Estimation of reducing sugar by DNS method
Aim: Colorimetric estimation of glucose by DNS method.
Principle: 3, 5-dinitro salicylic acid reacts with reducing sugar to give 3-amino,
5-nitro salicylic acid. Glucose is oxidised to gluconic acid. The red colour product
produced will have maximum absorption (ʎ max) at 540 nm. Therefore, the
concentration of glucose is found by measuring optical density (O.D.) at 540 nm.
Concentration range: 0- 2000µg
Volume range: 0- 2ml
Preparation of Glucose stock solution:
Exactly 1 gm of glucose (commercially called Dextrose/ D-Glucose) is weighed
accurately and transferred into a 100 ml standard flask carefully. Slowly add
distilled water and shake well for uniform concentration. Make up to volume to
100ml mark. Now, 1ml of this standard solution contain 100 mg of glucose.
Preparation of working standard solution:
Exactly 10 ml of the glucose stock solution is pipetted out into a 100 ml standard
flask. The solution is diluted up to the mark and shaken well for uniform
concentration. 1 ml of working standard contain 1mg (1000µg) of glucose.
Procedure for estimation of glucose in the given unknown sample using
standard curve.
Exactly 0 to 2 ml of working standard glucose solution in the volumes of 0, 0.4,
0.8, 1.2, 1.6 and 2 ml is pipetted out into different test tubes which are numbered
from 1 to 6.
Make up the volume of each tube to 2 ml adding appropriated volume of distilled
water.
Given unknown samples is taken in triplicates (tubes 7, 8 and 9). The unknown
concentration is measured 2-3 times to know the consistency.
Add 2 ml of DNS reagent to all the tubes and keep in boiling water bath for 10
min.
The tubes are cooled to room temperature (if required equal amount of distilled
water can be added to all the tubes after cooling). Mix well and record the
absorbance at 540 nm.
A standard graph taking concentrations of glucose along X-axis and
absorbance/O.D. on Y-axis is drawn. Using the standard graph, the concentration
of given unknown glucose solution is determined.
Result: The concentration of glucose in unknown sample is ------- / ml.
Reagents required:
1. Working standard of Glucose- 1mg/ ml of distilled water in standard flask.
2. DNS reagent:
Solution-1: 2 gm DNS dissolved in 40 ml of 2 N NaOH.
Solution-2: 10 g of Sodium Potassium Tartrate in 100 ml of water.
Solution-1 and Solution-2 is mixed and volume is made up to 200 ml using
distilled water.
Table for glucose standard curve:
Sl.no. Volume (ml) & Volume DNS Water Absorbance
Concentration of Water reagent at 540 nm
(μg) of Glucose Incubate
the
1 0.0 (0) 2.0 Cool the
tubes in
2 0.4 (400) 1.6 tubes to
boiling
3 0.8 (800) 1.2 room
water
2 ml to temperature.
4 1.2 (1200) 0.8 for 10
each
5 1.6 (1600) 0.4 minutes
tube If required
6 2.0 (2000) 0.0 5 ml to each
7 Uk1 tube
8 Uk2
9 Uk3
