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UV-Vis Simplifies Chemical Analysis.

What do plating chemicals, sugars, environmental samples, phamaceuticals, and food samples have in common? Researchers can use a spectrophotometer to quantitatively analyze all of them. Spectrophotometry is routinely used for industrial, research, and quality-control applications and provides analysts with valuable qualitative and quantitative analytical data.

In spectrophotometry, analysts measure the light absorption by dissolved ions, molecules, or other chemical species. A spectrophotometer optically measures the absorbance or transmittance of specific light wavelengths by a dissolved chemical species. This instrument generally consists of a light source; a monochromator; a combination of slits, lenses, and mirrors; a sample cell; a photodetector; and a readout device.

Many spectrophotometers measure absorption of ultraviolet and visible light, which have wavelengths in the range of 200-900 nm. UV-Vis spectrophotometers typically have a deuterium lamp for the UV range and a tungsten bulb for the visible region. Analysts can expand the wavelength range in the UV and the IR regions by adding a mercury lamp and a photomultiplier-tube detector.

The lamps in a UV/Vis spectrophotometer direct an intense light beam through the monochromator, which separates light into its component wavelengths using diffraction gratings, prisms, or holographic gratings. Slits, lenses, and mirrors isolate a wavelength band 0.05-20 nm wide. This light band passes through the sample cell, which is also called a cuvette. The round or square sample cell is made of glass, quartz, or plastic. The light then travels to the photodector, a measuring device that converts light energy to electrical energy that can be displayed on a meter or a digital readout.

The dissolved ions and compounds absorb part of the light that passes through the sample. Absorption depends on ion concentration and wavelength--each chemical species absorbs specific light wavelengths characteristic of its atomic structure.

Plotting light absorption as a function of wavelength gives a unique spectrum for each chemical species. Analysts use this plot to identify dissolved compounds or ions. They also calculate concentrations from absorption values at a particular wavelength using the Beer-Lambert law--A = [Epsilon] cd. A is absorbance, [Epsilon] is a molar absorption coefficient specific to the compound and wavelength, c is concentration, and d is the distance through the solution that the light travels.

Key parameters for UV/Vis spectrophotometers are low stray light, resolution, bandwidth, and wavelength range. These and the suitability of an instrument for an application define UV/Vis spectrophotometer performance.

Stray light is radiant energy outside the bandpass that is specifically transmitted by the monochromator. If analysts don't correct for stray light, it introduces serious errors in calculated concentrations based on calibration curves. Imperfections in a diffraction grating or prism, scattered light, light leaks from outside the spectrophotometer, and second-order effects cause stray light. Recent improvements in grating technologies have made high-resolution, low-stray-light spectrophotometers readily available.

The resolution--the ability of a spectrophotometer to detect absorbance changes in a narrow wavelength range--depends on the bandwidth. Ideally, the bandwidth of the spectrophotometer should be as narrow as possible.

When choosing a spectrophotometer, you should consider the applications for which it will be used.

UV/Vis instruments have a single-beam or a double-beam configuration. In a single-beam instrument, the solvent or blank absorption is stored in memory and subtracted by a microprocessor. In a double-beam spectrophotometer, the light beam is split in half. The two halves pass separately through the sample and a reference solution. This gives a direct reading of absorbance differences between the sample and the blank. Analysts often use double-beam instruments to produce absorbance spectra of unknown compounds, because they correct for solvent contributions over the entire spectral range.

Educational, biology, chemistry, and industrial quality-control and validation laboratories that use UV/Vis spectrophotometers to measure and display concentrations should consider low-cost models designed for quantitative analysis. Biochemistry, molecular biology, analytical chemistry, clinical, and pharmaceutical laboratories that perform enzyme or reaction kinetics assays need more sophisticated, computer-interfaced instruments with optional features such as sample cell temperature regulation, flow cells, and data manipulation/analysis software.
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Author:Karet, Gail
Publication:R & D
Article Type:Brief Article
Geographic Code:1USA
Date:Mar 1, 1999
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