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 in connection with 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. Recently, colorimetric analyses developed for colorimeters have been adapted for use with plate readers to speed up analysis and reduce the waste stream.

Non-enzymatic methods

Examples

;Calcium
Calcium + o-cresolphthalein complexone ----> colored complex
;Copper
Copper + bathocuproin disulfonate ----> colored complex
;Creatinine
Creatinine + picrate ----> colored complex
;Iron
Iron + bathophenanthroline disulfonate ---> colored complex
;Phosphate
Phosphate + ammonium molybdate + ammonium metavanadate ----> colored complex

Enzymatic methods

In enzymatic analysis 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 to provide the optimum conditions for the enzymes to act. Examples follow.

Examples

;Cholesterol
  1. Cholesterol + oxygen ----> cholestenone + hydrogen peroxide
  2. Hydrogen peroxide + 4-aminophenazone + phenol ----> colored complex + water
;Glucose
  1. Glucose + oxygen + water ----> gluconate + hydrogen peroxide
  2. Hydrogen peroxide + ABTS ----> colored complex
In this case, both stages of the reaction are catalyzed by enzymes.
;Triglycerides
  1. Triglycerides + water ----> glycerol + carboxylic acid
  2. Glycerol + ATP ----> glycerol-3-phosphate + ADP
  3. Glycerol-3-phosphate + oxygen -- --> dihydroxyacetone phosphate + hydrogen peroxide
  4. Hydrogen peroxide + 4-aminophenazone + 4-chlorophenol ----> colored complex
;Urea
  1. Urea + water ----> ammonium carbonate
  2. Ammonium carbonate + phenol + hypochlorite ----> colored complex
In this case, only the first stage of the reaction is catalyzed by an enzyme. The second stage is non-enzymatic.
;Abbreviations
In ultraviolet 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 and its reduced form.

Examples

;Pyruvate
Pyruvate + NADH ----> L-lactate + NAD