What is a Spectrophotometer?
A spectrophotometer is a measuring instrument that samples reflective or transmissive objects with discrete wavelengths. Due to the different molecular structures of different objects, the ability to absorb light of different wavelengths is also different. Therefore, each object has a specific absorption spectrum. A spectrophotometer separates each monochromatic light from a mixture of wavelengths and measures its intensity.
Spectrophotometry is a development of colorimetry. The colorimetric method is limited to the visible light region, and the spectrophotometric law can be extended to the ultraviolet light region and the infrared light region. Spectrophotometry requires that it is close to true monochromatic light, and its spectral bandwidth does not exceed 3-5nm at most, and it can be below 1nm in the ultraviolet region, which comes from prisms or gratings, and has high precision. Spectrophotometer is an instrument for quantitative and qualitative analysis of substances by spectrophotometry. Spectrophotometers can be divided into ultraviolet spectrophotometers, visible light spectrophotometers (or colorimeters), infrared spectrophotometers or atomic absorption spectrophotometers.
Spectrophotometers have become routine instruments in modern molecular biology laboratories. It is commonly used for nucleic acid, protein quantification and quantification of bacterial growth concentration. The spectrophotometer is mainly composed of a light source, a monochromator, a sample chamber, a detector, a signal processor, and a display and storage system.
It includes the visible light region with wavelengths ranging from 400 to 760 nm and the ultraviolet region with wavelengths ranging from 200 to 400 nm. Different light sources have their own unique emission spectrum, so different luminous bodies can be used as the light source of the instrument.
Emission spectrum of tungsten lamp: After the spectral light of 400-760nm wavelength emitted by the tungsten lamp light source is refracted by a prism, a continuous chromatogram consisting of red-orange, yellow-green, blue indigo and violet can be obtained. These chromatograms can be used as light sources for visible spectrophotometers.
Emission spectrum of hydrogen lamp (or deuterium lamp): Hydrogen lamp can emit a spectrum of 185-400 nm wavelength, which can be used as the light source of UV VIS spectrophotometer.
The absorption spectrum of the substance: 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. It appears several dark lines, that is, a certain part of the emission spectrum of the light source. Some wavelengths of light disappear due to absorption by the solution. This is called the absorption spectrum of the solution.
Different substances has different absorption spectrum. Therefore, according to the absorption spectrum, the substances contained in the solution can be identified. When light passes through a solution of a substance, the intensity of the transmitted light is reduced, because part of the light is reflected or dispersed on the surface of the solution, part of the light is absorbed by the substances that make up the solution, and only part of the light can pass through the solution.