This document describes a procedure to quantify glucose concentration using a spectrophotometric method with 3,5-dinitrosalicylic acid (DNSA). A series of glucose solutions ranging from 0-500 mg/ml are prepared. DNSA is added to samples and heated, causing a color change proportional to glucose concentration. Absorbance is measured at 540nm and a standard curve is generated according to the Beer-Lambert law. The unknown sample is then tested to determine its glucose concentration based on the standard curve. The results demonstrate increasing absorbance with higher glucose concentration, verifying the Beer-Lambert law.

1. Quantitative identification of glucose using DNSA with spectroscopy.
In this experiment we will find the amount of glucose by a spectrophotometric method. The
method is based upon the color .glucose has no color. Color forms when one mole of sugar will
react with one mole of 3,5-dinitrosalicylic acid. Amount of reduced DNS proportional to amount
of glucose. By applying the beer lambert’s law we measure the concentration of glucose by
detecting the reducing end of the DNSA which absorbs at 540nm .through this technique we can
estimate sugar present in the blood
Procedure:
Preparation of DNSA(3,5 dinitrosalicylic acid) solution:30g of sodium potassium tatrate
dissolved in 50ml of distilled water. Added 20ml of 2m NaOH and shake well. Add 1.0g DNS
powder and adjust the volume to 100 ml by adding distilled water.
Stock solution of 1000ppm glugose: 1ppm =1mg/1 liter ; 1000ppm = 1000mg/1 liter
Prepare a series of test tubes with glucose a concentration ranging from 0-500 mg/ml.
C1V1=C2V2 (c1 stock solu. ; v1 volume stock solu. use ; c2 conc. need ;v2 vol. need)
For 500 ppm. 1000* V1=500*10 ; V1=500*10/1000 ; V1 = 5
No of
obs.
Water
(ml)
Stock solution
(ml)
Total volume
(ml)
0 ppm 1 0 10
1 ppm 9 V1= 100*10/1000 = 1 10
2 ppm 8 V1= 200*10/1000 = 2 10
3 ppm 7 V1= 300*10/1000 = 3 10
4 ppm 6 V1= 400*10/1000 = 4 10
5 ppm 5 V1= 5 10
Added 1ml of the total vol.and 1ml of DNSA solu. to each tube and heat for ten min in boiling
water.Set the Spectrophotometer to 0 absorb at 540nm.

2. Observation:
Conc. 0 100 200 300 400 500 unknown
absorbance 0 0.348 0.765 1.08 1.526 1.641 0.67
Discussion: Result proof the beer lamberd’s law as we increase the concentration absorbance
also increase. Minimum 4ml is required to fill the cell in UV spectrometer but each group made
2 ml so we combined two groups.
Reference:
Lab manual in biochemistry by Arti Nigam.
0
0.5
1
1.5
2
0 200 400 600
absor.