Visible light absorption spectrophotometry

Upstone SL. Ultraviolet/visible light absorption spectrophotometry in clinical chemistry. In Meyers RA, ed. Encyclopedia of analytical chemistry Applications, theory, and instrumentation. New York John Wiley Sons, 2000 1699-713. 

Upstone, S.L., 2000. In Meyers, R.A. (Ed.), Ultraviolet/Visible Light Absorption Spectrophotometry in Clinical Chemistry. Encyclopedia of Analytical Chemistry. John Wiley Sons Ltd, Chichester, pp. 1699-1714. 

Ultraviolet-Visible Spectroscopy Ultraviolet-visible (UV-VIS) molecular absorption spectrophotometry (often called light absorption spectrophotometry or just UV-visible spectrophotometry) is a technique based on measuring the absorption of near-UV or visible radiation (180-770 nm) by molecules in solution.35,36 Reference standard characterization by UV-VIS spectophotometry includes determining the absorption spectra and the molar extinction coefficient. These two spectral characterizations are used as identifiers of reference standards. 

Chapter 1 is an introduction to the field of molecular fluorescence, starting with a short history of fluorescence. In Chapter 2, the various aspects of light absorption (electronic transitions, UV-visible spectrophotometry) are reviewed. 

Spectrophotometry is any technique that uses light to measure chemical concentrations. A procedure based on absorption of visible light is called colorimetry. The most-cited article in the journal Analytical Chemistry from 1945 to 1999 describes a colorimetric method by which biochemists measure sugars.4... 

Developments in traditional forms of spectrophotometry, as well as new methods, could find greater use in ocean measurements. Spectroscopy based on absorption of visible light may have reached a limit in its traditional form, however. Spectrophotometry will remain in wide use due to its ease, low cost, and great versatility. In many ways it remains the first choice for analysis, but its low sensitivity makes it useful for a rather limited spectrum of analytes. 

Spectrophotometry (the measurement of light absorption or transmission), is one of the most valuable analytical techniques available to biochemists. Unknown compounds may be identified by their characteristic absorption spectra in the ultraviolet, visible, or infrared. Concentrations of known compounds in solutions may be determined by measuring the light absorption at one or more wavelengths. Enzyme-catalyzed reactions frequently can be followed by measuring spectrophotometrically the appearance of a product or disappearance of a substrate. 

There are two commonly used sources of light in UV-visible absorption spectrophotometry, hydrogen or deuterium discharge lamps and incandescent filament... 

IR is an absorption method. Analysis utilizing absorption measurements can also be done with spectrophotometry and atomic absorption spectrophotometry (AAS). In the former method a monochromatic light (visible or ultraviolet) is passed through a solution containing a compound of an element with unknown concentration. The light absorption is measured and converted to concentration. 

The technique of detection via light absorption [9] exploits results from UV-visible spectrophotometry, especially in the use of monochromatic radiation and a reference system. This requires the presence of at least two fibers, to separate the outward and inward light beams, and so this method is rarely used. In contrast, fluorescence [10] can be measured directly using a single fiber and very low concentrations can be detected. Furthermore, coupling the immobilized enzyme on an optical fiber with a fluorescent cc actor, such as NADH, opens up greater possilnlities. The system is even simpler in the case of bio/chemiluminescence because the reference and excitation beams are obsolete, and the light can be emitted directly to the sensitive component [11]. The excellent quantum yield of bioluminescence also facilitates the detection of low concentrations. 

For a compound to be analyzed by spectrophotometry, it must absorb light, and this absorption should be distinguishable from that due to other substances in the sample. Because most compounds absorb ultraviolet radiation, measurements in this region of the spectrum tend to be inconclusive, and analysis is usually restricted to the visible spectrum. If there are no interfering species, however, ultraviolet absorbance is satisfactory. Proteins are normally assayed in the ultraviolet region at 280 nm because the aromatic groups present in virtually every protein have an absorbance maximum at 280 nm. 

Ultraviolet spectrophotometers cont.), single-beam, 225 standardisation, 226 Ultraviolet spectrophotometry, 221-232 absorption cells, 226 colorimetry, 228 derivative, 230 difference method, 229 dual-wavelength, 229 identification by, 231 influence of pH, 224 influence of solvent, 224 laws of absorption, 222 quantitative applications, 227 stray-light effects, 224 Ultraviolet-visible detector, 202 multiwavelength, 211 Unicontin, 1011 Unidiarea, 474 Unidone, 356 Uniflu, 557, 893 Unilobin, 709 Unimycin, 846 Uniphyllin, 1011 Uniprofen, 677 Unisom, 576... 

The most widespread use of UV and visible spectroscopy in biochemistry is in the quantitative determination of absorbing species (chromophores), known as spectrophotometry. All spectrophotometric methods that measure absorption, including various enzyme assa3rs, detection of proteins, nucleic acids and different metabolites, reside upon two basic rules, which combined are known as the Beer-Lambert law. Lambert s law states that the fraction ofli t absorbed by a transparent medium is independent of the incident li intensity, and each successive layer of the medium absorbs an equal fraction of the li t passing throu it. This leads to an exponential decay of the light intensity along the light path in the sample, which can be expressed mathematically, as follows ... 

Derivative and dual-wavelength spectrophotometry have also proved particularly useful for extracting ultraviolet-visible absorption spectra of analytes present in turbid solutioas, where light scattering obliterates the details of an absorption spectrum. For example. 

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