Fehling’s Test- Definition, Principle, Procedure, Result, Uses

Carbohydrates are one of the most important biomolecules that are involved in various metabolic processes and provide energy to living organisms. Carbohydrates can be classified into different types based on their structure and function, such as monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Some carbohydrates have the ability to reduce other substances by donating electrons or hydrogen atoms. These are called reducing sugars and they include glucose, fructose, lactose, maltose, etc. Other carbohydrates do not have this property and are called non-reducing sugars. These include sucrose, starch, cellulose, etc.

Fehling’s test is a simple and widely used chemical test that can differentiate between reducing and non-reducing sugars. It can also be used to identify the presence of aldehydes or ketones in carbohydrates. Fehling’s test is based on the oxidation-reduction reaction between the carbohydrate and a copper (II) complex in an alkaline medium. The test produces a characteristic reddish-brown precipitate of copper (I) oxide if the carbohydrate is a reducing sugar or an aldehyde. If the carbohydrate is a non-reducing sugar or a ketone, the test remains blue and no precipitate is formed.

Fehling’s test has many applications in biochemistry, food science, medicine, and forensic science. It can be used to detect the presence of glucose in urine, which is an indicator of diabetes mellitus. It can also be used to determine the purity of honey by detecting the presence of added sugars. Furthermore, it can be used to identify unknown carbohydrates by comparing their reactions with known standards.

In this article, we will discuss the definition, principle, procedure, result, uses, and limitations of Fehling’s test in detail. We will also explain the reaction involved in Fehling’s test and the requirements for performing it. We hope that this article will help you understand the concept and significance of Fehling’s test better.

Fehling’s test is a chemical test that can be used to identify and differentiate between reducing and non-reducing sugars in a solution. Reducing sugars are carbohydrates that can donate electrons to another substance, while non-reducing sugars are those that cannot. Fehling’s test can also distinguish between ketone and aldehyde functional groups in carbohydrates, as ketones are less reactive than aldehydes in this test.

The main objectives of Fehling’s test are:

• To detect the presence or absence of carbohydrates in a solution.
• To differentiate between reducing and non-reducing sugars based on their ability to reduce copper (II) ions to copper (I) oxide.
• To identify the type of functional group (ketone or aldehyde) in carbohydrates based on their oxidation products.

The principle of Fehling’s test is based on the oxidation-reduction reaction between the reducing sugars and the copper (II) ions present in the Fehling’s solution. Reducing sugars are carbohydrates that have a free or potentially free aldehyde or ketone group that can donate electrons to other molecules. Examples of reducing sugars are glucose, fructose, lactose, maltose, etc. Non-reducing sugars are carbohydrates that do not have a free or potentially free aldehyde or ketone group and cannot donate electrons to other molecules. Examples of non-reducing sugars are sucrose, starch, cellulose, etc.

The Fehling’s solution is a deep blue liquid that consists of two components: Fehling’s solution A and Fehling’s solution B. Fehling’s solution A is a solution of copper (II) sulfate (CuSO4), which provides the copper (II) ions for the reaction. Fehling’s solution B is a solution of potassium sodium tartrate (KNaC4H4O6) and sodium hydroxide (NaOH), which acts as a complexing agent and a base, respectively. The potassium sodium tartrate forms a bistartarocuprate (II) complex with the copper (II) ions, which prevents them from forming insoluble copper (II) hydroxide (Cu(OH)2) in the alkaline environment. The sodium hydroxide maintains the pH of the solution above 7, which is necessary for the reaction to occur.

When the Fehling’s solution is heated with a reducing sugar, the following reaction takes place:

$$\text{Reducing sugar} + 2\text{Cu}^{2+} + 5\text{OH}^- \rightarrow \text{Aldonic acid} + \text{Cu}_2\text{O} + 3\text{H}_2\text{O}$$

In this reaction, the reducing sugar is oxidized to an aldonic acid by losing electrons, while the copper (II) ions are reduced to copper (I) oxide (Cu2O) by gaining electrons. The copper (I) oxide forms a reddish-brown precipitate that indicates a positive result for the presence of reducing sugars. The aldonic acid is a carboxylic acid that has an aldehyde group at one end and a hydroxyl group at the other end.

If the Fehling’s solution is heated with a non-reducing sugar or in the absence of any sugar, no reaction takes place and the solution remains deep blue in color. This indicates a negative result for the presence of reducing sugars.

Some ketone sugars, such as fructose and alpha-hydroxy-ketones, can also give a positive result in Fehling’s test because they can tautomerize to form aldehydes under alkaline conditions. Tautomerization is a process in which a molecule can switch between two different structures that have the same chemical formula but different arrangements of atoms. In this case, the ketone group can change to an aldehyde group by shifting a hydrogen atom and a double bond.

The principle of Fehling’s test can be summarized as follows:

• Fehling’s test is used to detect and differentiate between reducing and non-reducing sugars.
• Reducing sugars can oxidize copper (II) ions to copper (I) oxide, forming a reddish-brown precipitate.
• Non-reducing sugars cannot oxidize copper (II) ions and leave the solution deep blue in color.
• Some ketone sugars can also give a positive result because they can tautomerize to form aldehydes under alkaline conditions.

The reaction involved in Fehling’s test depends on the type of carbohydrate present in the sample. The carbohydrates that can act as reducing sugars are those that have free or potentially free carbonyl groups (aldehyde or ketone). These include monosaccharides (such as glucose and fructose) and some disaccharides (such as maltose and lactose). Non-reducing sugars are those that do not have free or potentially free carbonyl groups, such as sucrose and starch.

The Fehling’s solution consists of two components: Fehling’s solution A and Fehling’s solution B. Fehling’s solution A is a blue-colored solution of copper (II) sulfate (CuSO4), which provides the copper (II) ions for the reaction. Fehling’s solution B is a clear solution of potassium sodium tartrate (KNaC4H4O6) and sodium hydroxide (NaOH), which acts as a complexing agent and a base, respectively.

When the two solutions are mixed together, they form a deep blue-colored complex of copper (II) ions and tartrate ions, called bistartarocuprate (II) complex. This complex has the formula 2- and can be written as:

$$\text{Cu}^{2+} + 2\text{OH}^- + \text{C}_4\text{H}_4\text{O}_6^{2-} \rightarrow \text{}^{2-} + \text{H}_2\text{O}$$

When the Fehling’s solution is heated with a reducing sugar, the bistartarocuprate (II) complex acts as an oxidizing agent and oxidizes the aldehyde or ketone group of the sugar to a carboxylic acid group. In the process, the copper (II) ions of the complex are reduced to copper (I) ions, which form an insoluble reddish-brown precipitate of copper (I) oxide (Cu2O). The overall reaction can be written as:

$$\text{}^{2-} + \text{reducing sugar} \rightarrow \text{Cu}_2\text{O} + \text{carboxylic acid} + \text{C}_4\text{H}_4\text{O}_6^{2-}$$

The amount of copper (I) oxide formed is proportional to the amount of reducing sugar present in the sample. Therefore, the intensity of the reddish-brown color indicates the degree of reduction.

If the Fehling’s solution is heated with a non-reducing sugar, no reaction occurs and the blue color of the bistartarocuprate (II) complex remains unchanged.

Some ketone sugars, such as fructose and alpha-hydroxy-ketones, can also react with Fehling’s solution because they can tautomerize to form aldehyde groups under alkaline conditions. However, the reaction is slower and less complete than with aldehyde sugars. The reaction can be written as:

$$\text{}^{2-} + \text{keto sugar} \rightarrow \text{keto-enol tautomerization} \rightarrow \text{}^{2-} + \text{aldo sugar}$$

$$\text{}^{2-} + \text{aldo sugar} \rightarrow \text{Cu}_2\text{O} + \text{keto acid} + \text{C}_4\text{H}_4\text{O}_6^{2-}$$

The keto acid formed in this reaction can further undergo decarboxylation to yield shorter chains of acids.

To perform Fehling’s test, you will need the following reagents, materials and equipment:

• Reagents: Fehling’s solution A and B, which are prepared by dissolving copper sulfate and potassium sodium tartrate in water, respectively. You will also need the sample solution that you want to test for reducing sugars. The sample should have a concentration of 5% (w/v).
• Materials: Pipettes, test tubes, test tube stand and a vortex mixer.
• Equipment: Water bath.

The procedure of Fehling’s test involves mixing equal volumes of Fehling’s solution A and B just before use and adding a few drops of the mixture to the sample solution in a test tube. The test tube is then heated in a water bath for a few minutes and the color change is observed. A positive result is indicated by the formation of a reddish-brown precipitate of cuprous oxide, while a negative result is indicated by the persistence of the deep blue color of the Fehling’s solution.

• Take 1 ml of a given sample in a clean, dry test tube. The concentration of the test samples should be 5% (w/v).
• Take control of 1 ml of distilled water in another tube.
• Add about 2-3 drops of Fehling’s reagent to both the tubes and mix them in a vortex.
• Keep the test tubes in the water bath for 1-2 minutes.
• Observe the appearance of color in the test tubes.
• Note down the appearance of color seen in the test tubes.

The procedure is simple and straightforward. It involves mixing the sample with Fehling’s reagent and heating it in a water bath. The color change indicates whether the sample contains reducing sugars or not. The control tube with distilled water serves as a negative control to compare the results.

• The result of Fehling’s test depends on the appearance of a reddish-brown precipitate of cuprous oxide (Cu2O) in the test tube after heating the sample with Fehling’s solution.
• The presence of the reddish-brown precipitate indicates a positive result and confirms that the sample contains reducing sugars. Reducing sugars are carbohydrates that can reduce copper (II) ions to copper (I) ions in an alkaline environment.
• The intensity of the color of the precipitate may vary depending on the concentration and type of reducing sugar present in the sample. For example, glucose gives a more intense color than maltose, and fructose gives a more intense color than glucose.
• The absence of the reddish-brown precipitate or the persistence of the deep blue color of Fehling’s solution indicates a negative result and suggests that the sample does not contain reducing sugars. Non-reducing sugars are carbohydrates that cannot reduce copper (II) ions to copper (I) ions in an alkaline environment.
• A negative result may also occur if the sample contains very low concentrations of reducing sugars or if the sample is acidic and prevents the oxidation of copper (II) ions. In such cases, it may be necessary to neutralize or dilute the sample before performing the test.
• Fehling’s test can be used to qualitatively detect and differentiate between different types of carbohydrates based on their ability to reduce copper (II) ions. However, it cannot be used to quantify the amount of reducing sugars present in a sample or to identify specific carbohydrates. For these purposes, other methods such as Benedict’s test, Barfoed’s test, or chromatography may be used.

Fehling’s test is a simple and reliable method to detect and differentiate between reducing and non-reducing sugars in various samples. Some of the uses of Fehling’s test are:

• To identify carbohydrates in food products: Fehling’s test can be used to determine the presence and amount of carbohydrates in food products such as honey, milk, fruit juices, etc. This can help to assess the nutritional value and quality of the food products.
• To distinguish between aldehydes and ketones in organic compounds: Fehling’s test can be used to distinguish between aldehydes and ketones in organic compounds as aldehydes are oxidized by Fehling’s solution while ketones are not. This can help to identify the functional groups and structures of organic compounds.
• To detect glucose in urine: Fehling’s test can be used to detect glucose in urine as glucose is a reducing sugar that reacts with Fehling’s solution. This can help to diagnose diabetes mellitus, a condition where the blood glucose level is abnormally high and glucose is excreted in urine.
• To measure the concentration of reducing sugars in solutions: Fehling’s test can be used to measure the concentration of reducing sugars in solutions by titrating the solution with Fehling’s solution until the end point is reached. The end point is indicated by the disappearance of the blue color or the formation of a brick-red precipitate. The amount of Fehling’s solution used can be used to calculate the concentration of reducing sugars in the solution.

• Fehling’s test cannot detect all types of reducing sugars. Some sugars, such as sucrose and maltose, do not react with Fehling’s solution or react very slowly. This can lead to false negative results or inaccurate quantification of reducing sugars.
• Fehling’s test cannot distinguish between different types of reducing sugars. For example, glucose and fructose both give a positive result with Fehling’s test, but they have different structures and properties. Therefore, Fehling’s test cannot be used to identify the specific sugar present in a sample.
• Fehling’s test is sensitive to the concentration and temperature of the sample and the reagent. If the sample is too dilute or the reagent is too old, the reaction may not occur or may be incomplete. If the temperature is too high or too low, the reaction rate may vary or the precipitate may dissolve. Therefore, Fehling’s test requires careful control of these factors to ensure reliable results.
• Fehling’s test can be affected by interfering substances present in the sample. Some substances, such as ascorbic acid (vitamin C), uric acid, and some amino acids, can also reduce copper (II) ions and produce a reddish-brown precipitate. This can lead to false positive results or overestimation of reducing sugars. Therefore, Fehling’s test should be performed on pure samples or after removing interfering substances by appropriate methods.

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