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 reagent that produces a colored product. 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

Copper

Creatinine

Iron

Phosphate (inorganic)

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 (CHOD-PAP method)

  1. Cholesterol + oxygen ----> cholestenone + hydrogen peroxide
  2. Hydrogen peroxide + 4-aminophenazone + phenol ----> colored complex + water

    Glucose (GOD-Perid method)

  3. Glucose + oxygen + water ----> gluconate + hydrogen peroxide
  4. Hydrogen peroxide + ABTS ----> colored complex
In this case, both stages of the reaction are catalyzed by enzymes.

Triglycerides (GPO-PAP method)

  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

  5. Urea + water ----> ammonium carbonate
  6. 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