ANALYTICAL cHEMISTRY - Iv

Principle

• The phenol sulfuric acid method is a simple and rapid colorimetric method to determine total carbohydrates in a food sample. The method detects virtually all classes of carbohydrates including mono-, di-, oligo-, and polysaccharides. Although the method detects almost all carbohydrates, the absorptivity of different carbohydrates varies. Thus, unless a sample is known to contain only one carbohydrate, the result may be expressed arbitrarily in terms of one carbohydrate.

• In this method, the concentrated sulfuric acid breaks down any polysaccharides, oligosaccharides and disaccharides to monosaccharides. Pentose sugar (containing five carbon atoms) is then dehydrated to furfural and hexose sugar (containing six carbon atoms) is dehydrated to the hydroxymethylfurfural. These compounds then react with phenol to produce a yellow-gold color. For products that are very high in xylose (a pentose sugar) content such as, wheat barn or corn barn, xylose (instead of glucose) 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 absorption is measured at 490 nm. The color of this reaction is stable for several hours and the accuracy of the method is within ±2 % under proper conditions.

• Carbohydrates are the major sources of calories in soft drinks, beer and fruit juices, supplying 4 kcal/g of carbohydrate. In this experiment, standard curve is prepared using glucose solution and this is used to determine the carbohydrate concentration of soft drinks and then the caloric content of those beverages is calculated.

About Carbohydrates:

• Carbohydrates are chemical compounds that contain only oxygen, hydrogen and carbon. They are made up of joined-up sugars. Sugars have the general formula C_m(H₂O)_n and are also known as saccharides. Certain carbohydrates are an important storage and transport form of energy in most organisms, including plants and animals.

• There are four types of carbohydrates named by number of sugar molecules they contain.

1. Simple saccharides with one or two sugar molecules.

Monosaccharides - single sugar

Example: glucose, Fructose

Disaccharides - two saccharides

Example: Sucrose, lactose

2. Longer chain saccharides

Oligosaccharides (shorter chains) often linked to amino acids or lipids. They play a special role in cell membrane. Polysaccharides (Long chains) are complex carbohydrates with linear chains of sugars branched clusters. Their function is either energy storage (starch, glycogen) or building structures (cellulose, chitin).

• Carbohydrates are the most common source of energy for human body. But if a person eats more than the needed amount of carbohydrates, the extra is changed into fat. If necessary, humans can live without eating carbohydrates because the human body can change proteins into carbohydrates.

• Food materials, having high levels of carbohydrates, are bread, pasta, potatoes, cereals, rice, etc.

Principle of phenol - sulfuric acid method:

• Carbohydrates are destroyed by heat and acid. They are particularly sensitive to strong acids and high temperatures under these conditions, a series of complex reactions takes place, beginning with a simple dehydration reaction. Continued heating in presence of acid produces various furan derivatives like furfural, 5-methylfurfural, 5-hydroxymethylfurfural, 1-(furan-2-yl)-2-hydroxyethanone, etc.

• These products then condense with themselves and other products to produce brown and black substances. They will also condense with various phenolic compounds such as phenol, resorcinol, orcinol, α-naphthol and naphthoresorcinol and various nitrogen containing compounds particularly nitrogen heterocycles to produce colored compounds that are useful for carbohydrate analysis.

• The most widely used method is with phenol. This method is simple, rapid, sensitive, accurate, specific for carbohydrates and widely applied. Virtually all classes of sugars including sugar derivatives and oligo- and poly saccharides undergo reaction I.e. hydrolysis in presence of hot, strong acid releasing mono-saccharides. A stable color is produced and results are reproducible. In this method, the result is never stoichiometric so standard curve must be used.

Procedure

1. For preparation of calibration curve: Use the 100 ppm standard glucose solution to prepare various sets as shown in the table below:

2. Decarbonate the beverages - for this take the sample in a beaker and stir it till no observable carbon dioxide bubbles appear.

3. Sample tubes- Pipette out 5 mL of the beverage in a 100 mL volumetric flask and dilute it up to the mark using distilled water. From this 1:20 diluted solution, pipette out 1mL of the solution in a 100 mL volumetric flak and dilute it up to the mark using distilled water. Pipette out 1 mL of this 1:2000 diluted solution in a test tube and add 1 mL water to make a volume of 2 mL in total.

4. Phenol addition- To each tube add 0.5 mL of 2.5 % phenol solution.

5. Concentrated H₂SO₄ addition - To each tube add 5 mL concentrated H₂SO₄. The reaction is driven by the heat produced upon the addition of H₂SO₄ to aqueous sample. So, the rate of addition H₂SO₄ must be standardized. Let the tubes stand for about half an hour to reach room temperature.

6. Measure the absorbance at 490 nm.

Observation

Calculations