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Colorimetric analysis is a method of determining the concentration of a chemical element or chemical compound in a solution with the aid of a color reagent.It is applicable to both organic compounds and inorganic compounds and may be used with or without an enzymatic stage.
Colorimetric assays use reagents that undergo a measurable color change in the presence of the analyte. They are widely used in biochemistry to test for the presence of enzymes, specific compounds, antibodies, hormones and many more analytes. For example, para-Nitrophenylphosphate is converted into a yellow product by alkaline phosphatase enzyme.
A colorimeter is a device used in colorimetry that measures the absorbance of particular wavelengths of light by a specific solution. [1] [2] It is commonly used to determine the concentration of a known solute in a given solution by the application of the Beer–Lambert law, which states that the concentration of a solute is proportional to the absorbance.
Colorimetry is "the science and technology used to quantify and describe physically the human color perception". [1] It is similar to spectrophotometry , but is distinguished by its interest in reducing spectra to the physical correlates of color perception, most often the CIE 1931 XYZ color space tristimulus values and related quantities.
It is done in one step where the Bradford reagent is added to a test tube along with the sample. After mixing well, the mixture almost immediately changes to a blue color. When the dye binds to the proteins through a process that takes about 2 minutes, a change in the absorption maximum of the dye from 465 nm to 595 nm in acidic solutions ...
This colorimetric method is ineffective when comparable amounts of arsenate are present in solution with phosphate. This is due to the strong chemical likeness of arsenate and phosphate. The resultant molybdenum blue for arsenate, using the same procedure, does produce a slightly different spectral signature, however. [11]
Automated color measurement is a newer method of liquid color measurement. In this case the sample is contained in a test tube; the tube is inserted into the instrument, and the color properties of the liquid read out on a screen. This method can provide objective measurements, but is far more expensive than a set of color standards.
The method combines the reactions of copper ions with the peptide bonds under alkaline conditions (the Biuret test) with the oxidation of aromatic protein residues. The Lowry method is based on the reaction of Cu +, produced by the oxidation of peptide bonds, with Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid in the Folin–Ciocalteu reaction).