Experiment No.
ESTIMATION OF REDUCING SUGARS
Date: BY DNSA REAGENT
Aim
To estimate the amount of reducing sugars present in the given sample by
DNSA method.
Principle
Sugars with reducing property arising out of the presence of a potential
aldehyde or keto group are called reducing sugars. Ex. Glucose, galactose,
lactose, maltose etc. 3,5 dinitrosalicylic acid (DNSA) is reduced to 3-amino-
5-nitrosalicylic acid in the presence of reducing sugars in an alkaline
solution. The reducing sugar is oxidized to sugar acid and Rochelle’s salt
reduces the tendency to dissolve oxygen by increasing the ion concentration in the solution.
Phenol increases the amount of color produced during the color-developing reaction. Sodium
bisulphite stabilizes the color obtained and reacts with any oxygen present in the medium.
Alkaline medium (NaOH) is required for the red-ox reaction between DNSA and glucose, or
other reducing sugars.
Materials
Dinitrosalicylic acid reagent (DNSA reagent): Dissolve by stirring 1 g
dinitrosalicylic acid, 200 mg crystalline phenol and 50 mg sodium
sulphite in 100 ml of 1% NaOH. Store at 4ºC.
40% Rochelle salt solution (Potassium sodium tartarate).
0.1N NaOH
Reagents Required:
Sodium potassium tartrate (Rochelle salt solution): Dissolve 45 gms of sodium
potassium tartrate in 75 mL of H2O.
3,5-DNS Reagent: Dissolve 1.5 gm of DNS reagent in 30 mL of 2 M/liter NaOH.
Standard glucose solution (1mg/ml)
Procedure:
Take several clean, dry and sterile test tubes.
Pipette out standard sugar solution in the range of 0 to 3 mL in different test tubes and
make up the volume of all test tubes to 3 ml with distilled water.
.Add 1 ml DNS reagent to all the test tubes and mix plug the test tube with cotton or
marble and keep the test tube in a boiling water bath for 15 minutes.
.After cooling to room temperature in a cold-water bath, add 1 ml of a 40% potassium
sodium tartrate (Rochelle salt) solution to stabilize the colour.
Finally, record the absorbance with a spectrophotometer at 540nm.
Please note that all the tubes must be cooled to room temperature before reading, since
the absorbance is sensitive to temperature.
Prepare standard curves of the sugars provided and use them to estimate the
concentration of the unknowns provided.
Prepare standard curves of the sugars provided and use them to estimate the
concentration of the unknowns provided.
Volume Volume Volume 40%
Absorbance
(ml) of Concentration (ml) of of DNS Rochelle
S. No (540nm)
Standard of glucose distilled reagent ( salt
O.D.
glucose water ml) solution
keep
the test
tube in
a
boiling
1 Blank – 3 1 1 ml –
water
bath
for 5-
15
minutes
keep
the test
tube in
0.3 100 ug/ml
a
boiling
2 2.7 1 1 ml –
water
bath
for 5-
15
minutes
3 0.6 200 ug/ml 1 keep 1 ml –
2.4 the test
tube in
a
boiling
water
bath
for 5-
15
minutes
keep
the test
tube in
a
boiling
4 0.9 300 ug/ml 2.1 1 1 ml –
water
bath
for 5-
15
minutes
keep
the test
tube in
a
boiling
5 1.2 400 ug/ml 1.8 1 1 ml –
water
bath
for 5-
15
minutes
keep
the test
tube in
a
boiling
6 1.5 500 ug/ml 1.5 1 1 ml –
water
bath
for 5-
15
minutes
Test Test – – 1 keep 1 ml –
solution solution the test
A (3ml) tube in
a
boiling
water
bath
for 5-
15
minutes
keep
the test
tube in
a
Test Test
boiling
solution solution – – 1 1 ml –
water
B (3ml)
bath
for 5-
15
minutes
Results and Interpretation:
Prepare a standard curve using known concentrations of glucose.
Plot the absorbance values against the concentrations to create a linear standard curve.
Use the standard curve to determine the concentration of glucose in your sample based on
its absorbance.
If you performed dilutions during the sample preparation, ensure to account for these
dilutions in your final concentration calculation.