Colorimetric analysis
Encyclopedia
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 compound
s and inorganic compound
s 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
.
, some cuvette
s and a suitable color reagent. The process may be automated, e.g. by the use of an AutoAnalyzer
or by Flow injection analysis
.
Calcium
+ o-cresolphthalein complexone ----> colored complex
Copper
Copper
+ bathocuproin disulfonate ----> colored complex
Creatinine
Creatinine
+ picrate
----> colored complex
Iron
Iron
+ bathophenanthroline disulfonate ---> colored complex
Phosphate (inorganic)
Phosphate
+ ammonium molybdate
+ ammonium metavanadate
----> colored complex
) 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.
Glucose (GOD-Perid method)
In this case, both stages of the reaction are catalyzed by enzymes.
Triglycerides (GPO-PAP method)
Urea
In this case, only the first stage of the reaction is catalyzed by an enzyme. The second stage is non-enzymatic.
Abbreviations
(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).
Pyruvate + NADH --(enzyme lactate dehydrogenase
)--> L-lactate
+ NAD
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...
of a chemical element
Chemical element
A chemical element is a pure chemical substance consisting of one type of atom distinguished by its atomic number, which is the number of protons in its nucleus. Familiar examples of elements include carbon, oxygen, aluminum, iron, copper, gold, mercury, and lead.As of November 2011, 118 elements...
or chemical compound
Chemical compound
A chemical compound is a pure chemical substance consisting of two or more different chemical elements that can be separated into simpler substances by chemical reactions. Chemical compounds have a unique and defined chemical structure; they consist of a fixed ratio of atoms that are held together...
in a solution
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
with the aid of a color reagent. It is applicable to both organic compound
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...
s and inorganic compound
Inorganic compound
Inorganic compounds have traditionally been considered to be of inanimate, non-biological origin. In contrast, organic compounds have an explicit biological origin. However, over the past century, the classification of inorganic vs organic compounds has become less important to scientists,...
s and may be used with or without an enzymatic stage. The method is widely used in medical laboratories
Medical laboratory
A medical laboratory or clinical laboratory is a laboratory where tests are done on clinical specimens in order to get information about the health of a patient as pertaining to the diagnosis, treatment, and prevention of disease.-Departments:...
and for industrial purposes, e.g. the analysis of water samples in connection with industrial water treatment
Industrial water treatment
Industrial Water Treatment can be classified into the following categories:* Boiler water treatment* Cooling water treatment* Wastewater treatment...
.
Equipment
The equipment required is a colorimeterColorimeter
For articles on Colorimeter see:* Colorimeter * Tristimulus colorimeter...
, some cuvette
Cuvette
A cuvette is a small tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz and designed to hold samples for spectroscopic experiments. The best cuvettes are as clear as possible, without impurities that might affect a spectroscopic reading...
s and a suitable color reagent. The process may be automated, e.g. by the use of an AutoAnalyzer
AutoAnalyzer
AutoAnalyzer is an automated analyzer using a special flow technique named "continuous flow analysis " first made by the Technicon Corporation. The instrument was invented 1957 by Leonard Skeggs, PhD and commercialized by Jack Whitehead's Technicon Corporation...
or by Flow injection analysis
Flow injection analysis
Flow injection analysis is an approach to chemical analysis that is accomplished by injecting a plug of sample into a flowing carrier stream...
.
Examples
CalciumCalcium
Calcium
Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
+ o-cresolphthalein complexone ----> colored complex
Copper
Copper
Copper
Copper is a chemical element with the symbol Cu and atomic number 29. It is a ductile metal with very high thermal and electrical conductivity. Pure copper is soft and malleable; an exposed surface has a reddish-orange tarnish...
+ bathocuproin disulfonate ----> colored complex
Creatinine
Creatinine
Creatinine
Creatinine is a break-down product of creatine phosphate in muscle, and is usually produced at a fairly constant rate by the body...
+ picrate
Picrate
A picrate is a salt or an ester of picric acid . But it could also be an additional compound which picric acid forms with many aromatic hydrocarbons, aromatic amines, aliphatic amines, alkalines, and other compounds. These additional compounds are also called picrates even though they are not a...
----> colored complex
Iron
Iron
Iron
Iron is a chemical element with the symbol Fe and atomic number 26. It is a metal in the first transition series. It is the most common element forming the planet Earth as a whole, forming much of Earth's outer and inner core. It is the fourth most common element in the Earth's crust...
+ bathophenanthroline disulfonate ---> colored complex
Phosphate (inorganic)
Phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
+ ammonium molybdate
Ammonium molybdate
Ammonium heptamolybdate is an odourless crystalline compound ranging in colour from white to yellow-green. It is usually encountered as the tetrahydrate, whose chemical formula is 6Mo7O24·4H2O...
+ ammonium metavanadate
Ammonium metavanadate
Ammonium metavanadate, NH4VO3, is a yellow crystalline solid which is water soluble inorganic acid that acts as insulin mimic. It functions as a catalyst to certain reactions and is known to have toxic effects in certain species. It has been found in slags and fly ash from coal burning which has...
----> colored complex
Enzymatic methods
In enzymatic analysis (which is widely used in medical laboratoriesMedical laboratory
A medical laboratory or clinical laboratory is a laboratory where tests are done on clinical specimens in order to get information about the health of a patient as pertaining to the diagnosis, treatment, and prevention of disease.-Departments:...
) the color reaction is preceded by a reaction catalyzed by an enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
. 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
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...
at a specified temperature (usually 37°C) to provide the optimum conditions for the enzymes to act. Examples follow.
Examples
Cholesterol (CHOD-PAP method)- CholesterolCholesterolCholesterol is a complex isoprenoid. Specifically, it is a waxy steroid of fat that is produced in the liver or intestines. It is used to produce hormones and cell membranes and is transported in the blood plasma of all mammals. It is an essential structural component of mammalian cell membranes...
+ oxygenOxygenOxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
--(enzyme cholesterol oxidaseCholesterol oxidaseIn enzymology, a cholesterol oxidase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are cholesterol and O2, whereas its two products are cholest-4-en-3-one and H2O2....
)--> cholestenone + hydrogen peroxideHydrogen peroxideHydrogen peroxide is the simplest peroxide and an oxidizer. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent... - Hydrogen peroxide + 4-aminophenazoneAminophenazoneAminophenazone — a pyrazolone with analgesic, anti-inflammatory, and antipyretic properties but has risk of agranulocytosis. A breath test with 13C-labeled aminopyrine has been used as a non-invasive measure of cytochrome P-450 metabolic activity in liver function tests....
+ phenolPhenolPhenol, also known as carbolic acid, phenic acid, is an organic compound with the chemical formula C6H5OH. It is a white crystalline solid. The molecule consists of a phenyl , bonded to a hydroxyl group. It is produced on a large scale as a precursor to many materials and useful compounds...
--(enzyme peroxidasePeroxidasePeroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
)--> colored complex
Glucose (GOD-Perid method)
- GlucoseGlucoseGlucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
+ oxygen + waterWaterWater is a chemical substance with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at ambient conditions, but it often co-exists on Earth with its solid state, ice, and gaseous state . Water also exists in a...
--(enzyme glucose oxidaseGlucose oxidaseThe glucose oxidase enzyme is an oxido-reductase that catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone. In cells, it aids in breaking the sugar down into its metabolites....
)--> gluconate + hydrogen peroxide - Hydrogen peroxide + ABTSABTSIn biochemistry, 2,2'-azino-bis or ABTS is chemical compound used to observe the reaction kinetics of specific enzymes...
--(enzyme peroxidasePeroxidasePeroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
)--> colored complex
In this case, both stages of the reaction are catalyzed by enzymes.
Triglycerides (GPO-PAP method)
- Triglycerides + water --(enzyme esteraseEsteraseAn esterase is a hydrolase enzyme that splits esters into an acid and an alcohol in a chemical reaction with water called hydrolysis.A wide range of different esterases exist that differ in their substrate specificity, their protein structure, and their biological function.- EC classification/list...
)--> glycerolGlycerolGlycerol is a simple polyol compound. It is a colorless, odorless, viscous liquid that is widely used in pharmaceutical formulations. Glycerol has three hydroxyl groups that are responsible for its solubility in water and its hygroscopic nature. The glycerol backbone is central to all lipids...
+ carboxylic acidCarboxylic acidCarboxylic acids are organic acids characterized by the presence of at least one carboxyl group. The general formula of a carboxylic acid is R-COOH, where R is some monovalent functional group... - Glycerol + ATPAdenosine triphosphateAdenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
--(enzyme glycerol kinaseGlycerol kinaseGlycerol kinase is a phosphotransferase enzyme involved in triglycerides and glycerophospholipids synthesis.Glycerol kinase catalyzes the transfer of a phosphate from ATP to glycerol thus forming glycerol phosphate . Adipocytes lack glycerol kinase so they cannot metabolize the glycerol produced...
)--> glycerol-3-phosphate + ADPAdenosine diphosphateAdenosine diphosphate, abbreviated ADP, is a nucleoside diphosphate. It is an ester of pyrophosphoric acid with the nucleoside adenosine. ADP consists of the pyrophosphate group, the pentose sugar ribose, and the nucleobase adenine.... - Glycerol-3-phosphate + oxygen --(enzyme glycerol-3-phosphate oxidaseGlycerol-3-phosphate oxidaseIn enzymology, a glycerol-3-phosphate oxidase is an enzyme that catalyzes the chemical reactionThus, the two substrates of this enzyme are sn-glycerol 3-phosphate and O2, whereas its two products are glycerone phosphate and H2O2....
) --> dihydroxyacetone phosphate + hydrogen peroxide - Hydrogen peroxide + 4-aminophenazoneAminophenazoneAminophenazone — a pyrazolone with analgesic, anti-inflammatory, and antipyretic properties but has risk of agranulocytosis. A breath test with 13C-labeled aminopyrine has been used as a non-invasive measure of cytochrome P-450 metabolic activity in liver function tests....
+ 4-chlorophenolChlorophenolA chlorophenol is any organochloride of phenol that contains one or more covalently bonded chlorine atoms. Chlorophenols are produced by electrophilic halogenation of phenol with chlorine. Most chlorophenols have a number of different isomers...
--(enzyme peroxidasePeroxidasePeroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
)--> colored complex
Urea
- UreaUreaUrea or carbamide is an organic compound with the chemical formula CO2. The molecule has two —NH2 groups joined by a carbonyl functional group....
+ water --(enzyme ureaseUreaseUrease is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia. The reaction occurs as follows:In 1926, James Sumner showed that urease is a protein. Urease is found in bacteria, yeast, and several higher plants. The structure of urease was first solved by P.A...
)--> ammonium carbonateAmmonium carbonateAmmonium carbonate is a commercial salt with the chemical formula 2CO3. It is used when crushed as a smelling salt. It can be crushed when needed in order to revive someone who has fainted... - Ammonium carbonate + phenolPhenolPhenol, also known as carbolic acid, phenic acid, is an organic compound with the chemical formula C6H5OH. It is a white crystalline solid. The molecule consists of a phenyl , bonded to a hydroxyl group. It is produced on a large scale as a precursor to many materials and useful compounds...
+ hypochloriteHypochloriteThe hypochlorite ion, also known as chlorate anion is ClO−. A hypochlorite compound is a chemical compound containing this group, with chlorine in oxidation state +1.Hypochlorites are the salts of hypochlorous acid...
----> colored complex
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 ultravioletUltraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
(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
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide, abbreviated NAD, is a coenzyme found in all living cells. The compound is a dinucleotide, since it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base and the other nicotinamide.In metabolism, NAD is involved...
(NAD) and its reduced
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
form (NADH).
Examples
PyruvatePyruvate + NADH --(enzyme lactate dehydrogenase
Lactate dehydrogenase
Lactate dehydrogenase is an enzyme present in a wide variety of organisms, including plants and animals.Lactate dehydrogenases exist in four distinct enzyme classes. Two of them are cytochrome c-dependent enzymes, each acting on either D-lactate or L-lactate...
)--> L-lactate
Lactic acid
Lactic acid, also known as milk acid, is a chemical compound that plays a role in various biochemical processes and was first isolated in 1780 by the Swedish chemist Carl Wilhelm Scheele. Lactic acid is a carboxylic acid with the chemical formula C3H6O3...
+ NAD
See also
- Blood sugarBlood sugarThe blood sugar concentration or blood glucose level is the amount of glucose present in the blood of a human or animal. Normally in mammals, the body maintains the blood glucose level at a reference range between about 3.6 and 5.8 mM , or 64.8 and 104.4 mg/dL...
- MBAS assayMBAS assayA methylene blue active substances assay, or MBAS assay, is a colorimetric analysis test method that uses methylene blue to detect the presence of anionic surfactants in a sample of water...
, an assayAssayAn assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
that indicates anionic surfactantSurfactantSurfactants are compounds that lower the surface tension of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid...
s in water with a bluing reaction. - Nessler cylinderNessler cylinderNessler cylinders are laboratory tubes with a fixed volume. On the walls there are marks of the nominal stroke volume and possibly one half-way mark...