Explain the theory of UV spectroscopy and its applications in biology.
The UV spectroscopy is the type of absorption spectroscopy in which light of ultraviolet region is absorbed by the molecule. This results in the excitation of electrons from the ground state to the higher energy state. The energy of the ultraviolet radiation that are absorbed is equal to the energy difference between the ground state and higher energy state.
Principle of UVspectroscopy: It is based on the principle of Beer-Lambert law. It states that when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with the thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution.
The expression of Beer-Lambert law is A= log (l0/l) = Ecl
Where A is absorbance, l0 is intensity of light incident upon sample cell, l is the intensity of light leaving sample cell, C is the molar concentration of solute, L is the length of sample and E is molar absorptivity.
Instrumentation: The modern UV spectrometers consist of following parts:
Light source: The tungsten filament lamps and hydrogen-deuterium lamps are most widely used that covers the whole UV region.
Monochromator: It is generally composed of prism and slits. The most of the spectrometers are double beam spectrometers. The radiation emitted from the primary source is dispersed with the help of rotating prism. The various wavelength of the light source which are separated by the prism is selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism.
Sample and reference cell: one of the two divided beams is passed through the sample solution and second beam is passed through the reference solution. Both the sample and reference solution are contained in the cells. These cells are made up if silica or quartz.
Detector: Generally two photocells serve the purpose of detector in the UV spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.
Amplifier: The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled to a small servometer. Generally current generated in the photocell is of very low intensity. Its main purpose is to amplify the signals many times to record signals.
Recording device: the amplifier is coupled to a pen recorder which is connected to the computer.
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