Anthrone Test- Definition, Principle, Procedure, Result, Uses

Anthrone test is a group test for carbohydrates that provides a rapid and convenient method for quantification of carbohydrates that are either free or bound to any lipids or proteins. Carbohydrates are organic compounds that consist of carbon, hydrogen and oxygen atoms in a fixed ratio. They are the main source of energy for living organisms and are involved in various biological processes. Carbohydrates can be classified into different types based on their size and structure, such as monosaccharides, disaccharides, oligosaccharides and polysaccharides. Monosaccharides are the simplest and smallest carbohydrates that cannot be hydrolyzed into smaller units. Examples of monosaccharides are glucose, fructose and galactose. Disaccharides are composed of two monosaccharide units linked by a glycosidic bond. Examples of disaccharides are sucrose, lactose and maltose. Oligosaccharides are composed of 3 to 10 monosaccharide units linked by glycosidic bonds. Examples of oligosaccharides are raffinose, stachyose and fructooligosaccharides. Polysaccharides are composed of more than 10 monosaccharide units linked by glycosidic bonds. Examples of polysaccharides are starch, glycogen, cellulose and chitin.

Some carbohydrates are free in solution, while others are bound to lipids or proteins to form complex molecules such as glycoproteins and glycolipids. Glycoproteins are proteins that have one or more carbohydrate chains attached to them. They play important roles in cell recognition, signaling, adhesion and immunity. Glycolipids are lipids that have one or more carbohydrate chains attached to them. They are mainly found in the cell membrane and function as receptors, antigens and markers.

Anthrone test is a chemical test that can detect and measure the amount of carbohydrates in a given solution, regardless of whether they are free or bound to lipids or proteins. Anthrone test is based on the reaction of carbohydrates with anthrone reagent in the presence of concentrated sulfuric acid. Anthrone reagent is a yellow crystalline compound that contains naphthol as its main component. Anthrone test can be used for both qualitative and quantitative analysis of carbohydrates in various samples such as blood serum, milk, urine, plant extracts and food products.

The main objectives of anthrone test are:

• To detect the presence of carbohydrates in a given solution. Carbohydrates are organic compounds that consist of carbon, hydrogen and oxygen atoms. They are important sources of energy and structural components for living organisms. Carbohydrates can be classified into monosaccharides, disaccharides, oligosaccharides and polysaccharides based on the number and type of sugar units they contain. Anthrone test can detect all types of carbohydrates that can be hydrolyzed to monosaccharides by concentrated sulfuric acid.
• To quantify the concentration of free and bound carbohydrates in a solution. Free carbohydrates are those that are not attached to any other molecules, such as glucose or fructose. Bound carbohydrates are those that are linked to lipids or proteins, such as glycolipids or glycoproteins. Anthrone test can measure the total amount of carbohydrates in a solution by converting them to a blue-green complex that absorbs light at 620 nm wavelength. The intensity of the color is proportional to the concentration of carbohydrates in the solution. By using a standard curve of known concentrations of glucose, the concentration of unknown samples can be calculated.

The principle of anthrone test is based on the formation of a blue-green complex between furfural or hydroxyl furfural and naphthol from the anthrone reagent. The furfural or hydroxyl furfural are produced by the dehydration of monosaccharides in the presence of concentrated sulfuric acid. The monosaccharides are derived from the hydrolysis of polysaccharides, glycoproteins or glycolipids by the same acid. The intensity of the blue-green color is proportional to the concentration of carbohydrates in the solution and can be measured by a spectrophotometer or a colorimeter at 620 nm wavelength. The amount of carbohydrates can be calculated by using a standard curve of absorbance versus glucose concentration.

The following diagram illustrates the principle of anthrone test:

graph LR
A --Hydrolysis--> B
B --Dehydration--> C
C --Reaction with Anthrone Reagent--> D
D --Absorbance at 620 nm--> E

The Anthrone test involves three main steps of reaction:

1. Hydrolysis of polysaccharides to monosaccharides: The concentrated sulfuric acid in the Anthrone reagent breaks down the complex carbohydrates (polysaccharides or oligosaccharides) into their simple sugar units (monosaccharides) by adding water molecules. For example, starch is hydrolyzed to glucose, and sucrose is hydrolyzed to glucose and fructose.

2. Dehydration of monosaccharides to furfural derivatives: The concentrated sulfuric acid also catalyzes the removal of water molecules from the monosaccharides, resulting in the formation of furfural derivatives. Depending on the type of monosaccharide, either furfural (from pentoses) or hydroxymethylfurfural (from hexoses) is formed. For example, xylose is dehydrated to furfural, and glucose is dehydrated to hydroxymethylfurfural.

3. Condensation of furfural derivatives with naphthol: The furfural derivatives react with two molecules of naphthol from the Anthrone reagent to form a blue-green complex. The complex has a characteristic absorption peak at 620 nm wavelength, which can be measured by a spectrophotometer or a colorimeter. The intensity of the color is proportional to the concentration of carbohydrates in the solution.

The following equations illustrate the reactions involved in the Anthrone test:

• Hydrolysis of polysaccharides to monosaccharides

$$\text{Polysaccharide} + \text{H}_2\text{O} \xrightarrow{\text{H}_2\text{SO}_4} \text{Monosaccharides}$$

• Dehydration of monosaccharides to furfural derivatives

$$\text{Monosaccharide} \xrightarrow{\text{H}_2\text{SO}_4} \text{Furfural derivative} + \text{H}_2\text{O}$$

• Condensation of furfural derivatives with naphthol

$$\text{Furfural derivative} + 2\text{Naphthol} \xrightarrow{\text{H}_2\text{SO}_4} \text{Blue-green complex}$$

The materials required for Anthrone test are as follows:

• Equipment:
  • UV Spectrophotometer
  • Vortex mixer
  • Mantle heater/Water Bath.
• Chemicals/Reagents:
  • Anthrone Reagent
  • Glucose
  • Other carbohydrates if desired
  • Sample
• Glasswares and other equipment:
  • Test tubes, Test tube stand, Pipettes, Beaker, Ice Test tube caps, Tissue paper, Wash bottle.

The details of each material are explained below:

• UV Spectrophotometer: This is a device that measures the absorbance of light by a solution at a specific wavelength. It is used to quantify the concentration of carbohydrates in the solution by comparing it with a standard curve.
• Vortex mixer: This is a device that creates a vortex in a liquid by spinning it rapidly. It is used to mix the solution and the anthrone reagent thoroughly before heating.
• Mantle heater/Water Bath: This is a device that provides a constant temperature for heating the solution and the anthrone reagent. It is used to facilitate the reaction between the carbohydrates and the anthrone reagent to form a blue-green complex.
• Anthrone Reagent: This is a chemical reagent that contains anthrone and concentrated sulfuric acid. It reacts with furfural or hydroxyl furfural produced from the dehydration of monosaccharides to form a blue-green complex. The intensity of the color depends on the amount of carbohydrates present in the solution.
• Glucose: This is a simple sugar that is used as a standard for quantifying the concentration of carbohydrates in the solution. It is prepared as a stock solution of 200 µg glucose per mL distilled water and then diluted to different volumes for making a standard curve.
• Other carbohydrates: These are optional samples that can be used to test for the presence and concentration of carbohydrates in them. They should be prepared as solutions of the same concentration as glucose (200 µg/mL) and then diluted to different volumes for testing.
• Sample: This is the unknown solution that contains carbohydrates that need to be detected and quantified. It can be any biological fluid or extract that contains free or bound carbohydrates.
• Test tubes, Test tube stand, Pipettes, Beaker, Ice Test tube caps, Tissue paper, Wash bottle: These are common laboratory glasswares and equipment that are used for holding, transferring, measuring, and washing the solutions. They should be clean and dry before use.

• Pipette out different volumes (50 µl, 100 µl, and so on) of glucose solution from the supplied stock solution (200µg /ml) into a series of test tubes and make up the volume to 1 mL with distilled water.
• Take a tube labeled as one as blank containing 1ml of just distilled water and the rest of the tubes labeled 2 to 9 for construction of a standard curve. Tubes 10-15 are for the unknown samples.
• Add 5 ml of the anthrone reagent To each tube and mix well by vortexing.
• Cool the tubes.
• Cover the tubes with caps on top and incubate at 90°C for 17 minutes or boiling water bath for 10 minutes.
• Cool the tubes to room temperature and measure the optical density of the solutions at 620 nm against a blank.
• Prepare a standard curve of absorbance against glucose concentration.
• Determine the amount of glucose in the unknown sample by plotting a standard curve of A620 on the Y-axis and concentration of Glucose on the X-axis.

• The presence of a blue-green complex indicates the presence of carbohydrates in the given solution .
• The intensity of the blue-green color is proportional to the concentration of carbohydrates in the solution.
• The absorbance of the blue-green complex can be measured at 620 nm wavelength in a spectrophotometer or in a red filter colorimeter .
• A standard curve of absorbance against glucose concentration can be prepared by using known concentrations of glucose as standards .
• The amount of glucose in the unknown sample can be determined by interpolating the absorbance value on the standard curve .
• The amount of carbohydrate present in 100 mg of the sample can be calculated by using the formula:

$$\text{Amount of carbohydrate present in 100 mg of the sample} = \frac{\text{mg of glucose}}{\text{Volume of test sample}} \times 100$$

Anthrone test is a useful method for the detection and quantification of carbohydrates in various samples. Some of the applications of anthrone test are:

• Anthrone test can be used to measure the total carbohydrate content in food products, such as cereals, fruits, vegetables, dairy products, etc. This can help in assessing the nutritional value and quality of food.
• Anthrone test can also be used to determine the carbohydrate content in biological samples, such as blood serum, urine, saliva, etc. This can help in diagnosing and monitoring various diseases and disorders related to carbohydrate metabolism, such as diabetes, glycogen storage diseases, etc.
• Anthrone test can be used to estimate the amount of carbohydrates in plant materials, such as leaves, stems, roots, seeds, etc. This can help in studying the photosynthesis and respiration processes in plants and their response to environmental factors.
• Anthrone test can be used to analyze the carbohydrate composition of polysaccharides, such as starch, cellulose, glycogen, etc. This can help in understanding the structure and function of these macromolecules and their role in various biological processes.

• Anthrone test gives a negative result in samples that are carbohydrates like D-glucose phenylosazone and D-glucose phenylosotriaole but do not form furfural or hydroxyfurfural in the dehydration step .
• Anthrone test is not specific for carbohydrates as other compounds like glycerol, amino acids, and aldehydes can also react with anthrone reagent and give a positive result.
• Anthrone test is sensitive to temperature and time variations as the color intensity of the complex depends on these factors. Therefore, the test tubes should be heated uniformly and for the same duration to avoid errors in measurement.
• Anthrone test requires a large amount of sample (1 mL) and reagent (5 mL) for each test tube, which might not be feasible for some samples or situations.
• Anthrone test is affected by the presence of interfering substances like iron, copper, and nitrate ions that can reduce the color intensity of the complex. Therefore, the sample should be free from these contaminants or treated with appropriate methods to remove them before performing the test.

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