Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
EXPERIMENT 6
COLORIMETRIC ANALYSIS OF REDUCING SUGAR
Aim: To learn the method used in the determination of the reducing sugar based on the
colorimetric analysis. Two method will be used namely the Somogyi Nelson method and
DNS method.
1
Lab Manual CBB20303 BIOCHEMISTRY
Experiment Procedures
A. Somogyi Nelsons Method
1. Prepare the copper reagent by adding 12 g of sodium tartarate and 24g sodium
carbonate in 250ml of distilled water. This is followed by the addition of 4 g of
copper sulphate and 16 g sodium hydrogen carbonate. Mix gently until all the
salts have dissolved. Dissolve 180g of sodium sulphate in 250 ml of distilled
water. Mix the 2 solutions and top up with distilled water to 1000ml.
2. The Nelson reagent is prepared by adding 25g of ammonium molydate in 450ml
of distilled water and slowly add 21ml of concentrated sulphuric acid. At the
same time add 3g of disodium hydrogen arseno sulphate in 25 ml of distilled
water. Mix the two solutions and top up to the total volume of 500ml.
3. Add 1.0ml of the sample to 1.0ml of the copper reagent and incubate in the
water bath at the temperature of 60oC for 10 minutes.
4. Cool down the reaction mixture to room temperature and add 1.0ml of the
Nelsons reagent, followed by 10 ml of distilled water. Allow it to stand for 15
min.
5. Determine the optical density at 660nm of the reaction mixture.
6. Prepare a standard curve for glucose by using the concentration of pure glucose
in the range 0-100mg/L (Note: The concentration of pure glucose stock solution
is 1.00g/L). Then continue with the steps 3 - 5.
7. Use the standard curve to determine of unknown glucose concentration.
2
Lab Manual CBB20303 BIOCHEMISTRY
Report
1. Compare the results from the two methods.
2. Check the reliability and accuracy of the methods