Spectrophotometer, also known as spectrometer (spectrometer), is the composition of the complex light, broken down into spectral lines of scientific instruments. The measurement range generally includes a visible light region with a wavelength range of 400-760 nm and a UV region with a wavelength range of 200-400 nm. Different light sources have their own emission spectra, so different light emitters can be used as the light source of the instrument.
Tungsten lamp emission spectrum: the tungsten light source issued by the 400 ~ 760nm wavelength of light through the prism refraction, can be obtained by the red orange, yellow green, blue indigo, purple composition of the continuous chromatography; the chromatogram can be used as a visible spectrophotometer light source.
Spectrophotometry is the specific wavelength or a certain wavelength range of light absorption, the material qualitative or quantitative analysis. Commonly used wavelength range: (1) 200 ~ 400nm ultraviolet region, (2) 400 ~ 760nm visible light area, (3) 2.5 ~ 25μm (according to wave number of 4000cm <-1> ~ 400cm <-1>) Of the infrared zone. The instrument used for the ultraviolet spectrophotometer, visible spectrophotometer (or colorimeter), infrared spectrophotometer or atomic absorption spectrophotometer. In order to ensure the accuracy and accuracy of the measurement, all instruments shall be regularly calibrated in accordance with the national metrological verification procedures or the provisions of this appendix.
Spectrophotometers have become conventional instruments for modern molecular biology laboratories. Commonly used in nucleic acid, protein
Quantitative and quantitative determination of bacterial growth concentration.
The instrument is mainly composed of light source, monochromator, sample room, detector, signal processor and display and storage system.
Including a visible light region with a wavelength range of 400-760 nm and a UV region with a wavelength range of 200 to 400 nm. Different light sources have their own emission spectra, so different light emitters can be used as the light source of the instrument.
Tungsten lamp emission spectrum: the tungsten light source issued by the 400 ~ 760nm wavelength of light through the prism refraction, can be obtained by the red orange, yellow green, blue indigo, purple composition of the continuous chromatography; the chromatogram can be used as a visible spectrophotometer Light source.
Hydrogen lamp (or deuterium lamp) emission spectrum: hydrogen lamp can send 185 ~ 400 nm wavelength spectrum can be used as a UV light source.
The 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 is no longer the spectrum of the light source, and there are several dark lines, that is, the light of some wavelengths in the light emission spectrum The solution absorbs and disappears, and the spectrum absorbed by the solution is called the absorption spectrum of the solution.
The absorption spectra of different substances are different, so the material contained in the solution can be identified according to the absorption spectrum.
The absorption spectrum of matter (2)
The intensity of the light transmitted through the solution of a certain substance is weakened because a portion of the light is reflected or dispersed on the surface of the solution, and a portion of the light is absorbed by the substance constituting the solution to absorb only a portion of the light through the solution.
Incident light = reflected light + scattered light + absorption light + through light
If we use distilled water (or the solvent that makes up the solution) as a “blank” to correct the loss of incident light due to factors such as reflection, dispersion, etc.:
Incident light = absorb light ten through light
The spectrophotometer uses a light source that can produce multiple wavelengths through a series of light splitting devices to produce a specific wavelength of light. After passing through the tested sample, part of the light is absorbed and the absorbance of the sample is calculated to convert the sample into the concentration The The absorbance of the sample is proportional to the concentration of the sample.
When the monochromatic radiation passes through the solution of the substance to be measured, the amount absorbed by the substance is proportional to the concentration of the substance and the thickness (optical path length) of the liquid layer,
A = -lg (I / I.) = – lgT = kLc
Where: A is the absorbance;
I. For the incident monochromatic light intensity;
I is the transmitted monochromatic light intensity;
T is the transmittance of matter;
K is the molar absorption coefficient;
L is the optical path of the substance to be analyzed, that is, the side of the cuvette
C is the concentration of the substance
The selective absorption wavelength of the material for light, and the corresponding absorption coefficient, is the physical constant of the substance. When the pure substance is known to be the absorption coefficient under certain conditions, the test substance can be formulated into the solution under the same conditions and the absorbance can be determined. The content of the substance in the test sample can be calculated from the above formula. In the visible area, in addition to some substances to absorb light, many substances themselves do not absorb but can be added under certain conditions, coloring reagent or after processing to color and then measured, it is also known as colorimetric analysis. As the color of the impact of color factors are more, and often use monochromatic light purity of the instrument, so the application of standard or reference materials at the same time operation.