Colorimetric analysis
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 prior to industrial water treatment.
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.
Non-enzymatic methods
Examples
- Calcium
Calcium + o-cresolphthalein complexone ----> colored complex[1]
- Copper
Copper + bathocuproin disulfonate ----> colored complex[2]
- Creatinine
Creatinine + picrate ----> colored complex[3]
- Iron
Iron + bathophenanthroline disulfonate ---> colored complex[4]
- Phosphate (inorganic)
Phosphate + ammonium molybdate + ammonium metavanadate ----> colored complex[5]
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)
- Cholesterol + oxygen --(enzyme cholesterol oxidase)--> cholestenone + hydrogen peroxide
- Hydrogen peroxide + 4-aminophenazone + phenol --(enzyme peroxidase)--> colored complex[6]
- Glucose (GOD-Perid method)
- Glucose + oxygen + water --(enzyme glucose oxidase)--> gluconate + hydrogen peroxide
- Hydrogen peroxide + ABTS --(enzyme peroxidase)--> colored complex[7]
In this case, both stages of the reaction are catalyzed by enzymes.
- Triglycerides (GPO-PAP method)
- Triglycerides + water --(enzyme esterase)--> glycerol + carboxylic acid
- Glycerol + ATP --(enzyme glycerokinase)--> glycerol-3-phosphate + ADP
- Glycerol-3-phosphate + oxygen --(enzyme glycerol-3-phosphate oxidase) --> dihydroxyacetone phosphate + hydrogen peroxide
- Hydrogen peroxide + 4-aminophenazone + 4-chlorophenol --(enzyme peroxidase)--> colored complex[8]
- Urea
- Urea + water --(enzyme urease)--> ammonium carbonate
- Ammonium carbonate + phenol + hypochlorite ----> colored complex[9]
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
- Perid = peroxidase
See also
References
- ^ Ray Sarkar and Chauhan (1967) Anal. Biochem. 20:155
- ^ Zak, B. (1958) Clin. Chim. Acta. 3:328
- ^ Hawk, Oser and Summerson, Practical Physiological Chemistry, Churchill, London, 1947, pp 839-844
- ^ Reference to follow
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- ^ Rey and Wielinger (1970) Z. analyt. chem. 252:224
- ^ Reference to follow
- ^ Fawcett and Scott (1960) J. Clin. Pathol. 13:156