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Lab Manual CBB20303 BIOCHEMISTRY

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.

Apparatus and chemicals


1. Copper sulphate
2. Sodium bicarbonate
3. Sodium sulphate
4. Concentrated sulphuric acid
5. Ammonium molydate
6. Distilled water
7. Glucose
8. Disodium hydrogen arsenosulphate (sodium argenate)
9. 3,5-dinitrosalicylic acid
10. Potassium sodium tartarate
11. Sodium hydroxide
12. Sodium carbonate
13. Measuring cylinders
14. A balance
15. Spatulas
16. 2-L beakers
17. unknown glucose concentration

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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.

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Lab Manual CBB20303 BIOCHEMISTRY

B. DNS (3,5 dinitrosalicylic acid) method.

1. Prepare the DNS reagent by dissolving 5g of DNS and 8g of NaOH in 100ml of


distilled water and top the volume to 300ml. Add 150g of potassium sodium
tartarate slowly until all the DNS dissolve and top up the volume to 500ml.
2. Add 1.5ml of sample (sugar) in a test tube and add 1.5ml of the DNS reagent.
3. Boil the reaction mixture for 5 min and allow to cool down.Add 10ml of istilled
water.
4. Read the absorbance at 750nm.
5. Prepare a standard curve for glucose using the concentration of pure glucose in
the range 0-100mg/L by repeating steps 2-4.
6. Use the standard curve to determine of unknown glucose concentration.

Report
1. Compare the results from the two methods.
2. Check the reliability and accuracy of the methods