How does a spectrophotometer work?
- 1 decade agoFavorite Answer
Light is quantized into tiny packets called photons, the energy of which can be transferred to an electron upon collision. However, transfer occurs only when the energy level of the photon equals the energy required for the electron to get promoted onto the next energy state, for example from the ground state to the first excitation state (Boyer, 1993). This said process is the basis for absorption spectroscopy, the technique used in the experiment to estimate the quantity and quality of DNA in solution. Generally, light of a certain wavelength and energy is illuminated on the sample, which absorbs a certain amount of energy from the incident light. The energy of the light transmitted from the sample afterwards is measured using a photodetector, which registers the absorbance of the sample.
The spectrophotometer is a complex instrument used in measuring the absorbance of biomolecules within the ultraviolet and visible light spectrum, similar to the one found in the laboratory. It is a conglomerate of light sources, wavelength selectors, optical systems, sample chambers, photodetectors, and meters functioning together to perform a specific task – to measure the absorbance of a sample.
There are two existing light sources within a UV-VIS spectrophotometer – one for each (UV and visible light) spectrum. The usual light source used to generate visible light is the tungsten-halogen lamp emitting 200-340 nm wavelengths (Boyer, 1993). The UV source can be either a high-pressure hydrogen lamp or deuterium lamp, the latter of which is the one found in the lab. When measuring absorbance at the UV spectrum, the other lamp has to be turned off. The same goes when measuring visible light absorbance. This is to prevent interference of unnecessary wavelengths in the incident light on the sample. Following the light source is a monochromator, the purpose of which is to filter light and select a specific wavelength by using either a prism or a diffraction grating. After the monochromator is a series of lenses, slits, mirrors, and filters that act as an optical system to concentrate, increase spectral purity of, and direct monochromatic light towards the sample chamber with cuvettes containing solutions to be tested. In the laboratory, the sample chamber is equipped with multiple slots to allow for continuous measurements of several sample replicates at a particular wavelength. However, since the instrument has only a single beam, every time the wavelength has to be changed a blank reading must precede any sample reading. With regards to cuvettes, which contain the sample solutions, there are three kinds available for use today. The first is made of glass and is often utilized for reading absorbance at wavelengths greater than 340 nm due to its undesirable absorption of UV light. The second is made of fused silica or quartz and is the one used in the experiment. It can be utilized in absorbance measurement throughout the UV-VIS spectrum (200 nm to 800 nm) because of its high grade of transparency. The last class is of disposable cuvettes, the material of which can vary. One example is made of polymethacrylate and is used only for measurement at 280 nm to 800 nm (Potter, 1995).
The light-sensitive detector follows the sample chamber and measures the intensity of light transmitted from the cuvettes and passes the information to a meter that records and displays the value to the operator on an LCD screen. Two kinds are of use in UV/VIS spectrophotometry today – the phototube and the photomultiplier tube. The phototube or photocell functions by generating an electric current. When a photon hits the cathode of the cell, an electron is ejected from the cathode and directed to the anode. This flow of electron produces a current, the magnitude of which is proportional to the energy of the photon. The photomultiplier tube, which is more sensitive, relies on Planck’s Photoelectric Effect. Photons hitting the tube’s photosensitive surface eject primary electrons, which then collide with another surface and release secondary electrons. These secondary electrons hit several other surfaces and eject more secondary electrons, which eventually get caught by an anode and produce an electric current. The current generated, however, is several-fold amplified so that even a single photon with very low energy can be detected and registered (Christian, 2004).
(1) Boyer, Rodney F. Modern Experimental Biochemistry. 2nd ed. California: Benjamin/Cummings Publishing, 1993.
(2) Christian, Gary D. Analytical Chemistry. 6th ed. New Jersey: John Wiley & Sons, Inc., 2004.
(3) Potter, Geoffrey W.H. Analysis of Biological Molecules: an introduction to principles, instrumentation, and techniques. Oxford: Alden Press, 1995.
- Anonymous4 years ago
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- 1 decade ago
It works by a light passing through a solution, the higher the M concentration of the solution the more light is absorbed. The percent of transmittance will help analysis the M concentration
- Anonymous1 decade ago
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