Qualitative analysis of Carbohydrate

The qualitative analysis of carbohydrate is the significant test for the detection and classification of the carbohydrate on the basis of colour change followed by the chemical reaction.

Carbohydrate is an organic biomolecule which is having the chemical formula Cm (H2O) n. The carbohydrate consists of three atoms viz. carbon, hydrogen and oxygen as the main constituent, where the ratio between the hydrogen and the oxygen is 2:1.

Carbohydrates are the major source of energy for all the living organisms which classifies majorly into mono, di, oligo and polysaccharides.

Content: Qualitative analysis of Carbohydrate

  1. Definition of Qualitative analysis of Carbohydrate
  2. Methods for Qualitative analysis of Carbohydrate

Definition of Qualitative analysis of Carbohydrate

The qualitative analysis of carbohydrate is detected on the basis of utilization of the reagent and the reaction between the test sample and reagent. On the reaction with the test sample with the chemical reagents, gives a significant colour which detects the presence or absence of carbohydrate.

Methods for Qualitative analysis of Carbohydrate

There are several methods to detect the presence of carbohydrates which are of the following types:

Molisch’s Test

It is the most common method for the detection of carbohydrates. Molisch’s test makes the use of Molisch’s solution (contains α-naphthol in 95% alcohol) and concentrated H2SO4.

Principle: Molisch’s test detects the carbohydrate presence, on the basis of dehydration reaction where the carbohydrate in the sample is dehydrated into aldehyde by the addition of concentrated H2SO4.

The aldehyde formed is either furfural (produced by the dehydration of pentoses or pentosans) or hydroxymethylfurfural (produced by the dehydration of hexoses or hexosans). Then the α-naphthol in the molisch reagent reacts with the aldehyde and form purple condensation product.

Procedure:

  1. Take a sample and add water to make 2ml of solution.
  2. Add 2drops of Molisch’s reagent.
  3. Then, pour 5ml of concentrated H2SO4 from the side of the test tube in an inclined position.
  4. Observe the colour change in the tube.

Result interpretation:

Molisch test

Positive result: Violet ring appears at the junction of two layers i.e. sugar and acid.

Negative result: Green or brown colour appears.

Application: It detects the presence of all carbohydrates.

Benedict’s Test

This method is also used for the analysis of carbohydrate, as reducing sugar. Reducing sugar consists of a free aldehyde or ketone group. Benedict’s test makes the use of Benedict’s solution as a reagent which contains copper sulphate, sodium citrate, sodium carbonate with a pH of 10.5.

Principle: The reducing sugar will reduce into the enediols by the reaction with alkaline reagent i.e. Benedict’s solution. The reducing sugar gives green to brick red colour precipitate depending upon the sugar concentration.

The colour change is due to the reduction reaction of copper (II) to copper (I) in the solution to form a red coloured precipitate. Which is water insoluble. The sodium carbonate provides the alkaline condition or maintains the alkalinity in the solution for the redox reaction to occur. Sodium citrate binds with the copper- II ions to prevent the reduction into copper I during storage.

Procedure:

  1. Prepare 1ml of a solution by adding a test sample and water.
  2. Add 2ml of Benedict’s solution in the test tube.
  3. Heat the solution in the water bath for 3minutes.
  4. Observe the test tube for the colour change.

Result interpretation:

benedict's test

Positive result: Green to brick-red precipitate forms.

Negative result: Colour remains unchanged.

Application: It detects the presence of reducing carbohydrates.

Fehling’s Test

This method is also used for the analysis of reducing sugar. It makes the use of Fehling’s solution A and as a reagent. Fehling’s solution A contains copper sulphate pentahydrate in 1L of distilled water. Fehling’s solution B contains potassium sodium tartrate and sodium hydroxide with 1L of distilled water.

Principle: In the Fehling’s test, a reduction reaction occurs in between the aldehyde or keto groups of the reducing sugar and the alkaline cupric hydroxide which on heating is reduced into cuprous oxide. This cuprous oxide gives a brick red coloured precipitate in the solution.

Procedure:

  1. Add 2ml of the test sample in a test tube.
  2. Then add Fehling’s A and B solution in equal proportion.
  3. After that, place the test tube in a hot water bath for 3minutes.
  4. At last, observe the colour change in a test tube.

Result interpretation:

fehling's test

Positive result: Brick-red precipitate forms.

Negative result: Colour remains unchanged.

Application: It detects the presence of reducing carbohydrates.

Barfoed’s Test

This method is also used for the analysis of monosaccharide reducing sugar. Barfoed’s test makes the use of Barfoed’s solution which contains copper acetate in the dilute acetic acid with a pH of 4.6.

Principle: In Barfoed’s test, the reducing monosaccharide is oxidized by the copper ion in the solution to form a carboxylic acid and copper (I) oxide which results into the formation of a red coloured precipitate.

Procedure:

  1. Add 1ml of the test sample in a test tube.
  2. Then add 2ml of Barfoed’s solution in the test tube.
  3. Place the tube in a water bath for boiling up to 1min.
  4. At last, observe the colour change in a test tube.

Result interpretation:

barfoed's test

Positive result: Gives red coloured precipitate.

Negative result: Colour remains unchanged.

Application: Used for the detection of reducing monosaccharides like glucose.

Bial’s Test

It is most commonly used for the detection of pentose sugars or pentose. By this method, we can also differentiate pentoses from the hexoses by the means of colour change. Bial’s test makes the use of Bial’s solution which contains orcinol, hydrochloric acid and ferric chloride.

Principle: In the Bial’s test, pentoses react with the Bial’s reagent and convert it into furfural derivatives as a result of dehydration by HCl. Then, orcinol and furfural condense in the presence of ferric ion to form a green coloured compound.

Procedure:

  1. Take 2ml of test solution in the test tube.
  2. Then, add 5ml of Bial’s reagent.
  3. Heat the solution in a water bath for 1min.
  4. Cool and observe the solution for the colour change.

Result interpretation:

bial's test

Positive result: Gives green coloured precipitate.

Negative result: Gives green coloured precipitate

Application: Used for the detection for the pentose monosaccharides like ribose.

Seliwanoff’s Test

It is most commonly used for the detection of Keto-sugars or Ketoses. By this method, we can also differentiate Ketoses from the Aldoses by the means of colour change. Seliwanoff’s test makes the use of Seliwanoff’s reagent which contains 0.5g of resorcinol per litre in 10% HCl.

Principle: In the Seliwanoff’s test, ketoses react with the HCl of the Seliwanoff’s reagent which yields furfural derivatives as a result of dehydration. Then, resorcinol and furfural react to give a deep red colour to the solution.

Procedure:

  1. Take 1ml of test solution in the test tube.
  2. Then, add 3ml of Seliwanoff’s reagent.
  3. Heat the solution in a water bath for 1min.
  4. Cool and observe the solution for the colour change.

Result interpretation:

seliwanoff's test

Positive result: Gives a deep red colour to the solution.

Negative result: Colour remains unchanged.

Application: Used for the detection of fructose, sucrose etc.

Iodine Test

It is widely used for the detection of starch in the solution. In an iodine test, iodine acts as an “Indicator”.

Principle: In the Iodine test, an iodine solution reacts with the starch (containing a polymer of α-amylose and amylopectin. This reaction of starch and iodine results in a starch-iodine complex which gives blue-black colour to the solution.

Procedure:

  1. Take 2ml of test solution in the test tube.
  2. Then, add 2 drops of iodine solution.
  3. Observe the solution for the colour change.

Result interpretation:

iodine test

Positive result: Gives blue-black colour to the solution.

Negative result: Gives a brown-yellow colour to the solution.

Application: Used for the detection of polysaccharides like starch.

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