Spectrophotometer, also known as spectrometer (spectrometer), is a scientific instrument that decomposes light into complex spectral lines. The measurement range generally includes the visible light range from 400 to 760 nm and the ultraviolet range from 200 to 400 nm. Different light sources have their own specific emission spectrum, so different light emitters can be used as the light source of the instrument.
Tungsten lamp emission spectrum: Tungsten light source issued by the 400 ~ 760nm wavelength spectrum of light through the prism refracted, available by the red orange, yellow green, indigo, purple continuous chromatography; the chromatographic can be used as a visible light spectrophotometer light source.
Luminosity definition
Spectrophotometry is the absorption of light at a specific wavelength or within a certain wavelength range, and the material is qualitatively or quantitatively analyzed. Commonly used wavelength ranges are: (1) UV light at 200-400 nm, (2) visible light at 400-760 nm, (3) 2.5-25 μm (4000 cm <-1> to 400 cm <-1> by wave number) Infrared zone. Instruments used for UV spectrophotometer, visible spectrophotometer (or colorimeter), infrared spectrophotometer or atomic absorption spectrophotometer. In order to ensure the precision and accuracy of measurement, all instruments should be regularly calibrated in accordance with the national metrological verification procedures or the provisions of this appendix.
Instrument composition
Spectrophotometers have become routine molecular biology laboratories. Commonly used in nucleic acid, protein
Quantification and quantification of bacterial growth concentration.
The instrument is mainly composed of light source, monochromator, sample chamber, detector, signal processor and display and storage system.
Spectral range
Including the wavelength range of 400 ~ 760 nm in the visible region and the wavelength range of 200 ~ 400 nm ultraviolet region.Each different light sources have their own unique emission spectrum, so you can use a different light emitter as the instrument light source.
Tungsten lamp emission spectrum: Tungsten light source issued by the 400 ~ 760nm wavelength spectrum of light through the prism refracted, available by the red orange, yellow green, indigo, purple continuous chromatography; the chromatographic can be used as a visible light spectrophotometer Light source.
Emission spectrum of hydrogen lamp (or deuterium lamp): hydrogen lamp emits the spectrum of 185 ~ 400 nm wavelength and can be used as the light source of the ultraviolet photometer.
Absorption spectrum of matter (1)
If a solution of a substance is placed between the light source and the prism, the spectrum displayed on the screen at this time is no longer the spectrum of the light source. It appears several dark lines, that is, the light source at some wavelengths in the emission spectrum Absorption of the solution and disappear, the absorption of the solution is called the absorption spectrum of the solution.
Absorption spectra of different substances are different, so based on the absorption spectrum, substances contained in the solution can be identified.
Absorption spectrum of substances (2)
As the light passes through a solution of a substance, the intensity of the transmitted light diminishes, and as a portion of the light is reflected or scattered on the surface of the solution, a portion of the light is absorbed by the substance that makes up the solution and only a portion of the light is transmitted through the solution.
Incident light = reflected light + dispersed light + absorbed light + transmitted light
If we use distilled water (or the solvent that makes up the solution) as a "blank" to correct for the loss of incident light due to reflections, dispersion, etc. then:
Incident light = absorbed light Ten Transmitted light
Spectrophotometer using a light source can produce multiple wavelengths through a series of light splitting device to produce a specific wavelength of the light source, the light transmitted through the test sample, the part of the light is absorbed, calculate the absorbance of the sample, which is converted to sample concentration . The absorbance of the sample is proportional to the concentration of the sample.
Monochromatic light radiation through the measured substance solution, the amount of material absorbed by the substance concentration and the liquid layer thickness (optical path length) is proportional to the relationship as follows:
A = -lg (I / I.) = - lgT = kLc
Where: A is the absorbance;
I. Is the incident monochromatic light intensity;
I is the transmission of monochromatic light intensity;
T is the material transmittance;
k is the molar absorption coefficient;
L is the optical path length of the analyte, that is, the side length of the cuvette
c is the concentration of the substance
The wavelength of selective absorption of light by matter, and the corresponding absorption coefficient, is the physical constant of the substance. When knowing the absorption coefficient of a certain substance under certain conditions, the test article can be formulated into a solution under the same conditions to determine its absorbance. The content of the substance in the test article can be calculated from the above formula. In the visible region, in addition to some substances on the light absorption, many substances themselves are not absorbed, but under certain conditions can be added to the color reagent or after treatment to color and then measured, it is also known as colorimetric analysis. As the color development of the factors affecting the color depth, and often use monochromatic optical purity of the instrument, so the determination of the application of standard or reference at the same time operation.
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