Regardless of the Photometer, Colorimeter, or Spectrophotometer, the basic structural principles are similar, both by light source, monochromator, slit, absorber cup, and detector system. Composition (Figure 2-3).
(a) light source
A good light source requires high luminous intensity, stable light, wide spectral range and long service life. Almost all photometers use a regulated tungsten lamp (Tungsten Lamp). It is suitable for light sources in the 340-900nm range. A more advanced spectrophotometer is equipped with a hydrogen-regulated hydrogen lamp. It is suitable for use as a light source for UV spectroscopic analysis of 200-360 nm.
(2) Monochromator
Spectrophotometric determination of the absorbance of a substance needs to be carried out at a specific wavelength. The role of the monochromator is to select monochromatic light of a certain wavelength range as needed. It is difficult to select a single wavelength of light in actual work. Monochromatic light refers to the maximum emission at this wavelength, while the emission energy in the adjacent longer and shorter wavelength ranges is less. The narrower the wavelength range of monochromatic light, the higher the sensitivity of the instrument and the more reliable the measurement results.
The simplest monochromator is the filter (a certain color of glass) used on the photoelectric colorimeter. Since the spectral range of the passing light is wide, the resolution of the photoelectric colorimeter is poor, but the comparative color analysis can still obtain a satisfactory effect. Diffraction Grating of Prism is a better monochromator. They separate light from a single wavelength over a wide spectral range (Figure 2-4).
(three) slit
The intensity of the emitted light passing through the monochromator may be too strong or too weak to facilitate further detection. The slit is a slit formed by a pair of separators on the light path. The incident monochromatic light intensity is adjusted by adjusting the size of the slit and the incident light is formed into parallel rays to accommodate the needs of the detector. The slit of the photoelectric colorimeter is fixed, while the slit size of the photometer and spectrophotometer is adjustable.
(four) absorption cup
The absorption cup, also called the Sample Cell, is one of the most important parts of the photometric system. An optical glass absorber cup is used for measurement in the visible range, and a quartz absorber cup is used for measurement in the ultraviolet range. Note that protecting the quality of the absorption cup is one of the important conditions for obtaining good analytical results. Do not touch the absorption cup with rough or hard materials. Do not hold the optical surface of the absorption cup with your fingers. Rinse with water after use. Do not leave any test solution, especially protein and nucleic acid solution.
(5) Detector system
Photoelectric elements such as selenium photocells, photocells or photomultiplier tubes are commonly used as light receptors to convert the energy of light (I) through the absorption cup into electrical energy. The current generated is further measured by an appropriate method. The selenium photocell for the photoelectric colorimeter is used as a light receiver. Selenium photocells have low light sensitivity. It cannot detect very weak light. Further, it is insensitive to light waves having a wavelength of 270 nm or less and 700 nm or more.
More sophisticated spectrophotometers use vacuum phototubes or photomultiplier tubes as receivers, and use amplification to increase sensitivity. Although the energy of a monochromatic light with a narrow spectral range is much weaker than that of a wide range, such a sensitive detection system with an amplified line may still be accurately detected.
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