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  Section: Biotechnology Methods » Biochemistry
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Determination of Blood Glucose by the O-Toluidine Method

Proteins in blood are precipitated with trichloro acetic acid, because they interfere with estimation. Contents are filtrated obtained is known as protein-free filtrate. It contains glucose whose concentrate is to be determined. Equal volumes of protein-free filtrate and glucose solution are treated simultaneously with o-toluidine reagent (in acetic acid) and kept in a boiling-water bath. A blue-green N-glycosylamine derivative is formed. The intensity of blue-green is proportional to the amount of glucose present. The optical density values of all 3 solutions are read in a photoelectric colorimeter using a red filter (625 nm) and the amount of glucose present in 100 mL of blood is calculated


  1. O-toluidine reagent: 90 mL of o-toluidine was added to 5 gms thiourea, and diluted to 1 liter with glacial acetic acid stored in brown bottle and the reagent was kept in a refrigerator.
  2. 10% Trichloro acetic acid (TCA).
  3. Glucose standard solution (0.1 mg/mL): 10 mg of glucose were dissolved in about 50 mL of distilled water in a 100 mL volumetric flask. To this 30 mL of 10% TCA was added and make up the volume to 100 mL with distilled water.
  4. Blank solution: 30 mL of 10% TCA was diluted to 100 mL.
Preparation of protein-free filtrate: 3 mL of distilled water and 0.5 mL of blood were taken in a dry test tube and mixed well. 1.5 mL of 10% TCA was added, thoroughly mixed, and allowed to stand for 10 minutes before it was filtered into a dry test tube.

Development of color: Standard glucose solutions were taken in 6 test tubes in the range of 0.2 to 1 mL, 1 mL of protein-free filtrate was taken in a seventh test tube. To all these tubes, 5 mL of o-toluidine was added and mixed thoroughly.

The tubes were kept in boiling water bath for 10 minutes, cooled, and the optical density read at 620 mm.

Result: The concentration of blood glucose in a given sample is .................... mg/mL.


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