Colorimetric analysis

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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. The method is widely used in medical laboratories and for industrial purposes, e.g. the analysis of water samples in connection with industrial water treatment.

Contents

Equipment

The equipment required is a colorimeter, some cuvettes and a suitable color reagent. The process may be automated, e.g. by the use of an AutoAnalyzer or by flow injection analysis. Recently, colorimetric analyses developed for colorimeters have been adapted for use with plate readers to speed up analysis and reduce the waste stream. [1]

Non-enzymatic methods

Examples

Calcium

Calcium + o-cresolphthalein complexone → colored complex [2]

Copper

Copper + bathocuproin disulfonate → colored complex [3]

Creatinine

Creatinine + picrate → colored complex [4]

Iron

Iron + bathophenanthroline disulfonate → colored complex [5]

Phosphate (inorganic)

Phosphate + ammonium molybdate + ascorbic acid → blue colored complex [6]

Enzymatic methods

In enzymatic analysis (which is widely used in medical laboratories) the color reaction is preceded by a reaction catalyzed by an enzyme. As the enzyme is specific to a particular substrate, more accurate results can be obtained. Enzymatic analysis is always carried out in a buffer solution at a specified temperature (usually 37°C) to provide the optimum conditions for the enzymes to act. Examples follow.

Examples

Cholesterol (CHOD-PAP method)

  1. Cholesterol + oxygen --(enzyme cholesterol oxidase)--> cholestenone + hydrogen peroxide
  2. Hydrogen peroxide + 4-aminophenazone + phenol --(enzyme peroxidase)--> colored complex + water [7]

Glucose (GOD-Perid method)

  1. Glucose + oxygen + water --(enzyme glucose oxidase)--> gluconate + hydrogen peroxide
  2. Hydrogen peroxide + ABTS --(enzyme peroxidase)--> colored complex [8]

In this case, both stages of the reaction are catalyzed by enzymes.

Triglycerides (GPO-PAP method)

  1. Triglycerides + water --(enzyme esterase)--> glycerol + carboxylic acid
  2. Glycerol + ATP --(enzyme glycerol kinase)--> glycerol-3-phosphate + ADP
  3. Glycerol-3-phosphate + oxygen --(enzyme glycerol-3-phosphate oxidase) --> dihydroxyacetone phosphate + hydrogen peroxide
  4. Hydrogen peroxide + 4-aminophenazone + 4-chlorophenol --(enzyme peroxidase)--> colored complex [9]

Urea

  1. Urea + water --(enzyme urease)--> ammonium carbonate
  2. Ammonium carbonate + phenol + hypochlorite ----> colored complex [10]

In this case, only the first stage of the reaction is catalyzed by an enzyme. The second stage is non-enzymatic.

Abbreviations

  • CHOD = cholesterol oxidase
  • GOD = glucose oxidase
  • GPO = glycerol-3-phosphate oxidase
  • PAP = phenol + aminophenazone (in some methods the phenol is replaced by 4-chlorophenol, which is less toxic)
  • Perid = peroxidase

Ultraviolet methods

In ultraviolet (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 nicotinamide adenine dinucleotide (NAD) and its reduced form (NADH).

Examples

Pyruvate

Pyruvate + NADH --(enzyme lactate dehydrogenase)--> L-lactate + NAD [11]

See also

Related Research Articles

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<span class="mw-page-title-main">Glycolysis</span> Catabolic pathway

Glycolysis is the metabolic pathway that converts glucose into pyruvate, and in most organisms, occurs in the liquid part of cells, the cytosol. The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH). Glycolysis is a sequence of ten reactions catalyzed by enzymes.

<span class="mw-page-title-main">Catalase</span> Biocatalyst decomposing hydrogen peroxide

Catalase is a common enzyme found in nearly all living organisms exposed to oxygen which catalyzes the decomposition of hydrogen peroxide to water and oxygen. It is a very important enzyme in protecting the cell from oxidative damage by reactive oxygen species (ROS). Catalase has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of hydrogen peroxide molecules to water and oxygen each second.

A dehydrogenase is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+/NADP+ or a flavin coenzyme such as FAD or FMN. Like all catalysts, they catalyze reverse as well as forward reactions, and in some cases this has physiological significance: for example, alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde in animals, but in yeast it catalyzes the production of ethanol from acetaldehyde.

<span class="mw-page-title-main">Peroxidase</span> Peroxide-decomposing enzyme

Peroxidases or peroxide reductases are a large group of enzymes which play a role in various biological processes. They are named after the fact that they commonly break up peroxides.

<span class="mw-page-title-main">Heme</span> Chemical coordination complex of an iron ion chelated to a porphyrin

Heme, or haem, is a precursor to hemoglobin, which is necessary to bind oxygen in the bloodstream. Heme is biosynthesized in both the bone marrow and the liver.

In biochemistry, an oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. This group of enzymes usually utilizes NADP+ or NAD+ as cofactors. Transmembrane oxidoreductases create electron transport chains in bacteria, chloroplasts and mitochondria, including respiratory complexes I, II and III. Some others can associate with biological membranes as peripheral membrane proteins or be anchored to the membranes through a single transmembrane helix.

<span class="mw-page-title-main">Glucose oxidase</span> Class of enzymes

The glucose oxidase enzyme also known as notatin is an oxidoreductase that catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone. This enzyme is produced by certain species of fungi and insects and displays antibacterial activity when oxygen and glucose are present.

<span class="mw-page-title-main">ABTS</span> Chemical compound

In biochemistry, ABTS is a chemical compound used to observe the reaction kinetics of specific enzymes. A common use for it is in the enzyme-linked immunosorbent assay (ELISA) to detect the binding of molecules to each other.

The oxidase test is used to determine if an organism possesses the cytochrome c oxidase enzyme. The test is used as an aid for the differentiation of Neisseria, Moraxella, Campylobacter and Pasteurella species. It is also used to differentiate pseudomonads from related species.

<span class="mw-page-title-main">Horseradish peroxidase</span> Chemical compound and enzyme

The enzyme horseradish peroxidase (HRP), found in the roots of horseradish, is used extensively in biochemistry applications. It is a metalloenzyme with many isoforms, of which the most studied type is C. It catalyzes the oxidation of various organic substrates by hydrogen peroxide.

<span class="mw-page-title-main">Glycerol-3-phosphate dehydrogenase</span> Class of enzymes

Glycerol-3-phosphate dehydrogenase (GPDH) is an enzyme that catalyzes the reversible redox conversion of dihydroxyacetone phosphate to sn-glycerol 3-phosphate.

Berthelot's reagent is an alkaline solution of phenol and hypochlorite, used in analytical chemistry. It is named after its inventor, Marcellin Berthelot. Ammonia reacts with Berthelot's reagent to form a blue product which is used in a colorimetric method for determining ammonia. The reagent can also be used for determining urea. In this case the enzyme urease is used to catalyze the hydrolysis of urea into carbon dioxide and ammonia. The ammonia is then determined with Berthelot's reagent.

<span class="mw-page-title-main">Molybdenum blue</span> Pigment

Molybdenum blue is a term applied to:

In enzymology, a manganese peroxidase (EC 1.11.1.13) is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">NADH peroxidase</span>

In enzymology, a NADH peroxidase (EC 1.11.1.1) is an enzyme that catalyzes the chemical reaction

The Trinder glucose activity test is a diagnostic test used in medicine to determine the presence of glucose or glucose oxidase. The test employs the Trinder reagent, and is a colour change test resulting from the Trinder reaction.

<span class="mw-page-title-main">Resazurin</span> Chemical compound

Resazurin is a phenoxazine dye that is weakly fluorescent, nontoxic, cell-permeable, and redox‐sensitive. Resazurin has a blue to purple color and is used in microbiological, cellular, and enzymatic assays because it can be irreversibly reduced to the pink-colored and highly fluorescent resorufin (7-Hydroxy-3H-phenoxazin-3-one). At circum-neutral pH, resorufin can be detected by visual observation of its pink color or by fluorimetry, with an excitation maximum at 530-570 nm and an emission maximum at 580-590 nm.

<span class="mw-page-title-main">Lactoperoxidase</span> Mammalian protein found in Homo sapiens

Lactoperoxidase is a peroxidase enzyme secreted from mammary, salivary and other mucosal glands including the lungs, bronchii and nose that functions as a natural and the first line of defense against bacteria and viruses. Lactoperoxidase is a member of the heme peroxidase family of enzymes. In humans, lactoperoxidase is encoded by the LPO gene.

<span class="mw-page-title-main">Galactose oxidase</span>

Galactose oxidase is an enzyme that catalyzes the oxidation of D-galactose in some species of fungi.

References

  1. ↑ 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.
  2. ↑ Ray Sarkar and Chauhan (1967) Anal. Biochem. 20:155
  3. ↑ Zak, B. (1958) Clin. Chim. Acta. 3:328
  4. ↑ Hawk, Oser and Summerson, Practical Physiological Chemistry, Churchill, London, 1947, pp 839-844
  5. ↑ Reference to follow
  6. ↑ 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.
  7. ↑ Reference to follow
  8. ↑ Rey and Wielinger (1970) Z. analyt. chem. 252:224
  9. ↑ Reference to follow
  10. ↑ Fawcett and Scott (1960) J. Clin. Pathol. 13:156
  11. ↑ Reference to follow
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