Experiment 3

An o-toluidine method for blood glucose determination

Objective ： to grasp the principle and procedure of the o-toluidine method for glucose determination.

principle ： o-Toluidine, a primary aromatic amine, in acetic acid solution reacts relatively selectively with glucose, the condensation of the aldehyde group of glucose with the amino group of o-toluidine to give a green chromogen,yielding a stable green color (Schiff base) .

The concentration of the glucose is proportionally to to the intensity of colored derivatives.

procedure ：

1. Centrifuge the fresh and anticoagulant whole blood specimen at 2500rpm for 5 min and separate the blood serum . (The step is finished before the class.)

2.marked 6 big test tubes and manipulate according to the following table

Arrangement Every group of the long table make the standard curve (blank 1 2 3 4 )

And others of the long table use the direct Calculation to get the concentration of the specimen. Use the tubes blank , one of the (1 2 3 4 ) and specimen.

3. Put tubes in a 100℃ waterbath for 10 min; then cool them with tap water.

4. Measure the absorbance of the solvent in the test tubes in a spectrophotometer at 620nm ,against the blank tube adjusted to 100 per cent transmittance(A620nm=0).

Reagent /ml Test tube No

Blank specimen 1 2 3 4

Blood serum --- 0.10 --- --- --- ---

Standard solvent of --- --- 0.25 0.50 0.75 1.00

Glucose(0.2mg/ml)

distilled water 1.0 0.9 0.75 0.50 0.25 ---

O-toluidine reagent 2.0 2.0 2.0 2.0 2.0 2.0

5. Draw a standard curve with the X axis represents the glucose content of tubes from No.1 to No.4 respectively; and the Y axis the absorbance of the four tubes.

6. Obtain the glucose content of 0.1ml specimen directly from the standard curve , calculate the serum glucose level in milligrams per 1000ml.

7. Calculating the glucose content per liter according to the following formula.

  Glucose concentration (mmol/L) = ×Cstandard×

The normal glucose contain with empty stomoach using this method is between 3.9mmol/L and 5.6mmol/L.

ODsample 

ODstandard

1000

0.1×180

Result:

Tube No

blank 1 2 3 4

Abs 0

0

722 λ=620nm

C(mg)

A

bl ood gl ucose srandard curve

0

0. 2

0. 4

0. 6

0. 8

1

1. 2

0 0. 05 0. 1 0. 15 0. 2 0. 25

content

A

722 λ=620nm

0 0

0.05

0.21

0.10.4

7

0.15

0.66

0.20.9

6

x0.6

5

content A

Notes:

Please not use water to wash the cuvette.

Pay attention to use the solvent accurately, some of those are poisonous and strong caustic.

Please use the blank tube as the control and not to use the water as the control.