The phenol-sulfuric acid technique is a fast and easy method for determining the amount of carbohydrates present in an experiment sample. It can detect virtually all kinds of carbohydrates including di-, mono-the -, and even oligo and polysaccharides. Although the method is able to detect nearly every type of carbohydrates, their absorptivity for the various carbohydrates differs. So, unless the sample is proven to have only one type of carbohydrate, the results should be calculated arbitrarily using one carbohydrate.

In this process, the concentrated sulfuric acid is able to break down all polysaccharides and disaccharides and oligosaccharides into monosaccharides. These pentoses (5-carbon substances) are later dehydrated to furfural and the hexoses (6-carbon substances) to the hydroxymethyl furfural. The compounds react with phenol, resulting in an orange-gold hue. If the product is extremely rich in xylose (a pentose) like bran from wheat or corn it is suggested to use xylose to create the standard curve used in the test, and then determine the absorption at 480 nanometers. If the product is high in hexose sugars, glucose is often used to build the standard curve. its absorption can be measured as 490nm. The color produced by this reaction lasts for a long time and the precision of the procedure is within +-2% in appropriate conditions.

Objective

• Find out the total amount of carbohydrates in the sample.

Principle

Carbohydrates (simple sugars, oligosaccharides, polysaccharides, and their derivatives) react in the presence of strong acid and heat to generate furan derivatives that condense with phenol to form stable yellow-gold compounds that can be measured spectrophotometrically at 490 nm.

Requirements

• Equipment’s: Spectrophotometer, Test tube, Test tube stand, Vortex, Water bath
• Reagents: Phenol 5%, Sulphuric acid 96% reagent grade., Glucose

Reagents Preparation

1. Phenol 5%: Redistilled (reagent grade) phenol (50g) dissolved in water and diluted to one liter.
2. Stock standard glucose solution: 100 milligrams of glucose mixed with 50ml double-distilled water. Make up up to 100 mL.
3. Working standard glucose solution: Take 10mL of the stock solution and build the total to 100 mL using double-distilled water.

Procedure

1. You’ll require seven clean, dry test tubes for the phenol sulfuric acid. 
2. Prepare dilutions using glucose standards using concentrations of 40,80, 120, 160, 200 and Ug/200 ul by transferring the proper volume of glucose from the glucose solution standard (1 mg per milliliter) then diluting using the distillation water to make the total volume of 200 ul.
3. The tubes should be filled with 0.2 milliliters of a 5 percent solution of phenol. 
4. Each tube should be filled with 1 millilitre of pure Sulphuric acid, and thoroughly mix it. 
5. The contents in the tubes should be mixed after 10 minutes. Then put them in a bath at 25 to 30⁰C for 20 minutes.
6. Switch on the Spectrophotometer and select 490 nm for the wavelength. 
7. For the first time you must set your absorbance (OD) in Blank to zero. 
8. Create an absorbance standard curve at 490 nm and the concentration of glucose in g/200l along the “Y” axis.
9. Record the unidentified Value “x” from the graph which corresponds to the reading of OD from the sample test.

Calculation

Carbohydrate estimation using the phenol sulfuric acid method, which shows the increasing sugar concentration

Test Sample = Concentration of unknown “x” in µg/200 µl = …………. x 5 µg/ml

Interpretation

The method of phenol sulfuric acids involves mixing a set solutions that have known glucose concentrations and the method’s phenol sulfuric acid reagent. A standard curve may be made, and the sugar concentrations of untested samples can be determined.

Advantages

• The ease, sensitivity, and speed are the advantages of the phenol sulfuric acid method. 
• When compared to other procedures, the estimation of carbohydrates/glycogen/starch requires only a few standard reagents, making phenol sulfuric acid method significantly simpler and less expensive.

Limitations

• Despite the fact that method of phenol sulfuric acids can identify most carbohydrate, absorption for the different carbohydrates differs in the phenol sulfuric method.

References

• Viel, Marie & Collet, Florence & Lanos, Christophe. (2018). Chemical and multi-physical characterization of agro-resources’ by-product as a possible raw building material. Industrial Crops and Products. 120. 214-237. 10.1016/j.indcrop.2018.04.025. 
• Nielsen, S. Suzanne (2010). [Food Science Texts Series] Food Analysis Laboratory Manual || Phenol-Sulfuric Acid Method for Total Carbohydrates. , 10.1007/978-1-4419-1463-7(Chapter 6), 47–53. doi:10.1007/978-1-4419-1463-7_6
• https://biocyclopedia.com/index/plant_protocols/carbohydrates/phenol_suplhuric_acid_method_for_total_carbohydrate.php 
• https://noteshippo.com/phenol-sulfuric-acid-method/