Working principle and characteristics of spectrophotometer

A spectrophotometer, also known as a spectrometer, is a scientific instrument that decomposes light with complex components into spectral lines. The measurement range generally includes the visible light region with a wavelength range of 380 to 780 nm and the ultraviolet light region with a wavelength range of 200 to 380 nm. Different light sources have their own unique emission spectra, so different luminous bodies can be used as the light source of the instrument. The emission spectrum of a tungsten lamp: the spectrum light of 380-780nm wavelength emitted by a tungsten lamp is refracted by a prism to obtain a continuous chromatogram consisting of red, orange, yellow, green, blue, indigo, and violet; this chromatogram can be used as visible light The light source of the spectrophotometer.

The simple principle of 　　 spectrophotometer
The spectrophotometer adopts a light source that can generate multiple wavelengths. Through a series of spectroscopic devices, a light source of a specific wavelength is generated. After the light source passes through the sample under test, part of the light source is absorbed. The absorbance value of the sample is calculated and converted into the sample. concentration. The absorbance of the sample is directly proportional to the concentration of the sample.

The basic working principle of 　　 spectrophotometer
The basic working principle of a spectrophotometer is based on the selective absorption of light (wavelength of light) by substances. Different substances have their own absorption bands. Therefore, when the light-dispersed spectrum passes through a certain solution, Certain wavelengths of light will be absorbed by the solution. At a certain wavelength, there is a certain proportional relationship between the concentration of the substance in the solution and the degree of light energy weakening, which conforms to Beer's law.

T = I/Io lg(Io/I)=εcb

where T is the transmittance, Io is the intensity of the incident light, I is the intensity of the transmitted light, A is the extinction value (absorbance), ε is the absorption coefficient, b is the optical path length of the solution, and c is the concentration of the solution. It can be seen from the above formula that when the incident light, the absorption coefficient and the thickness of the solution are constant, the light transmittance changes according to the concentration of the solution.

The 　　 spectrophotometer has become a routine instrument in modern molecular biology laboratories. It is often used for nucleic acid, protein quantification and quantification of bacterial growth concentration.

The working principle and characteristics of spectrophotometer

Features of 　　 Spectrophotometer
Unique dual-path, dual-beam optical system, the instrument has higher resolution, lower stray light, stronger stability and reliability, and more accurate analysis;

adopts 320*240 dot matrix type high brightness 6" liquid crystal display, the display is clear and the information is complete;

Unique long optical path design makes the instrument resolution higher, especially suitable for micro testing. The powerful data processing function enables the test results to be fully applied, and the user editing is simpler and faster;

The suspension optical system design is adopted, and the overall optical path is independently fixed on a 16mm thick aluminum non-deformation base. The deformation of the bottom plate and external vibration have no effect on the optical system, which greatly improves the stability and reliability of the instrument ；

Using synchronous sine mechanism, high wavelength accuracy and good repeatability;

Using ARM system;

0.1/0.2/0.5/1.0/2.0/4.0 six levels of spectral bandwidth are automatically selectable to meet the measurement needs of different users;

24-bit high-speed, high-precision A/D conversion, the instrument has higher precision and faster response speed;

The main components adopt imported configuration, which makes the instrument stray light lower, more stable and more reliable;

More powerful functions, the host can independently complete functions such as photometric measurement, quantitative measurement, spectral scanning, kinetics, DNA/protein testing, multi-wavelength testing and data printing;

Fully considering the usage habits of different users, this series of instruments are equipped with the spectroscopic scanning software of MetaAnalysis. When operating online, in addition to all the test functions of the host, it can also achieve more powerful data processing functions and enable data storage. Reach infinity.

The working principle and characteristics of spectrophotometer

Wavelength selection
The choice of wavelength is generally to choose the wavelength (λmax) of the absorption peak of the substance to be measured. Because the absorbance and sensitivity are measured at λmax. When the absorbance is measured at the absorption peak wavelength, the wavelength change affects it; while at other wavelengths, the wavelength change has a great influence on the absorbance, and even the measured concentration-absorbance curve does not appear to be a straight line.

To choose the wavelength required to measure a certain solution, it is possible to use different wavelengths as the absorption spectrum curve of the solution, and select the appropriate wavelength from the curve to carry out the measurement of this solution. However, there are some special cases in the analysis work. Circumstances, one should not rely solely on this principle, but should conduct actual tests based on the following three principles, and then fully consider the pros and cons before making a selection.

  1. The tested solution should have an appropriate optical density. Generally speaking, the appropriate optical density is 0.1-0.7, and 0.2-0.6 is ideal. An optical density that is too low causes a large relative error due to the reading error of the instrument. On the contrary, an optical density that is too high often exceeds the linear range and introduces errors.

  2. The influence of interference should be reduced to the limit. In the reaction, if there is an interference color that is not easy to remove, a wavelength that is not sensitive to the interference color should be selected.

3. The standard curve should be close to a straight line in the largest possible range.

721 type spectrophotometer structure
721 type spectrophotometer allows the measurement wavelength range of 360 ~ 800nm, its structure is relatively simple, and the measurement sensitivity and precision are high. Therefore, it is widely used.

The continuous radiant light emitted from the light source lamp hits the condenser lens, and after being condensed, it passes through the plane mirror at a 90° angle and is reflected to the incident slit. As a result, when incident into the monochromator, the slit is located exactly on the focal plane of the spherical collimating objective lens. After the incident light is reflected by the collimating objective lens, it is directed to the prism with a parallel light beam. After the light enters the prism, it undergoes dispersion. The light returned after the dispersion is reflected by the collimator lens, and then is collected on the light-emitting slit, and then enters the cuvette after passing through the condenser. Part of the light is absorbed, and the transmitted light enters the photoelectric tube to generate a corresponding photocurrent. Read on the microammeter after magnification.

How to use the 721 spectrophotometer
① First turn on the power, turn on the power switch 1, the indicator light is on, open the cuvette dark box cover 8, and preheat for 20 minutes.

②Wavelength selection knob 6, select the desired wavelength of monochromatic light, and use sensitivity knob 2 to select the desired sensitivity level.

③Put the cuvette into the cuvette, turn the zero position knob 5 to zero, close the cuvette dark box cover, push the cuvette lever 3, make the reference cuvette at the blank calibration position, make the photoelectric tube see light, and rotate the transmittance Adjust knob 4 to make the pointer of microammeter 9 accurately at 100%. Adjust the zero position and 100% position several times in succession according to the above method, and then the measurement can be carried out.

The working principle and characteristics of spectrophotometer

Matters needing attention in the use and maintenance of 721 spectrophotometer:
①The continuous use of the instrument should not exceed 2 hours, and it should be used after an interval of 0.5 hours.

②After each cuvette is used, it should be washed with deionized water and dried upside down, and then stored in the cuvette box. In daily use, care should be taken to protect the light-transmitting surface of the cuvette to prevent damage or scratches, so as not to affect the light transmittance.

③The instrument should not be damp. In daily use, you should always pay attention to whether the moisture-proof silica gel (at the bottom of the instrument) on the monochromator has changed color. If the color of the silica gel has turned red, it should be removed and dried or replaced immediately.

④ When consigning or moving the instrument, please handle it carefully.