be used to detect nicotinic acid, gossypol, phosphine and methyl ester in feed analysis.
Routine maintenance
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Understand the daily maintenance of analytical instruments and simple test methods for main technical indicators, and often maintain and test the instruments to ensure that they work in the best state.
1、 Temperature and humidity are important factors affecting the performance of the instrument.They can cause corrosion of mechanical parts, reduce the smoothness of the metal mirror surface, and cause errors or performance degradation of the mechanical part of the instrument;The aluminum film of optical components such as grating, reflector, focusing mirror, etc. is rusted, resulting in insufficient light energy, stray light, noise, etc., and even the instrument stops working, thus affecting the service life of the instrument.It shall be corrected regularly during maintenance.The instrument room with constant humidity throughout the year shall be equipped with constant temperature equipment, especially the laboratory located in the south.
2、 The dust and corrosive gas in the environment will also affect the flexibility of the mechanical system, reduce the reliability of various limit switches, keys, and optoelectronic couplers, which is also one of the reasons for the corrosion of aluminum film on optical components.Therefore, it must be cleaned regularly to ensure the environment and the sanitary conditions in the instrument room and prevent dust.
3、 After the instrument is used for a certain period, a certain amount of dust will accumulate inside. It is better to open the instrument cover regularly by the maintenance engineer or under the guidance of the engineer to remove dust inside, and at the same time, retighten the radiators of each heating element, clean the sealing window of the optical box, calibrate the optical path if necessary, clean and lubricate the mechanical part,Finally, restore the original state, and then carry out some necessary tests, adjustments and records.
matters needing attention
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1. Before starting the machine, take out the desiccant in the sample chamber. It is forbidden to open the sample chamber cover during the self inspection of the instrument.
2. The solution in the cuvette should be 2/3~4/5 of the height of the cuvette, and should not be too full to prevent the overflow of liquid from corroding the instrument.The cuvette shall be kept clean during measurement, and the liquid drops on the pool wall shall be wiped dry with mirror wiping paper, and the transparent surface shall not be pinched by hand.Quartz cuvette shall be used to determine the UV wavelength.
3. It is forbidden to put reagents or liquid substances on the surface of the instrument during measurement. If the sample tank is dirty due to solution overflow or other reasons, it should be cleaned as soon as possible.
4. After the experiment, pour out the solution in the cuvette, wash the cuvette with distilled water or organic solvent until it is clean, and then stand upside down to dry.Turn off the power supply, put the desiccant into the sample room, cover the dust cover, make use registration, and leave after being approved by the management teacher.
Problem handling
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Problem handling of UV-VIS spectrophotometer:
1. If the instrument cannot be initialized, shut down and restart.
2. If the absorption value is abnormal, check in sequence whether the wavelength setting is correct (re adjust the wavelength and re zero), whether the measurement is zero adjusted (re zero if it is misoperated), whether the cuvette is used incorrectly (quartz cuvette is used when measuring the ultraviolet band), and whether the sample preparation is wrong (re prepare the sample if there is an error).
