Explain Procedure for nelson somogyi method for glucose estimation?

Carry out the procedure following the steps enumerated herewith:

1. Label a series of  Folins tubes as blank(B), standard (S1-S6) and sample (SA 1-4) and place them in a rack.

2. Preparation of Standard Glucose Solution: Weigh 50 mg of glucose and dissolve it in distilled water and make up the volume to 500 ml. This is your standard glucose solution. (0.1 mg/ml)

3. Colour Development for standard solution: Take six different known concentrations of standard glucose solution (0.4 ml. 0.6 ml, 0.8 ml, 1.0 ml, 1.2 ml and 1.4 ml) in the first six Folins tubes (S1-S6) to prepare a standard curve. Make the volume to 2 ml with distilled water in each tube. Add 2 ml of alkaline copper reagent. Mix well.

4. For preparation of blank(B): 2 ml of distilled water and 2 ml of alkaline copper reagent will be added to the tube B.

5. Preparation of Unknown Solution: Take the unknown solution given to you and make the volume to 100 ml mark with distilled water. Use 0.8 ml and 1.2 ml volumes of this in duplicate. Proceed just as you did for standard tubes in step 3.

6. Mix the contents of all the tubes, plug them tightly with cotton wool and put them in a boiling water bath for 20 minutes.

7. Remove the tubes from the water bath and cool to each tube.

8. Add 2 ml of arsenomolybdate reagent standard. Shake well and make the volume upto 25 ml mark with distilled water. Mix. Read at 540 nm.

9. Plot a graph between concentration of glucose in standared solution and the corresponding OD. Estimate the content of glucose in the unknown solution by using the standard curve as well as mean OD.