The Principle of a UV Spectrometer

Ultraviolet (UV)-visible spectrometers are used to analyze the interactions between radiation and matter in the UV-visible region of the electromagnetic spectrum. The principle involved is the measurable absorption of energy by chemical compounds when certain electronic transitions are excited between their molecular orbitals.

Spectrometer Principle
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A source of polychromatic light with a continuous UV spectrum is separated by a prism or diffraction grating into component UV wavelengths between 200 and 400 nm (nanometers) and visible wavelengths between 400 and 800 nm. Each wavelength is split into two equal-intensity beams by a half-mirrored device. The sample beam passes through a container of the compound being studied. A reference beam passes through an identical container of sample solvent.

Scanning and Detection

The spectrometer automatically scans all the component wavelengths passing through the containers. The intensity of each wavelength is measured by electronic detectors and compared. The reference beam intensities are subtracted to determine the absorption of energy by the sample at each wavelength.

Absorption Spectrum
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The spectrometer records all the wavelengths at which absorption occurs together with the amount of absorption at each wavelength. The instrument then creates a graph, called a spectrum, of absorbance versus wavelength.

UV-Visible Spectrometer Instruments

Cintra 101, for powerful performance at an entry level price Cintra 202, offering automation and has higher specifications than the 101 Cintra 303, a research grade instrument with variable slit width and enhanced UV performance Cintra 404, a true double monochromator with outstanding specifications for best performance

Powerful Software Cintral software is Windows XP based and offers full instrument and accessory control, data analysis, report generation and complete system performance validation and IQ/OQ. Cintral software is also available as an upgrade on any first or second generation GBC UV-Vis spectrometer. Automated Accessories A complete range of modular automated accessories is available, including auto sippers, batch sample changers, two total integrating spheres, autosamplers and a wide choice of ambient, water-thermostatable or peltier controlled cell holders. Changeover is quick and easy with the accessories being automatically recognized by the software.

Cintra 101

The Cintra 101 is a budget-priced, entry level instrument. It is a true double beam spectrometer, with low stray light and noise specifications. The Cintra 101 is now available with Windows XP-based Cintral software and is capable of wavelength

scanning, time scanning, fixed wavelength measurement, quantify application and an instrument validation application.

Cintra 202
The Cintra 202 will challenge many higher priced UV-Vis and UV-Vis-NIR instruments with its wavelength range of 190 to 1200nm and very low stray light and noise specifications. This true double beam spectrometer will suit routine lab work or more advanced applications reaching into the NIR regions. With a full range of accessories available including samples changers, auto samplers, peltier or water thermostattable temperature control and reflectance spheres capable of measurement up to 1150nm, this low cost instrument provides maximum flexibility and power for all applications.

Layout of a Spectrometer

The most basic design of a modern spectrometer is an assembly of a slitted screen, a diffraction grating and a photodetector. The screen allows a beam of light into the interior of the spectrometer, where the light passes through the diffraction grating. The grating splits the light into a beam of its component colors, similar to a prism. According to the University of Arizona (reference 1), many spectrometers also have a collimating mirror that makes the light waves parallel and coherent, thus making it more focused. This applies especially to spectrometers used in telescopes. The light then reflects onto a detector that picks up individual wavelengths.

working: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 Plancks Photoelectric Effect. Photons hitting the tubes 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).