Methodology for Carbohydrates
Phenol suplhuric acid method for total carbohydrate
The phenol sulphuric acid method to estimate total carbohydrates is described below:
In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This forms a green colored product with phenol and has absorption maximum at 490nm.
- Phenol 5%: Redistilled (reagent grade) phenol (50g) dissolved in water and diluted to one liter.
- Sulphuric acid 96% reagent grade.
- Standard Glucose: Stock – 100mg in 100mL of water. Working standard – 10mL of stock diluted to 100mL with distilled water.
- Weigh 100mg of the sample into a boiling tube.
- Hydrolyse by keeping it in boiling water bath for 3 hours with 5mL of 2.5 N-HCl and cool to room temperature.
- Neutralise it with solid sodium carbonate until the effervescence ceases.
- Make up the volume to 100mL and centrifuge.
- Pipette out 0.2, 0.4, 0.6, 0.8 and 1mL of the working standard into a series of test tube.
- Pipette out 0.1 and 0.2mL of the sample solution in two separate test tubes. Make up the volume in each tube to 1mL with water.
- Set a blank with 1mL of water.
- Add 1mL of phenol solution to each tube.
- Add 5mL of 96% sulphuric acid to each tube and shake well.
- After 10min shake the content in the tubes and place in a water bath at 25-30°C for 20min.
- Read the color at 490nm.
- Calculate the amount of total carbohydrate present in the sample solution using the standard graph
Absorbance corresponds to 0.1mL of the test = ‘x’ mg of glucose
100mL of the sample solution contains = (‘x’ ÷ 0.1) × 100mg of glucose = % of total carbohydrate present
- Dubois, M, Gilles, K A, Hamilton, J K, Rebers, P A and Smith, F (1956) Anal Chem 26 350.
- Krishnaveni, S, Theymoli Balasubramanian and Sadasivam, S (1984) Food Chem 15 229.