Phenol Sulfuric Acid Method

Among many colorimetric methods for carbohydrate analysis, the phenol-sulfuric acid method is the easiest and most reliable method.The method detects virtually all classes of carbohydrates, including mono-, di-, oligo-, and polysaccharides. Although the method detects almost all carbohydrates, the absorptivity of the different carbohydrates varies. Thus, unless a sample is known to contain only one carbohydrate, the results must be expressed arbitrarily in terms of one carbohydrate.

In this method, the concentrated sulfuric acid breaks down any polysaccharides, oligosaccharides, and disaccharides to monosaccharides. Pentoses (5-carbon compounds) are then dehydrated to furfural, and hexoses (6-carbon compounds) to hydroxymethyl furfural. These compounds then react with phenol to produce a yellow-gold color. For products that are very high in xylose (a pentose), such as wheat bran or corn bran, xylose should be used to construct the standard curve for the assay, and measure the absorption at 480 nm. For products that are high in hexose sugars, glucose is commonly used to create the standard curve, and the absorption is measured at 490 nm. The color for this reaction is stable for several hours, and the accuracy of the method is within ±2% under proper conditions.

For example, carbohydrates can be estimated using phenol Sulphuric acid method. 

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.