Colorimetry

Colorimetric assays use the optical density of the solution in the well or the kinetic change of the optical density as readout parameter. These assays are typically applied to study the functional activity of enzymes. With an appropriate substrate it is possible to measure the activity directly by recording the color change in the well that originates from the differences in the absorbance spectra between the educt and the product of the reaction. In cases where the educt and product are colorless, a further chemical reaction is carried out to produce a colored final product 

In one type of colorimetric assays, the absorbance assays, the substrate absorbs light while the reaction product has no absorbance at the test wavelength, or vice versa. An example is NADH, which absorbs at 340 run, while NAD has no absorbance at this wavelength. NADH is a substrate for dehydrogenases with NAD+ being the product and hence the activity of these enzymes can be directly assessed by absorbance assays at 340 nm. 

Chromogenic assays are colorimetric assays which use a substrate containing a chromophore. The substrate itself is designed such that it is colorless, and during the enzymatic reaction the chromophore is released and the color change is measured by a plate reader. This technique is applied for certain enzyme reactions for which a conversion of assays to fluorescent readout for HTS is Hkely to be problematic or even impossible, e.g., when it would require labehng of a small substrate with a large fluorophore. 

Phosphomolybdate - Malachite Green complex with absorbance at 620 nm 

Due to their simple performance, and especially the potential ability to eliminate the use of analytical instruments, more and more colorimetric sensors are being developed, including DNA sensors (Elghanian et al., 1997), pH sensors (Gazda et al., 2004), metal ion sensors (Ghosh et al., 2006), and now, aptasensors (Huang et al., 2005 Lu and Liu, 2006, 2007 Wang et al., 2006 Zhao et al., 2007). 

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Colorimetry, in which a sample absorbs visible light, is one example of a spectroscopic method of analysis. At the end of the nineteenth century, spectroscopy was limited to the absorption, emission, and scattering of visible, ultraviolet, and infrared electromagnetic radiation. During the twentieth century, spectroscopy has been extended to include other forms of electromagnetic radiation (photon spectroscopy), such as X-rays, microwaves, and radio waves, as well as energetic particles (particle spectroscopy), such as electrons and ions. ... 

Numerous methods for the deterrnination of monomer purity, including procedures for the deterrnination of saponification equivalent and bromine number, specific gravity, refractive index, and color, are available from manufacturers (68—70). Concentrations of minor components are deterrnined by iodimetry or colorimetry for HQ or MEHQ, by the Kad-Eisher method for water, and by turbidity measurements for trace amounts of polymer. 

National Institute of Standards and Technology (NIST). The NIST is the source of many of the standards used in chemical and physical analyses in the United States and throughout the world. The standards prepared and distributed by the NIST are used to caUbrate measurement systems and to provide a central basis for uniformity and accuracy of measurement. At present, over 1200 Standard Reference Materials (SRMs) are available and are described by the NIST (15). Included are many steels, nonferrous alloys, high purity metals, primary standards for use in volumetric analysis, microchemical standards, clinical laboratory standards, biological material certified for trace elements, environmental standards, trace element standards, ion-activity standards (for pH and ion-selective electrodes), freezing and melting point standards, colorimetry standards, optical standards, radioactivity standards, particle-size standards, and density standards. Certificates are issued with the standard reference materials showing values for the parameters that have been determined. 

Procedures for determining the quaUty of formaldehyde solutions ate outlined by ASTM (120). Analytical methods relevant to Table 5 foUow formaldehyde by the sodium sulfite method (D2194) methanol by specific gravity (D2380) acidity as formic acid by titration with sodium hydroxide (D2379) iron by colorimetry (D2087) and color (APHA) by comparison to platinum—cobalt color standards (D1209). 

Methods for iodine deterrnination in foods using colorimetry (95,96), ion-selective electrodes (94,97), micro acid digestion methods (98), and gas chromatography (99) suffer some limitations such as potential interferences, possibHity of contamination, and loss during analysis. More recendy neutron activation analysis, which is probably the most sensitive analytical technique for determining iodine, has also been used (100—102). 

Solvent extraction techniques are useful in the quantitative analysis of niobium. The fluoro complexes are amenable to extraction by a wide variety of ketones. Some of the water-insoluble complexes with organic precipitants are extractable by organic solvents and colorimetry is performed on the extract. An example is the extraction of the niobium—oxine complex with chloroform (41). The extraction of the niobium—pyrocatechol violet complex with tridodecylethylammonium bromide and the extraction of niobium—pyrocatechol—sparteine complex with chloroform are examples of extractions of water-soluble complexes. Colorimetry is performed on the extract (42,43). Colorimetry may also be performed directly on the water-soluble complex, eg, using ascorbic acid and 5-nitrosahcyhc acid (44,45). 

The nitroparaftins have been determined by procedures such as fractionation, titration, colorimetry, kifrared spectroscopy, mass spectrometry, and gas chromatography. The early analytical methods and uses of polynitroparaftins as analytical reagents have been reviewed (11). More recent quaHtative and quantitative methods have also been reviewed (83). 

ISO 7724-1-1984, CIE Standards Colorimetric Observers Paints and Varnishes—Colorimetry, Part 1, Principles-, ISO 7724-2-1984, Part 2, Colour Measurement, ISO, Geneva, Swit2erland. 

ISO 7724-3-1984, 1st ed.. Paints and Varnishes—Colorimetry, Part 3, Calculation of Colour Differences, ISO, Geneva, Switzerland. 

A number of simple, standard methods have been developed for the analysis of ammonium compounds, several of which have been adapted to automated or instmmental methods. Ammonium content is most easily deterrnined by adding excess sodium hydroxide to a solution of the salt. Liberated ammonia is then distilled into standard sulfuric acid and the excess acid titrated. Other methods include colorimetry (2) and the use of a specific ion electrode (3). 

Methods chemi = chemiluminescence color = colorimetry and fluor = fluorescence. Manual loading. 

Absorbance. Analyte measurements in clinical analyzers using Hquid reagents are most commonly performed by transmission of light, ie, by absorbance photometry or colorimetry (Fig. 3a). The Hquid to be analyzed is either held in a cuvette or passed through a flowceU having transparent walls. 

For colorimetric or gravimetric determination l-nitroso-2-naphthol can be used. For chromatographic ion exchange (qv), cobalt is isolated as the nitroso-(R)-salt complex. The cyanate complex is used for photometric determination and the thiocyanate for colorimetry. A rapid chemical analysis of... 

Cahbration with standard reflectance and transmittance samples should be routinely used for optimum results in spectrophotometry and colorimetry. Cahbration of the wavelength (32) and photometric (33) scales is also advisable. The cahbration of a white reflectance standard in terms of the perfect reflecting diffuse, T, has been discussed (34), as have diagnostic tiles for tristimulus colorimetry (35). A collaborative reference program is available on instmment performance (36). 

CIE Publication 15.2 Colorimetry, 2nd ed.. Central Bureau of CIE, Vienna, Austria, 1986 Available from U.S. National Committee CIE, c/o National Institute of Standards and Technology, Washington, D.C. 

Cyanide in Waters etc. (by Reflux Distillation followed by either Potentiometry using a Cyanide Selective Electrode or Colorimetry, or Continuous Elow Determination of Cyanide or Determination by Microdiffusion), 1988... 

Sulphate in Waters, Effluents and Solids (2nd Edition) [including Sulphate in Waters, Effluents and Some Solids by Barium Sulphate Gravimetry, Sulphate in waters and effluents by direct Barium Titrimetry, Sulphate in waters by Inductively Coupled Plasma Emission Spectrometry, Sulphate in waters and effluents by a Continuous Elow Indirect Spectrophotometric Method Using 2-Aminoperimidine, Sulphate in waters by Elow Injection Analysis Using a Turbidimetric Method, Sulphate in waters by Ion Chromatography, Sulphate in waters by Air-Segmented Continuous Elow Colorimetry using Methylthymol Blue], 1988... 

Spectrophotometry-Colorimetry One of most useful and versatile methods but can be time consuming 10- to 10- M (10- with pre-concentration)... 

Greater range of detection systems to which the desorbed gas can be subjected (e.g. chromatography, infra-red and ultraviolet spectroscopy, colorimetry) Limitations Certain resins undergo degradation even below 250°C Test sample may be thermally unstable Not all compounds readily desorb ... 

Tetra-alkyl lead compounds in air Personal monitoring with atomic absorption analysis or electrothermal atomization or X-ray fluorescence spectrometry or on-site colorimetry 9... 

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