Orthotolidine Methods for Blood Glucose

Orthotolidine methods for blood glucose  we have briefly explained about principle, requirements, procedure, and calculations.

Orthotolidine Method Definition

The O-toluidine method of blood glucose measurement is an older approach. Because O-toluidine method is thought to be a carcinogen, this approach is no longer utilised and has been superseded by enzymatic procedures. Because of its simplicity, sensitivity, and accuracy, O-toluidine method is still widely used.

Orthotolidine Method Principle

Tricholoroacetic acid is used to precipitate the proteins first. In a hot acidic media, glucose in a protein free filtrate reacts with O-toluidine (primary aromatic amine) to create N-glycosamine, a persistent green coloured compound. The o-toluidine reaction is stabilised by the presence of thiourea. Specphotometrically, the intensity of the colour generated is measured at 630nm, which is directly proportional to the glucose concentration in the fluid.



1. Graduated pipettes

2. Test tubes

3. Micropipettes

4. Water bath

5. Spectrophotometer


1. Benzoic acid

2. D-glucose

3. Glacial acetic acid

4. O-toluidine

5. Thiourea

6. Trichloroacetic acid


O-toluidine reagent

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.

Glucose standard

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.

Trichloro acid

Prepare a 100% (w/v) solution by dissolving 2.2 g of TCA in 1 mL of H2O. Then prepare a 10% solution by adding 0.1 mL of 100% TCA solution to 0.9 mL of H2O. Keep both solutions ice cold.

Benzoic acid

Dissolve 1 gm of benzoic acid in water and make up to 1 liter. Prepare the benzoic acid at least 24 hours before use.

Blood Sample

Collect 2-3 ml of blood in a fluoride tube. Use plasma for testing. Serum/Whole blood can also be used. Prepare protein-free filtrate if the sample is grossly hemolyzed or icteric. Add 100 µl specimens to 900 µl of 5gm/dl TCA, mix and centrifuge to get the filtrate.

Orthotolidine Method Procedure

Orthotolidine methods for blood glucose

1. 0.2ml of serum was mixed with 1.8ml of 10% TCA. Centrifuge and 1.0ml of supernatant also taken 0.5, 1.0, 1.5, 2.0, 2.5ml of standard glucose solution corresponding to the values 50, 100, 150, 200, 250 µg respectively and made upto 2.5ml of orthotoludine reagent and mixed thoroughly and keep the tubes in a boiling water bath for 10 minutes.

2. Remove the tubes and brought to room temperature by putting them in cold water bath for 10 minutes. Determine the optical density of solution in various standards and solution using the red filter using blank reagent read at 630nm in O-toluidine method.

Orthotolidine Method Calculation

The concentration of blood glucose in a given sample is ………………..mg/mL in O-toluidine method.