Methodology for Carbohydrates
Determination of reducing sugars by Nelson-Somogyi method
Sugars with reducing property (arising out of the presence of a potential aldehyde or keto groups) are called reducing sugars. Some of the reducing sugards 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.
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 color developed is compared with a set of standards in a colorimeter at 620nm.
- Alkaline Copper tartrate
- (A) Dissolve 2.54g anhydrous sodium carbonate, 2g sodium bicarbonate, 2.5g potassium sodium tartrate and 20g anhydrous sodium sulphate in 80mL water and make up to 100mL.
- (B) Dissolve 15g copper sulphate in a small volume of distilled water. Add one drop of sulphuric acid and make up to 100mL.
Mix 4mL of B and 96mL of solution A befire use.
- Arsenomolybdate Reagent: Dissolve 2.5g ammonium molybdate in 45mL water. Add 2.5mL sulphuric acid and mix well. Then add 0.3g disodium hydrogen arsenate dissolved in 25mL water. Mix well and incubate at 37°C for 24 to 48 hours.
- Standard Glucose Solution: Stock: 100mg in 100mL distilled water.
- Working Standard: 10mL of stock diluted to 100mL with distilled water [100mg/mL].
- Weigh 100mg of the sample and extract the sugars with the hot 80% ethanol twice (5mL each time)
- Collect the supernatant and evaporate it by keeping it on a water bath at 80°C.
- Add 10mL water and dissolve the sugars.
- Pipette out aliquots of 0.1 or 0.2mL to separate test tubes.
- Pipette out 0.2, 0.4, 0.6, 0.8 and 1mL of the working standard solution into a series of test tubes.
- Make up the volume in both standard and samp-le tubes to 2mL with distilled water.
- Pipette out 2mL distilled water in a separate tuibe to set a blank.
- Add 1mL of alkaline copper tartrate reagent to each tube.
- Place the tubes in a boiling waterfor 10 minutes.
- Cool the tubes and add 1mL of arsenomolybolic acid reagent to all the tubes.
Absorbance corresponds to 0.1mL of test = ‘x’ mg of glucose
10mL contains = (‘x’ ÷ 0.1) × 10mg of glucose = % of reducing sugars
- Somogyi, M (1952) J boil Chem200 245.
- Krishnaveni, S, Theymoli Balasubramanian and Sadasivam, S (1984) Food Chem 15 229.