physical Physical may refer to: *Physical examination In a physical examination, medical examination, or clinical examination, a medical practitioner examines a patient for any possible medical signs or symptoms of a medical condition. It generally co ...

and

analytical chemistry Analytical chemistry studies and uses instruments and methods to separate, identify, and quantify matter. In practice, separation, identification or quantification may constitute the entire analysis or be combined with another method. Separati ...

, colorimetry or colourimetry is a technique used to determine the concentration of colored compounds in

solution Solution may refer to: * Solution (chemistry), a mixture where one substance is dissolved in another * Solution (equation), in mathematics ** Numerical solution, in numerical analysis, approximate solutions within specified error bounds * Soluti ...

. A colorimeter is a device used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light (not to be confused with the

tristimulus colorimeter A tristimulus colorimeter, colloquially shortened to ''colorimeter'', is used in digital imaging to profile and calibrate output devices. It takes a limited number of wideband spectral energy readings along the visible spectrum by using filtered ...

used to measure colors in general). To use the colorimeter, different solutions must be made, including a control or reference of known concentration. With a visual colorimeter, for example the Duboscq colorimeter illustrated, the length of the light path through the solutions can be varied while

filtered Filtration is a physical separation process that separates solid matter and fluid from a mixture using a ''filter medium'' that has a complex structure through which only the fluid can pass. Solid particles that cannot pass through the filter m ...

light transmitted through them is compared for a visual match. The concentration times path length is taken to be equal when the colors match, so the concentration of the unknown can be determined by simple proportions.

Nessler tube Nessler cylinders (also named color comparison cylinders or color comparing cylinders) are laboratory tubes with a fixed volume, made of glass with optically plane bottom. On the walls, there are marks of the nominal stroke volume (usually 100 ...

s work on the same principle. There are also electronic automated colorimeters; before these machines are used, they must be calibrated with a

cuvette A cuvette ( French: cuvette = "little vessel") is a small tube-like container with straight sides and a circular or square cross section. It is sealed at one end, and made of a clear, transparent material such as plastic, glass, or fused quartz. ...

containing the control solution. The concentration of a sample can be calculated from the intensity of light before and after it passes through the sample by using the

Beer–Lambert law The Beer–Lambert law, also known as Beer's law, the Lambert–Beer law, or the Beer–Lambert–Bouguer law relates the attenuation of light to the properties of the material through which the light is travelling. The law is commonly applied t ...

. Photoelectric analyzers came to dominate in the 1960s. The color or wavelength of the filter chosen for the colorimeter is extremely important, as the wavelength of light that is transmitted by the colorimeter has to be the same as that absorbed by the substance being measured. For example, the filter on a colorimeter might be set to red if the liquid is blue.

Absorption colorimeter

A colorimeter is a device used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light. To use this device, different

s must be made, and a control (usually a mixture of

distilled water Distilled water is water that has been boiled into vapor and condensed back into liquid in a separate container. Impurities in the original water that do not boil below or near the boiling point of water remain in the original container. Thus, di ...

and another solution) is first filled into a

and placed inside a colorimeter to calibrate the machine. Only after the device has been calibrated you can use it to find the densities and/or concentrations of the other solutions. You do this by repeating the calibration, except with cuvettes filled with the other solutions. The filter on a colorimeter must be set to red if the liquid is blue. The size of the filter initially chosen for the colorimeter is extremely important, as the wavelength of light that is transmitted by the colorimeter has to be same as that absorbed by the substance.

Colorimetric assays

{{main, Colorimetric analysis Colorimetric assays use reagents that undergo a measurable color change in the presence of the

analyte An analyte, component (in clinical chemistry), or chemical species is a substance or chemical constituent that is of interest in an analytical procedure. The purest substances are referred to as analytes, such as 24 karat gold, NaCl, water, etc. ...

. 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 The enzyme alkaline phosphatase (EC 3.1.3.1, alkaline phosphomonoesterase; phosphomonoesterase; glycerophosphatase; alkaline phosphohydrolase; alkaline phenyl phosphatase; orthophosphoric-monoester phosphohydrolase (alkaline optimum), systematic ...

enzyme. *

Coomassie Blue Coomassie brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie brilliant blue G-250 differs from ...

once binding to proteins elicits a spectrum shift, allowing quantitative dosage. A similar colorimetric assay, the

Bicinchoninic acid assay The bicinchoninic acid assay (BCA assay), also known as the Smith assay, after its inventor, Paul K. Smith at the Pierce Chemical Company, now part of Thermo Fisher Scientific, is a biochemical assay for determining the total concentration of pro ...

, uses a chemical reaction to determine protein concentration. * Enzyme linked immunoassays use enzyme-complexed-antibodies to detect antigens. Binding of the antibody is often inferred from the color change of reagents such as

TMB TMB may refer to: Organizations *Tamilnad Mercantile Bank Limited, India *Texas Medical Board * Thai Military Bank * Transports Metropolitans de Barcelona, Spain Places *Kendall-Tamiami Executive Airport near Miami, Florida, US, IATA code *Tsing M ...

References

Analytical chemistry Absorption spectroscopy

Absorption colorimeter

Colorimetric assays

See also

References