Download Glucose

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:
1. Standard Glucose Solution:
0.1g anhydrous glucose is dissolved in distilled water and then raised the
volume to 100 ml with distilled water.
2. Dinitro salicylic acid reagent:
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:
1. Pipette in duplicate the following reagents into a series of dry-clean
and labelled test tubes and as indicated in the following table, take
Section A.
SECTION A
Tube
No.
ml. Stand.
Glucose.
ml.
H2 O
ml.
Dinitrosalicylic
reagent
SECTION B
ml. H2O
bbbbbB
BB
7.0
1
0.0
1.0
2.0
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
2. After replacing the above mentioned solutions as in section A in the
labelled tubes, shake well and then place them in a boiling water bath for
5 minutes.
3. Cool the tubes thoroughly and then add 7.0 ml of distilled water to
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.
4. Record the readings in section B, and plot the relationship between the
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.
5. Use the already prepared standard curve for the determination of the
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)
1
2
3
4
5
6
7
- 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:
Experiment 1:
Results Sheet
No.