Skip to content
Home » Phenol-Sulfuric Acid Method for Total Carbohydrates

Phenol-Sulfuric Acid Method for Total Carbohydrates

Phenol-Sulfuric Acid method for total carbohydrates post we briefly summarise about: principle, reagents requirements, procedure, result, application and limitations of Phenol-Sulfuric Acid method.

Phenol-Sulfuric Acid Method for Total Carbohydrates

Phenol sulfuric acid method is a colorimetric method for determining total carbohydrates in a sample that is simple and quick. The approach can identify monosaccharides, disaccharides, oligosaccharides, and polysaccharides. 

Despite the fact phenol sulfuric acid method identifies nearly all carbohydrates, the absorptivity of the various carbohydrates varies. Unless the presence of only one carbohydrate in a sample is known, the phenol sulfuric acid method results must be represented arbitrarily in terms of one carbohydrate.


Among the quantitative assays for carbohydrate measurement, the phenol-sulphuric acid method is the simplest and most dependable. Phenol sulfuric acid method most commonly used to determine the amount of neutral sugar in oligosaccharides, proteoglycans, glycoproteins, and glycolipids.

Glucose is dehydrated to hydroxymethyl furfural in a hot acidic media. When combined with phenol, this produces a yellow-brown compound with a maximum absorption wavelength of 490 nm. Phenol sulfuric acid method is one of the most accurate ways for calculating total carbohydrate content.

Phenol-Sulfuric Acid Method

Chemical Reaction for Phenol sulfuric acid method



  1. Spectrophotometer
  2. Test tube
  3. Test tube stand
  4. Vortex
  5. Water bath


  1. Phenol
  2. Sulphuric acid
  3. Glucose

Reagents Preparation

1. Phenol 5%

Phenol 5% Phenol, 5% by weight (add 95mL double distilled water to 5g reagent grade phenol, crystals).

2. Stock

Stock standard glucose solution: 100 mg of glucose in 50 ml double distilled water and make up the volume to 100 mL.

3. Standard

Working standard glucose solution: Take 10 mL from this stock solution and make up the volume to 100 mL with double distilled water.


  1. You’ll need 7 clean, dry test tubes for phenol sulfuric acid method. Make dilutions of glucose standards with concentrations of 40, 80, 120, 160, and 200 µg per 200 µl by transferring the appropriate amount of glucose from the standard glucose solution (1 mg/ml) and diluting it with distilled water to a total volume of 200 µl.
  2. Fill each tube with 0.2 mL of a 5 percent phenol solution. Fill each tube with 1 mL of pure Sulphuric acid and well mix it. Mix the contents of the tubes after 10 minutes and place in a water bath set at 25-300C for 20 minutes.
  3. Turn on the Spectrophotometer and choose 490 nm as the wavelength. To begin, set the absorbance (OD) of Blank to zero. Plot a standard curve of absorbance at 490 nm versus glucose concentration in g/200l on the “Y” axis.
  4. Record the Unknown value “x” from the graph that corresponds to the OD reading of the test samples.


Phenol-Sulfuric Acid Method

Carbohydrate Estimation by phenol sulfuric acid method showing increasing amount of sugar concentration

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

Phenol-Sulfuric Acid Method


The phenol sulfuric acid method involves mixing a set of solutions with known glucose concentrations with the phenol sulfuric acid method reagent. A standard curve can be created, from which the concentrations of unknown sugar samples can be calculated.


The simplicity, sensitivity, and speed of this approach are phenol sulfuric acid method benefits. 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.


Despite the fact that the phenol sulfuric acid method identifies almost all carbohydrates, the absorptivity of the various carbohydrates varies in phenol sulfuric acid method.

Further Readings


Join Our Telegram Channel!