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Tuesday, April 26, 2011

Determination of Reducing Sugars by Nelson-Somogyi Method


Sugars with reducing property (arising out of the presence of a potential aldehyde or keto group) are called reducing sugars. Some of the reducing sugars are glucose, galactose, lactose and maltose. The Nelson-Somogyi method is one of the classical and widely used methods for the quantitative determination of reducing sugars.

Determination of reducing sugar


PRINCIPLE
The reducing sugars when heated with alkaline copper tartrate reduce the copper from the cupric to cuprous state and thus cuprous oxide is formed. When cuprous oxide is treated with arsenomolybdic acid, the reduction of molybdic acid to molybdenum blue takes place. The blue colour developed is compared with a set of standards in a colorimeter at 620 nm.
MATERIALS
Alkaline Copper Tartrate
(i) Dissolve 2.5 g anhydrous sodium carbonate, 2 g sodium bicarbonate, 2.5 g potassium sodium tartrate and 20 g anhydrous sodium sulphate in 80 mL water and make up to 100 mL.
(ii) Dissolve 15 g copper sulphate in a small volume of distilled water. Add one drop of sulphuric acid and make up to 100 mL.
Mix 4 mL of B and 96 mL of solution A before use.
Arsenomolybdate reagent: Dissolve 2.5 g ammonium molybdate in 45 mL water. Add 2.5 mL sulphuric acid and mix well. Then add   0.3 g disodium hydrogen arsenate dissolved in 25 mL water. Mix well and incubate at 37°C for 24–48 hours.
Standard glucose solution: Stock: 100 mg in 100 mL distilled water.
Working standard: 10 mL of stock diluted to 100 mL with distilled water [100 µg/mL].
PROCEDURE
1. Weigh 100 mg of the sample and extract the sugars with hot 80% ethanol twice (5 mL each time).
2. Collect the supernatant and evaporate it by keeping it on a water bath at 80°C.
3. Add 10 mL water and dissolve the sugars.
4. Pipette out aliquots of 0.1 or 0.2 mL to separate test tubes.
5. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard solution into a series of test tubes.
6. Make up the volume in both sample and standard tubes to 2 mL with distilled water.
7. Pipette out 2 mL distilled water in a separate tube to set a blank.
8. Add 1 mL of alkaline copper tartrate reagent to each tube.
9. Place the tubes in a boiling water for 10 minutes.
10. Cool the tubes and add 1 mL of arsenomolybolic acid reagent to all the tubes.
11. Make up the volume in each tube to 10 mL with water.
12. Read the absorbance of blue colour at 620 nm after 10 min.
13. From the graph drawn, calculate the amount of reducing sugars present in the sample.
CALCULATION
Absorbance corresponds to 0.1 mL of test = x mg of glucose
10 mL contains  = x / 0.1 × 10 mg of glucose
     = % of reducing sugars

REFERENCES
1. Somogyi, M. (1952). J. Biol. Chem., 200, 245.
2. Krishnaveni, S.; Theymoli Balasubramanian and Sadasivam, S. (1984). Food Chem., 15, 229.
3. Internet Sources

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