Chromatographic separation technology is the basic experiment in the "Biochemical Analysis Experiment" of Chinese universities. In the late 1970s , the "nucleic acid protein detector" developed in China was qualitatively detected and separated using a certain wavelength ( 280 nm or 254 nm, etc.), and was recorded by a recorder for long-term use in university laboratories. In the efforts of science and technology personnel in China, in recent years, there have been "computer collectors", "computer nucleic acid protein detectors", "dual-wavelength computer nucleic acid protein detectors" and "chromatography - conductances" that can replace recorders. With systems and other products, it is rapidly applied to teaching experiments, scientific research and pharmaceutical production.
one, Detection principle
All UV absorption detector working principle is based on the absorption of light --- Lambert Law - Beer's Law. The law states that when a bundle of monochromatic light (λ) radiation passes through a solution of a dilute concentration material, if the solvent does not absorb light, the absorbance of the liquid is proportional to the concentration of the light absorbing material and the distance of the light passing through the solution. Its relationship is:
A(λ)=a(λ)bc
A=-LgT=Lg1/T
Second, the classification of chromatography equipment:
According to the trace method: recorder trace and computer trace (plus acquisition analyzer)
According to the detection wavelength: single wavelength detection and dual wavelength (multi-wavelength) synchronous detection
According to the tester: nucleic acid protein detection and nucleic acid protein detection, conductivity detection combined
Third, the traditional detector:
The traditional chromatographic experimental device consists of a single-wavelength nucleic acid protein detector ( plus a computer collector or a computer collector installed in the detector ) , a chromatography column, a constant current pump, a partial collector, and a recorder. . Experiments are carried out on known substances (proteins or nucleic acids, etc.) at a wavelength (such as 280 nm or 254 nm, etc.), and most of the chromatographic experimental devices in university laboratories fall into this category. Features are as follows:
1. Before the test, one wavelength ( 280nm , 254nm ) must be selected for detection, and the known components are separated by the characteristic wavelength of the substance.
2. Students are required to adjust the transmittance to 100% before the test, and then adjust the absorbance to 0 . Students often ask: although the transmission rate and absorbance at this point ( T=100% , A=0 ) are in line with the A=lg(1/T) relationship , how to ensure that the Lambert - Bill is met within the measurement range. law?
3 , the traditional detector to ensure that the measurement reading falls within the effective measurement range of the instrument, there is a sensitivity selection device on the instrument panel (
4. The measurement results of the traditional detector under different sensitivities are 0-10mv DC signals, and the signals are output to the recorder or computer collector. Obviously, the absorbance on the recording paper or computer screen is not the actual absorbance of the sample, but should also be modulated by the sensitivity of the instrument.
Fourth, the new detector
For the purpose of detecting, separating and purifying biological macromolecules such as proteins, nucleic acids and peptides , the computer nucleic acid protein detection system in the new chromatography experimental system has the following characteristics:
1. The system intelligently adjusts the absorbance to 0.000 and the light transmittance to 100% . And the absorbance and transmittance in the measurement range strictly conform to A=Lg(1/T) .
2 , single wavelength detection or dual wavelength synchronous detection.
3 , map editing and other functions. The degree is linear with the absorption coefficient, solution concentration and optical path. The system comes with a data acquisition workstation, integrated detection, acquisition, and software workstation integration. The analysis software has real-time tracing, analysis parameters, map preservation, map printing, map editing and other functions, providing peak height, peak width, peak area, normalization, retention time, half-width, peak width, area content, purity , column resolution and other parameters.
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