Table of Contents
Nelson Somogyi method for glucose estimation post we briefly summarises about: principle, reagents requirements, procedure, result, application and limitations of Nelson Somogyi method.
Nelson Somogyi Method
Nelson-Somogyi method is used to measure reducing sugar by using copper reagents and arsenolmolibdat. The principle of nelson Somogyi method is the amount of deposro oxide deposits that react with arsenomolibdate which is reduced to molybdine blue and the blue color is measured absorbance.
The free aldehyde and keto groups are routinely examined when reducing sugars. Glucose, galactose, lactose, and maltose are examples of reducing sugars. The cyclic form must first open the ring form to produce reactive aldehyde before oxidation may take place.
As a result, any sugar containing a hemiacetal is a reducing sugar, although glycosides containing acetals are not. Because ketones tautomerise to aldose via an enediol, they can also help with sugar reduction. The Nelson Somogyi method is a well-known and commonly used method for estimation of reducing sugars (glucose) quantitatively.
Nelson Somogyi Method Principle
Nelson Somogyi method uses copper and arsenomolybdate reagent and is a variation of Somogyi’s titrometric method for use with a colorimeter. When reducing sugars like glucose, galactose, lactose, and maltose are heated with alkaline copper tartrate, the copper is reduced from cupric to cuprous, resulting in the formation of cuprous oxide.
The reduction of molybdic acid to molybdenum blue occurs when cuprous oxide is treated with arsenomolybdate. The produced blue hue can be compared to known quantity standards in a colorimeter at 520nm. The test ranges from 5 to 600 µg of sugar.
2. Test tube Stand
3. Test tubes
4. Vortex mixer
5. Water bath
6. Weighing Machine
1. Nelson’s reagent
Arsenomolybadate reagent (Nelson’s reagent), 25g ammonium molybdate is dissolved in 450 ml of water and 21 ml of concentrated sulphuric acid is slowly added with stirring. Then 3 g of sodium hydrogen arsenate dissolved in 25 ml distilled water, is poured slowly with constant stirring. The solution is incubated at 370C for 24 hrs. Filtered and stored in amber coloured reagent bottle.
2. Copper reagent A
25 g of anhydrous sodium carbonate, 25 g of sodium potassium tartarate (Rochelle salt) and 20 g sodium bicarbonate are dissolved in about 700 ml of water, finally add 200 g of anhydrous sodium sulphate and dilute the solution to one litre, and allow keeping it for two days at room temperature. Collect the clean supernatant by filtering.
3. Copper reagent B
15 g Copper Sulfate Pentahydrate is dissolved in 100 ml water. Add 1 drop of concentrated Sulphuric Acid
4. Copper reagent C
Prepare fresh before use by mixing 25 ml of copper reagent A and 1 ml of copper reagent B.
5. Glucose standard
Dissolve 100 mg of glucose in 100 ml of distilled water. Working standard solution (100µg glucose per ml): From the standard stock solution of glucose, pipette 10 ml in 100 ml volumetric flask and make up the volume with distilled water. The working standard of glucose will be 100 µg per ml.
1. Weigh 100 to 500 mg of sample and extract the material with hot 80 % ethanol in mortar and pestle. Collect the supernatant and evaporate the ethanol on a water bath at 800C.
2. Add 10 ml water and dissolve the material. Pipette out aliquots of 0.1 to 2 ml in separate test tubes.
3. Also pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard glucose in another set of test tubes. Make the volume to 2 ml with distilled water.
4. Pipette out 2 ml of distilled water in a separate tube which can be used as blank. Add 2 ml of alkaline working copper reagent C to all the tubes. Mix well.
5. Heat the content in a boiling water bath for 10 min and cool to room temperature. Add 1 ml of arsenomolybdate reagent to all the tubes.
6. Mix well and make the volume to 10 ml with water. Read the blue colour at 520 nm in colorimeter/spectrophotometer.
7. Although the colour is stable determine the absorbance at a fixed time after the addition of arsenomolybdate reagent. Calculate the amount of reducing sugar glucose standard curve.
Glucose Standard Curve
Example graph for glucose standard curve
Absorbance corresponds to 0.1 ml of test sample = ——- mg of glucose
10 ml contains = (——–/0.1) × 10 mg of glucose = % of reducing sugars