Nelson-Somogyi method can be used to quantify the reduction of sugar using arsenolmolibdat and copper reagents. The principle behind the Nelson Somogyi method is the quantity of deposro oxide deposits which react with arsenomolibdate, which reduces to molybdine blue. The blue hue is determined by as absorbance.
The keto and aldehyde free groups are regularly studied when reducing sugars. Lactose, galactose and maltose are all examples of sugars that are reduced. The cyclic form has to first break the ring to create reactive aldehyde prior to the process of oxidation can begin.
In the end the sugar that has an hemiacetal is a reducing sugar, whereas glycosides that contain Acetals aren’t. Since ketones automerise into aldose by using an enediol they may also assist in reduction of sugar. Nelson Somogyi method is an incredibly popular and widely used method to estimate the reduction of sugars (glucose) quantitatively.
Principle of Nelson Somogyi Method
Nelson Somogyi method utilizes arsenomolybdate reagent and copper and is a variation on Somogyi’s method of titrometrics to be used with the colorimeter. When sugars that are reduced, such as lactose, galactose and maltose are heated by the alkaline copper tartrate the copper gets diminished from cupric and then cuprous which results in the creation the cuprous oxide.
The reduction of molybdic acids to molybdenum blue takes place when cuprous oxide is processed by arsenomolybdate. The blue hue that is produced can be compared with standard values of known quantities using a colorimeter with 522nm. The test can range from 5 to 600 ug of sugar.
- Test tube Stand
- Test tubes
- Vortex mixer
- Water bath
- Weighing Machine
1. Nelson’s reagent
- Arsenomolybadate Reagent (Nelson’s reagent), 25g ammonium molybdate dissolves in 450ml of water.
- Then 21ml of concentrated sulphuric acid is added while stirring.
- Then 3 grams of sodium hydrogen arsenate dissolved in 25ml of distilled water is poured in slowly with continuous stirring.
- The solution is then incubated with the temperature of 370C for 24 hours.
- The solution is then filtered and stored in an the amber colored bottles for reagents.
2. Copper reagent A
- 25 grams of anhydrous sodium carbonate, 25 g sodium potassium tartarate (Rochelle salt) and 20 g of sodium bicarbonate dissolve in around 700 ml of water.
- Then add 200 g of anhydrous sodium sulphate,
- Dilute the solution to 1 Liter, then allow to keep it for 2 days at room temperatures.
- The supernatant is collected by filtering.
3. Copper reagent B
- 15 grams of Copper Sulfate Pentahydrate is dissolvable in 100 ml of water.
- Then add add 1 ml of copper reagent B.
4. Copper reagent C
- Freshly prepare before use by mixing 25ml of copper reagent A with 1 milliliter of copper reagent B.
5. Glucose standard
- Dissolve 100 mg glucose in 100 ml distillate water.
- Standard working solution (100ug glucose per milliliter): From the standard solution of glucose pipette 10ml into a 100 ml volumetric flasks, and fill the remainder by using the distilled water. The glucose standard is 100 ug per milliliter.
Procedure of Nelson Somogyi Method
- Measure 100-500 grams of the sample. Then, extract the substance using hot ethanol of 80 percent using a morter and pestle. The supernatant should be collected and evaporate the ethanol over an 800C water bath.
- Mix 10 ml of water in order to dissolve the substance. Pipette out portions from 0.1 to 2 ml into distinct test tubes.
- Also , pipette the 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard glucose in a different tube set. Make the volume of 2 milliliters using distilled water.
- Pipette 2ml of distilled water into an additional tube that can be used as a blank. Add 2 ml alkaline working copper reagent C to all of the tubes. Mix thoroughly.
- Warm the contents in a boiling water bath for 10 minutes and then cool to ambient temperature. Add 1 ml arsenomolybdate to all of the tubes.
- Mix well, and then increase the volume to 10 ml by adding water. Read the blue colour at 520 nm in colorimeter/spectrophotometer.
- While the color is stable, analyze the absorbance over a predetermined time following the addition of arsenomolybdate as reagent. Calculate the amount of reduction Sugar glucose in the standard curve.
Absorbance corresponds to 0.1mL of test = ‘x’ mg of glucose
10mL contains = (‘x’ ÷ 0.1) × 10mg of glucose = % of reducing sugars