What does a spectrophotometer measure? Introduction to the principle of a spectrophotometer

What is a spectrophotometer

A spectrophotometer is a measuring instrument that uses discrete wavelengths to sample reflective or transmissive objects. Due to the different molecular structures of different objects, their absorption capacity for light of different wavelengths is also different. Therefore, each object has a specific absorption spectrum. An instrument that can separate each monochromatic light from a mixed light of various wavelengths and measure its intensity is called a spectrophotometer.

Spectrophotometry is the development of colorimetry. Colorimetry is limited to the visible light region, while spectrophotometry can be extended to the ultraviolet and infrared regions. Spectrophotometry requires nearly true monochromatic light, with a maximum spectral bandwidth of no more than 3-5nm, and can be less than 1nm in the ultraviolet region. It comes from a prism or grating and has high accuracy. Spectrophotometer? It is an instrument that uses spectrophotometry to perform quantitative and qualitative analysis of substances. Spectrophotometers can be divided into ultraviolet spectrophotometers, visible spectrophotometers (or colorimeters), infrared spectrophotometers or atomic absorption spectrophotometers.

Spectrophotometer components

Spectrophotometer has become a common instrument in modern molecular biology laboratories. It is often used for quantitative analysis of nucleic acids, proteins, and bacterial growth concentrations. The instrument is mainly composed of a light source, a monochromator, a sample chamber, a detector, a signal processor, and a display and storage system.

Spectrophotometer Features

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

Adopt 320*240 dot matrix high brightness 6” LCD display, with clear display and complete information;

The unique long optical path design makes the instrument have higher resolution, which is 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 entire optical path is independently fixed on a 16mm thick aluminum non-deformation base. The deformation of the base plate and external vibration have no effect on the optical system, which greatly improves the stability and reliability of the instrument;

Adopting synchronous sinusoidal mechanism, the wavelength has high accuracy and good repeatability;

Adopt ARM system;

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

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

The main components are imported, which makes the instrument have lower stray light, stronger stability and reliability;

The functions are more powerful. The host can independently complete photometric measurement, quantitative measurement, spectrum scanning, kinetics, DNA/protein testing, multi-wavelength testing and data printing.

Taking into full consideration the usage habits of different users, this series of instruments are equipped with Yuanxi's spectrum scanning software as standard. When operating online, in addition to realizing all the test functions of the host, it can also realize more powerful data processing functions and make data storage unlimited.

Spectrophotometer Principle

The spectrophotometer uses a light source that can generate multiple wavelengths, and then generates a light source of a specific wavelength through a series of spectroscopic devices. After the light passes through the sample being tested, part of the light is absorbed, and the absorbance value of the sample is calculated, which is then converted into the concentration of the sample. The absorbance value of the sample is proportional to the concentration of the sample.

When monochromatic light radiation passes through the solution of the substance being measured, the amount absorbed by the substance is proportional to the concentration of the substance and the thickness of the liquid layer (optical path length), and the relationship is as follows:

A=-lg(I/I0)=-lgT=kLc

Where: A is absorbance;

I0 is the incident monochromatic light intensity;

I is the intensity of the transmitted monochromatic light;

T is the transmittance of the substance; k is the molar absorption coefficient;

L is the optical path of the substance being analyzed, that is, the side length of the cuvette;

c is the concentration of the substance;

The wavelength of light selectively absorbed by a substance and the corresponding absorption coefficient are the physical constants of the substance. When the absorption coefficient of a pure substance under certain conditions is known, the sample can be prepared into a solution under the same conditions, and its absorbance can be measured. The content of the substance in the sample can be calculated by the above formula. In the visible light region, except for some substances that absorb light, many substances do not absorb light themselves, but can be measured after adding color reagents or treating them under certain conditions to develop color, so it is also called colorimetric analysis. Because there are many factors that affect the depth of color during color development, and instruments with poor monochromatic light purity are often used, standard or reference substances should be used for simultaneous operation during measurement.