Principle: – A very useful method for the colorimetric determination of carbohydrates is based on the reaction of an throne with carbohydrates in the presence of strong H2SO4. The conc. H2SO4 hydrolyses the glycoside i.e. bonds to give monosaccharide which are then a hydrated to give furfural and its derivatives which form complexes with an throne. Furfural reacts with an throne to give a blue-green co lour. The blue green co lour shows an absorption maximum at 8.50nm. This method can be used for the determination of hex uses, aldopentoses and hex agonic acids, either free or present in polysaccharides. The reaction is not suitable when protein containing large amount of Tryptophans are present.
Requirement: –
1) An thrones reagent. [0.2 % an throne in cons. H2SO4.]
2) Standard glucose solution [2 llgm / ml]
3) Distilled water.
Procedure: – Prepare a series of test tubes containing graded amount [0.5 llgm] of glucose solution. Make up the volume to 3ml with distilled water. Add slowly 5ml of an throne reagent [from the burette]. Mix thoroughly and neat the tubes on boiling water bath for 10 mins with a marble on top to prevent loss of water by evaporation. Cool and measure the extinction at 650nm against a reagent blank.
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