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Colorimetric analysis
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<h2 id="equipment">Equipment</h2> <p>The equipment required is a <a href="/facts/Colorimeter_(chemistry)/AfqldtCV">colorimeter</a>, some <a href="/facts/Cuvette/SpAJp2Nx">cuvettes</a> and a suitable color reagent. The process may be automated, e.g. by the use of an <a href="/facts/AutoAnalyzer/3zVE55hK">AutoAnalyzer</a> or by <a href="/facts/Flow_injection_analysis/JBUnAmaY">flow injection analysis</a>. Recently, colorimetric analyses developed for colorimeters have been adapted for use with <a href="/facts/Plate_readers/v7eN64Rd">plate readers</a> to speed up analysis and reduce the waste stream.<a class="footnote-ref" id="fnref:1" href="#fn:1"><sup>1</sup></a> </p> <h2 id="non-enzymatic-methods">Non-enzymatic methods</h2> <h3>Examples</h3> <h4>Calcium</h4> <a href="/facts/Calcium/hf252CC0">Calcium</a> + o-cresolphthalein complexone → colored complex<a class="footnote-ref" id="fnref:2" href="#fn:2"><sup>2</sup></a> <h4>Copper</h4> <a href="/facts/Copper/I8PEE8oX">Copper</a> + bathocuproin disulfonate → colored complex<a class="footnote-ref" id="fnref:3" href="#fn:3"><sup>3</sup></a> <h4>Creatinine</h4> <a href="/facts/Creatinine/gkebqj2L">Creatinine</a> + <a href="/facts/Picrate/q8JfQDUp">picrate</a> → colored complex<a class="footnote-ref" id="fnref:4" href="#fn:4"><sup>4</sup></a> <h4>Iron</h4> <a href="/facts/Iron/QK1JYy8E">Iron</a> + bathophenanthroline disulfonate → colored complex<a class="footnote-ref" id="fnref:5" href="#fn:5"><sup>5</sup></a> <h4>Phosphate (inorganic)</h4> <a href="/facts/Phosphate/FDGVCxUG">Phosphate</a> + <a href="/facts/Ammonium_molybdate/cN9qnHkp">ammonium molybdate</a> + <a href="/facts/Ascorbic_acid/IA2f1fTm">ascorbic acid</a> → blue colored complex<a class="footnote-ref" id="fnref:6" href="#fn:6"><sup>6</sup></a> <h2 id="enzymatic-methods">Enzymatic methods</h2> <p>In enzymatic analysis (which is widely used in <a href="/facts/Medical_laboratory/ov10GjQD">medical laboratories</a>) the color reaction is preceded by a reaction <a href="/facts/Catalyzed/LPBS7TQC">catalyzed</a> by an <a href="/facts/Enzyme/DSI1Fh7y">enzyme</a>. As the enzyme is specific to a particular <a href="/facts/Enzyme_substrate_(Biology)/6MnaV19h">substrate</a>, more accurate results can be obtained. Enzymatic analysis is always carried out in a <a href="/facts/Buffer_solution/zrLz9eaa">buffer solution</a> at a specified temperature (usually 37°C) to provide the optimum conditions for the enzymes to act. Examples follow. </p> <h3>Examples</h3> <h4>Cholesterol (CHOD-PAP method)</h4> <ol><li><a href="/facts/Cholesterol/TnfPlYc1">Cholesterol</a> + <a href="/facts/Oxygen/acyn6o8r">oxygen</a> --(enzyme <a href="/facts/Cholesterol_oxidase/HL20KEIT">cholesterol oxidase</a>)--> cholestenone + <a href="/facts/Hydrogen_peroxide/vju6HBIc">hydrogen peroxide</a></li> <li>Hydrogen peroxide + 4-<a href="/facts/Aminophenazone/5qER8WFE">aminophenazone</a> + <a href="/facts/Phenol/ACJs5NIU">phenol</a> --(enzyme <a href="/facts/Peroxidase/pEgHpmCV">peroxidase</a>)--> colored complex + water<a class="footnote-ref" id="fnref:7" href="#fn:7"><sup>7</sup></a></li></ol> <h4>Glucose (GOD-Perid method)</h4> <ol><li><a href="/facts/Glucose/oUSkoLCD">Glucose</a> + oxygen + <a href="/facts/Water/0lkXnJPK">water</a> --(enzyme <a href="/facts/Glucose_oxidase/6MM8Fmf8">glucose oxidase</a>)--> <a href="/facts/Gluconate/5s6xenLn">gluconate</a> + hydrogen peroxide</li> <li>Hydrogen peroxide + <a href="/facts/ABTS/v9qUAIMK">ABTS</a> --(enzyme <a href="/facts/Peroxidase/pEgHpmCV">peroxidase</a>)--> colored complex<a class="footnote-ref" id="fnref:8" href="#fn:8"><sup>8</sup></a></li></ol> <p>In this case, both stages of the reaction are catalyzed by enzymes. </p> <h4>Triglycerides (GPO-PAP method)</h4> <ol><li><a href="/facts/Triglycerides/BQmjv4A0">Triglycerides</a> + water --(enzyme <a href="/facts/Esterase/Ff2bb7LD">esterase</a>)--> <a href="/facts/Glycerol/sRQ6NBQQ">glycerol</a> + <a href="/facts/Carboxylic_acid/U4xuMosy">carboxylic acid</a></li> <li>Glycerol + <a href="/facts/Adenosine_triphosphate/nB9bwgcU">ATP</a> --(enzyme <a href="/facts/Glycerol_kinase/Huet40Rc">glycerol kinase</a>)--> <a href="/facts/Glycerol-3-phosphate/3upX66wU">glycerol-3-phosphate</a> + <a href="/facts/Adenosine_diphosphate/JEDnsCjn">ADP</a></li> <li>Glycerol-3-phosphate + oxygen --(enzyme <a href="/facts/Glycerol-3-phosphate_oxidase/3qSLhXHg">glycerol-3-phosphate oxidase</a>) --> dihydroxyacetone phosphate + hydrogen peroxide</li> <li>Hydrogen peroxide + 4-<a href="/facts/Aminophenazone/5qER8WFE">aminophenazone</a> + 4-<a href="/facts/Chlorophenol/sAfJNJqD">chlorophenol</a> --(enzyme <a href="/facts/Peroxidase/pEgHpmCV">peroxidase</a>)--> colored complex<a class="footnote-ref" id="fnref:9" href="#fn:9"><sup>9</sup></a></li></ol> <h4>Urea</h4> <ol><li><a href="/facts/Urea/6j84AoCG">Urea</a> + water --(enzyme <a href="/facts/Urease/qh6XKw4w">urease</a>)--> <a href="/facts/Ammonium_carbonate/UjMdwbTv">ammonium carbonate</a></li> <li>Ammonium carbonate + <a href="/facts/Phenol/ACJs5NIU">phenol</a> + <a href="/facts/Hypochlorite/XMJdjAEM">hypochlorite</a> ----> colored complex<a class="footnote-ref" id="fnref:10" href="#fn:10"><sup>10</sup></a></li></ol> <p>In this case, only the first stage of the reaction is catalyzed by an enzyme. The second stage is non-enzymatic. </p> <h4>Abbreviations</h4> <ul><li>CHOD = cholesterol oxidase</li> <li>GOD = glucose oxidase</li> <li>GPO = glycerol-3-phosphate oxidase</li> <li>PAP = phenol + aminophenazone (in some methods the phenol is replaced by <a href="/facts/4-Chlorophenol/dL3LrvKz">4-chlorophenol</a>, which is less toxic)</li> <li>POD = peroxidase</li></ul> <h2 id="ultraviolet-methods">Ultraviolet methods</h2> <p>In <a href="/facts/Ultraviolet/kncBUqn5">ultraviolet</a> (UV) methods there is no visible color change but the principle is exactly the same, i.e. the measurement of a change in the absorbance of the solution. UV methods usually measure the difference in absorbance at 340 nm wavelength between <a href="/facts/Nicotinamide_adenine_dinucleotide/BHdLW925">nicotinamide adenine dinucleotide</a> (NAD) and its <a href="/facts/Redox/Pyd5RVcj">reduced</a> form (NADH). </p> <h3>Examples</h3> <h4>Pyruvate</h4> <a href="/facts/Pyruvate/qJTgl4J9">Pyruvate</a> + NADH --(enzyme <a href="/facts/Lactate_dehydrogenase/FGuLGcXe">lactate dehydrogenase</a>)--> <a href="/facts/Lactic_acid/BsVRFx7s">L-lactate</a> + NAD<a class="footnote-ref" id="fnref:11" href="#fn:11"><sup>11</sup></a> <h2 id="see-also">See also</h2> <ul><li><a href="/facts/Blood_sugar/I4SkNzrm">Blood sugar</a></li> <li><a href="/facts/MBAS_assay/Nqp9HKv1">MBAS assay</a>, an <a href="/facts/Assay/fPUYjx1I">assay</a> that indicates <a href="/facts/Ion/3bePpDna">anionic</a> <a href="/facts/Surfactant/zgVX6g8o">surfactants</a> in water with a bluing reaction.</li> <li><a href="/facts/Nessler_cylinder/b47hpwFD">Nessler cylinder</a></li></ul> <h2 id="references">References</h2> <ol> <li id="fn:1"><p>Greenan, N. S., R.L. Mulvaney, and G.K. Sims. 1995. "A microscale method for colorimetric determination of urea in soil extracts". Commun. Soil Sci. Plant Anal. 26:2519-2529. <a href="#fnref:1" class="footnote-back-ref">↩</a></p></li> <li id="fn:2"><p>Ray Sarkar and Chauhan (1967) Anal. Biochem. 20:155 <a href="/wiki/Anal._Biochem." target="_blank">/wiki/Anal._Biochem.</a> <a href="#fnref:2" class="footnote-back-ref">↩</a></p></li> <li id="fn:3"><p>Zak, B. (1958) Clin. Chim. Acta. 3:328 <a href="/wiki/Clin._Chim._Acta." target="_blank">/wiki/Clin._Chim._Acta.</a> <a href="#fnref:3" class="footnote-back-ref">↩</a></p></li> <li id="fn:4"><p>Hawk, Oser and Summerson, Practical Physiological Chemistry, Churchill, London, 1947, pp 839-844 <a href="#fnref:4" class="footnote-back-ref">↩</a></p></li> <li id="fn:5"><p>Reference to follow <a href="#fnref:5" class="footnote-back-ref">↩</a></p></li> <li id="fn:6"><p>Heidari-Bafroui, Hojat; Ribeiro, Brenno; Charbaji, Amer; Anagnostopoulos, Constantine; Faghri, Mohammad (2020-10-16). "Portable infrared lightbox for improving the detection limits of paper-based phosphate devices". Measurement. 173: 108607. doi:10.1016/j.measurement.2020.108607. ISSN 0263-2241. S2CID 225140011. <a href="https://doi.org/10.1016%2Fj.measurement.2020.108607" target="_blank">https://doi.org/10.1016%2Fj.measurement.2020.108607</a> <a href="#fnref:6" class="footnote-back-ref">↩</a></p></li> <li id="fn:7"><p>Reference to follow <a href="#fnref:7" class="footnote-back-ref">↩</a></p></li> <li id="fn:8"><p>Rey and Wielinger (1970) Z. analyt. chem. 252:224 <a href="#fnref:8" class="footnote-back-ref">↩</a></p></li> <li id="fn:9"><p>Reference to follow <a href="#fnref:9" class="footnote-back-ref">↩</a></p></li> <li id="fn:10"><p>Fawcett and Scott (1960) J. Clin. Pathol. 13:156 <a href="/wiki/J._Clin._Pathol." target="_blank">/wiki/J._Clin._Pathol.</a> <a href="#fnref:10" class="footnote-back-ref">↩</a></p></li> <li id="fn:11"><p>Reference to follow <a href="#fnref:11" class="footnote-back-ref">↩</a></p></li> </ol>