Spectrophotometer

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Basic components of a Spectrophotometer consist of the exciter lamp, the entrance slit, the monochromator, the analytical cell or cuvette, and the photodltector. Components of a single beam spectrophometer. A, exiter lamp, B entrance slit C monochromator, D exit, E cuvetter, F photodetector and G meter. An exciter lamp provides electromagnetic radiation as visible, infrared, or UV light that will pass through the monochromator to be separated into discrete wavelengths. Light of a selected wavelength will be incident on the cuvette containing the solution of which the absorption is to be measured. For spectrophotometric work in the visible and near-infrared ranges, tungsten and halogen quartz lamps are good sources of radiant energy. Several types of vapor lamps are available for ultraviolet range. The hydrogen lamp is widely used. A mercury lamp is less desirable owing to its uneven emission spectrum. A xenon lamp gives a brilliant light that is ideal for applications requiring a narrow slit, but it is not suited for routine application owing to problems with stray light. For infrared spectrophotometry, a silicone carbide rod heated to 1200 degree Celsius works well. Collimating lenses are often inserted between the exciter lamp and the entrance slit to focus the light into a beam of parallel light rays.

The function of the entrance slit is to reduce stray light and prevent scattered light from entering the monochromator. If stray light were allowed to pass through the analytical cell, this would cause a deviation from the Beer-Lambert law. The result would cause a significant error in the measurement.

A monochromator is a device that produces light of specific wavelengths from a light source. Types of monochrdmators include prism, diffraction grating, and interference filter. Prisms are wedge haped pieces of glass, quartz, or sodium chloride. When white light strikes a prism, it is dispersed to form a spectrum due to different angles of refraction by different wavelength at the air-prism interface.
Diffraction gratings are made by cutting tiny grooves or slits into an aluminized surface of a flat piece of crown glass. These grooves arc cut at a precise angle and at an equal distance from each other. There are usually 1000 to 50,000 grooves to the inch. Each of these grooves acts both as a prism to refract white light and as a slit to diffract it into several spectra. Each spectrum is at a different angle from the grating. The brightest of these is called the first-order spectrum that is to be used for measurement. Usually, gratings are capable of better resolution than prisms. Gratings also have the additional advantage of covering all essential wavelengths, in contrast to the glass or quartz prisms, which cannot be used in the ultraviolet region. Because high-quality gratings can now be produced economically, most spectrophotometers incorporate diffraction gratings.

Interference filters are made by placing semitransparent silver films on both sides of a dielectric such as magnesium fluoride. When light perpendicular to the silvered surface enters the filter, it passes through the dielectric and is reflected from the second silvered surface back to the first surface. This process repeats itself until the light is finally transmitted through the filter and into the analytical cell. Constructive and destructive interferences occur as the light is reflected between the silver films. Interference filters allow transmission of 40 to 60 percent of the incident light, with a bandwidth between
10 nm and 20 nm. By definition, a bandpass is the range of wavelengths between the points at which transmittance is one half peak transmittance. The dielectric thickness may be varied to produce filters of different bandpasses.

The cuvette or analytical cell holds the solution of which the absorption is to be measured. Cuvettes are made of soft glass, borosilicate glass, quartz, or plastic. Soft glass cuvettes are preferable for acidic solutions that do not etch glass. Strong alkaline solutions should be measured in borosilicate cuvettes because of their high resistance to alkali. Only quartz or plastics that do not absorb ultraviolet radiation may be appropriate for wavelengths below 320 nm. A rectangular cuvette, which presents a flat surface to the incident light, has less radiant energy loss from reflection than does a round cuvette. For routine work, this loss is usually not significant, accounting for about 4 percent of the incident energy for most round cuvettes. Room light entering the cuvette may cause measurement errors. A light shield should be placed over the cuvette well when a spectrophotometric reading is being made. Types of photodetectors include barrier-layer cell, phototube, photomultiplier tube or PMT, and a variety of semiconductor photo detectors. All of these devices use photosensitive materials in their cathodes that release electrons when they are exposed to light energy. The anodes attract or collect electrons emitted from the cathode. If a closed electrical circuit is provided, the free electrons produce a current.

Barrier layer cells generate their own electrical output directly from light energy and do not need an external power source. Selenium coated with silver serves as the negative electrode while the iron base serves as the positive electrode. The spectral response of a barrier layer cell is in the range of 380 to 700 nm. These cells are found in older model colorimeters and spectrophotometers.

The widely used phototube has a curved sheet of photosensitive material that serves as the cathode and a positively charged thin tube that serves as the anode. A limitation of the phototube is the small amount of photocurrent generated.
PMTs consist of a photoemissive cathode, an anode, and an internal electron-multiplying series of dynodes. Many photomultiplier tubes have 9 to 16 photosensitive dynodes. All of these components are encased in a glass evacuated tube. When radiant energy strikes the cathode, the emitted electrons are attracted to the first adjacent dynode. On striking the dynode, each electron causes the emission of several other electrons. The electrons emitted from the first dynode are subsequently attracted to the second dynode, where the same emission cycle is repeated. This process continues through the entire series of dynodes, resulting in a multiplication of the number of electrons, until the anode is reached. The amplification factor achieved by a photomultiplier tube may be as high as 106. Because of their excellent sensitivity and rapid response, all stray light and room light must be carefully shielded from the photomultiplier tube to prevent bum out.

Semiconductor detectors including photo resistor, photodiode, and phototransistor have virtually replaced conventional phototubes in modem laboratory instruments. A semiconductor is used in an electrical circuit to regulate the current by changing its internal resistance. This is accomplished by changing the potential bias across its semiconductor junction. Unlike conventional semiconductors that respond to changes in voltage, semiconductor photo detectors respond to bias changes resulting from absorption of radiant energy.