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

Seliwanoff’s test is a chemical test that is used to distinguish between two types of sugars: ketoses and aldoses. Ketoses are sugars that have a ketone group (C=O) in their structure, while aldoses are sugars that have an aldehyde group (CHO) in their structure. Examples of ketoses are fructose, ribulose, and xylulose. Examples of aldoses are glucose, ribose, and xylose.

Seliwanoff’s test is based on the fact that ketoses react faster and more intensely with a reagent composed of resorcinol and concentrated hydrochloric acid (HCl) than aldoses. The reagent causes the dehydration of the sugars and the formation of a colored complex called xanthenoid. The color of the complex ranges from pink to cherry red depending on the type and concentration of the sugar. Ketoses produce a deep cherry red color within a minute, while aldoses produce a faint pink color after several minutes.

Seliwanoff’s test can be used to detect the presence of ketohexoses (six-carbon ketoses) in a given sample, such as fructose. It can also be used to differentiate ketoses from aldoses in a mixture of sugars. However, it cannot identify the specific type of ketose or aldose present in the sample. For that purpose, other tests are required.

The main objectives of Seliwanoff’s test are:

• To detect the presence of ketohexoses in a given sample. Ketohexoses are six-carbon sugars that have a ketone group at the second carbon atom, such as fructose and sorbose.
• To distinguish ketoses from aldoses. Aldoses are sugars that have an aldehyde group at the first carbon atom, such as glucose and galactose. Aldoses and ketoses have different chemical properties and biological functions.
• To identify the type of ketose based on the reaction time and intensity of color. Different ketoses react with Seliwanoff’s reagent at different rates and produce different shades of red color. For example, fructose reacts faster and gives a deeper red color than sorbose.

The principle of Seliwanoff’s test is based on the difference in the rate of dehydration of ketoses and aldoses in the presence of concentrated acid. Ketoses are more rapidly dehydrated than aldoses and form a compound called 5-hydroxymethyl furfural (HMF). This compound reacts with resorcinol, which is the main component of Seliwanoff’s reagent, to produce a cherry red-colored complex called xanthenoid. The intensity and the time of appearance of the color depend on the type and concentration of the sugar present in the sample. Ketohexoses, such as fructose, give a strong positive result within a minute, while aldohexoses, such as glucose, give a weak positive result after several minutes. Other carbohydrates that contain fructose as a component, such as sucrose and inulin, also give a positive result for this test. The reaction can be summarized as follows:

The principle of Seliwanoff’s test can be used to distinguish between ketoses and aldoses, as well as to estimate the concentration of ketoses in a given sample by measuring the absorbance of the colored complex at 480 nm. A standard curve can be prepared by using known concentrations of fructose and comparing them with the unknown sample. However, this method has some limitations, such as interference from other sugars and false-positive results due to prolonged boiling or high concentration of glucose. Therefore, other specific tests may be required to confirm the identity and quantity of the sugar present in the sample.

The reaction in Seliwanoff’s test involves the dehydration of ketoses and the condensation of the resulting furfural with resorcinol. The reaction can be summarized as follows:

• The test sample is mixed with Seliwanoff’s reagent, which consists of resorcinol and concentrated hydrochloric acid (HCl).
• The mixture is heated in a water bath for a short time (usually one minute).
• The heating causes the acid hydrolysis of polysaccharides and oligosaccharides in the sample to yield monosaccharides.
• Ketoses, such as fructose, are more rapidly dehydrated than aldoses, such as glucose, in the presence of concentrated acid. The dehydration reaction removes water molecules from the sugar and forms a cyclic compound called 5-hydroxymethyl furfural (HMF).
• HMF reacts with two molecules of resorcinol in a series of condensation reactions to produce a complex compound called xanthenoid. Xanthenoid has a deep cherry red color and is soluble in water.
• Aldoses may also react slightly with resorcinol to form a faint pink to cherry red color, but this reaction is much slower and requires prolonged heating.
• The intensity and timing of the color formation indicate whether the sample contains ketoses or aldoses. A positive result for ketoses is indicated by a rapid formation of a deep cherry red color, while a negative result for aldoses is indicated by no color change or a delayed formation of a faint color.

To perform Seliwanoff’s test, you will need the following:

• Reagent: Seliwanoff’s reagent is prepared by dissolving 0.05% resorcinol (m-hydroxybenzene) in 3 N HCl. To make 100 ml of this reagent, you will need to dissolve 50 mg of resorcinol in 33 ml of concentrated HCl and then dilute it with water.
• Test sample: You can use any carbohydrate solution that you want to test for the presence of ketoses. For example, you can use fructose, glucose, sucrose, inulin, etc. The concentration of the test sample should be around 1% to 5%.
• Distilled water: You will need distilled water as a blank or negative control to compare the results with the test sample.
• Materials required: You will need some basic laboratory materials such as test tubes, test tube stand, pipettes, etc. to perform the test.
• Equipment: You will need a water bath to heat the test tubes for a specific time and observe the color change.

• Take two clean, dry test tubes and add 1 ml of the test sample in one test tube and 1 ml of distilled water in another as blank.
• Add 2 ml of Seliwanoffs’ reagent to both the test tubes.
• Keep both the test tubes in a water bath for 1 min.
• Observe the formation of color and note it down.

The procedure of Seliwanoff’s test is simple and straightforward. It involves the following steps:

1. Preparation of test tubes: Two test tubes are used for this test, one containing the test sample and the other containing distilled water as a blank or control. The test sample can be any carbohydrate solution that needs to be tested for the presence of ketoses. The blank is used to compare the color change with the test sample. Both the test tubes are labeled accordingly.
2. Addition of Seliwanoff’s reagent: Seliwanoff’s reagent is a mixture of resorcinol and concentrated hydrochloric acid. It is added to both the test tubes in equal amounts (2 ml). The reagent acts as a dehydrating and oxidizing agent that reacts with ketoses to form a cherry red-colored complex. The reagent should be added carefully and gently mixed with the test sample or water.
3. Heating in water bath: Both the test tubes are then placed in a water bath at a temperature of about 100°C for 1 minute. The heating time is important as it determines the rate and extent of the reaction. Ketoses react faster than aldoses and produce a cherry red color within 1 minute. Aldoses may react slightly to produce a faint pink to cherry red color if the heating time is prolonged.
4. Observation of color change: After heating, both the test tubes are removed from the water bath and allowed to cool slightly. The color change in both the test tubes is then observed and noted down. A positive result is indicated by the formation of a cherry red-colored complex in the test tube containing the test sample. A negative result is indicated by the absence of such color or the appearance of the color after a prolonged period of time in the test tube containing the test sample.

The procedure of Seliwanoff’s test is summarized in the following table:

• The formation of the cherry red-colored complex indicates a positive result which means that the given sample contains ketoses.
• The absence of such color or the appearance of the color after a prolonged period of time indicates a negative result which means that the test sample doesn’t have ketoses.
• The intensity of the color depends on the concentration and type of ketose present in the sample. For example, fructose gives a more intense color than sucrose or inulin.
• The time required for the color development also varies with different ketoses. For example, fructose gives a positive result within 1 minute, while sucrose and inulin take about 5 minutes to show a faint color.
• The color reaction can be quantified by measuring the absorbance of the solution at 490 nm using a spectrophotometer. A standard curve can be prepared using known concentrations of fructose and used to calculate the unknown concentration of ketose in the sample.

Seliwanoff’s test is a useful method for detecting and distinguishing ketoses from aldoses in various samples. Some of the applications of this test are:

• Seliwanoff’s color reaction is used in the method for the colorimetric determination of fructose in fermentation media. Fructose is a common sugar produced by the fermentation of various substrates by microorganisms. The concentration of fructose in the fermentation broth can be estimated by measuring the intensity of the cherry red color produced by Seliwanoff’s reagent. The color intensity is proportional to the amount of fructose present in the sample. This method can be used to monitor the progress and efficiency of fermentation processes.

• A modified version of this test can be used for the determination of the concentration of ketoses in a given sample. By using a different acid concentration and reaction time, Seliwanoff’s test can be adapted to measure the concentration of other ketoses besides fructose, such as sorbose, tagatose, and ribulose. The modified test involves adding a known amount of resorcinol to the sample and measuring the absorbance at 480 nm after heating for a specific time. The absorbance is then compared to a standard curve prepared with known concentrations of ketoses.

• Seliwanoff’s test can also be used as a qualitative test for some carbohydrates that contain ketoses as their components. Some carbohydrates, such as sucrose and inulin, give a positive result for Seliwanoff’s test because they are hydrolyzed by acid to yield fructose as one of their products. This test can help to identify these carbohydrates from others that do not contain ketoses. However, this test is not specific for these carbohydrates and may give false-positive results with other compounds that react with Seliwanoff’s reagent.

• The high concentration of glucose or other sugar may interfere by producing similar colored compounds with Seliwanoff’s reagent.
• Prolonged boiling can transform glucose to fructose by the catalytic action of acid and form cherry red-complex giving a false-positive result.
• This test is a generalized test and doesn’t distinguish between specific ketoses, and a separate test is required for the particular ketose sugar identification.

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