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.
 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

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).
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.

ODsample  1000
  Glucose concentration (mmol/L) = ×Cstandard×
ODstandard 0.1×180

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

Tube No blank 1 2 3 4
Abs 0

A 722 λ=620nm

0
C(mg)
content A 722 λ=620nm

0 0
0.05 0.21 blood glucose srandard curve

0.1 0.47 1.2

1
0.15 0.66 0.8
0.6
A

0.2 0.96 0.4

0.2
x 0.65 0
0 0.05 0.1 0.15 0.2 0.25
content
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.