LAB EXPERIMENT 1___ _______

Calorimetric Determination of Glucose by the 3,5-

dinitrosalicylic acid Method.

:Principle

Several reagents have been employed which assay sugars by using their
reducing properties. This method tests for the presence of free carbonyl
group (C=O), the so-called reducing sugars. This involves the oxidation
of the aldehyde functional group present in, for example, glucose and the
ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic
acid (DNS) is reduced to 3-amino-5-nitrosalicylic acid under alkaline
:conditions, as illustrated in the equation below

The chemistry of the reaction is complicated since standard curves do not

always go through the origin and different sugars give different color
yields. The method is therefore not suitable for the determination of a
.complex mixture of reducing sugar

:Materials

:Standard Glucose Solution .1

0.1g anhydrous glucose is dissolved in distilled water and then raised the
.volume to 100 ml with distilled water
:Dinitro salicylic acid reagent .2
a. Solution "a" is prepared by dissolving 300g of sodium potassium
.tartarate in about 500 ml distilled water

b. Solution "b" is prepared by dissolving 10 g of 3,5-dinitrosalicylic acid

.in 200 ml of 2N NaOH solution

c. The dinitrosalycilate reagent is prepared by mixing solutions a & b and

.raising the final volume to 1 litre with distilled water

:Procedure

Pipette in duplicate the following reagents into a series of dry-clean .1

and labelled test tubes and as indicated in the following table, take
.Section A

SECTION A SECTION B
.Tube No ml. Stand. ml. H2O ml. Dinitrosalicylic bbbbbB
ml. H2O BB
.Glucose reagent

1 0.0 1.0 2.0 7.0

2 0.2 0.8 2.0 7.0
3 0.4 0.6 2.0 7.0
4 0.6 0.4 2.0 7.0
5 0.8 0.2 2.0 7.0
6 1.0 0.0 2.0 7.0

After replacing the above mentioned solutions as in section A in the .2

labelled tubes, shake well and then place them in a boiling water bath for
.5 minutes

Cool the tubes thoroughly and then add 7.0 ml of distilled water to .3
each tube as indicated in section B of the previous table, Read the
extinction (Optical density) of the colored solutions at 540 nm using the
.solution in tube 1 as a blank (control)
Note: All the tubes must be cooled to room temperature before reading
.since the extinction is sensitive to temperature change

Record the readings in section B, and plot the relationship between the .4
optical density and the concentration of glucose solution. See whether
there is a linear relationship between the concentrations of glucose
.solutions and their corresponding optical densities

Use the already prepared standard curve for the determination of the .5
unknown concentration of the glucose solution provided and tissue
.extract form exp.6 or any other unknown reducing sugar sample
Name: No.

Experiment 1:

Results Sheet
The concentration of standard glucose solution : mg/ml

: After conducting your test, fill the following table -

Tube Concentration Absorbance

No. )Mg/ml( )At 540 nm(

- Plot the standard curve of the absorbance (y- axis) against the
concentration ( x-axis )

Use this plot to estimate the concentration of your unknown glucose -

.sample

Express your results in mg/dl , mg% , g/ml and g/l -

Name: No.

Experiment 1:

Results Sheet