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Qualitative Tests for Carbohydrates (Biochemistry)

Qualitative tests for carbohydrates (biochemistry) post we briefly summarise about: principle, reagents requirements, procedure, result, application and limitations of Qualitative tests for carbohydrates.

Carbohydrates are the most frequent and one of the carbon, hydrogen, and oxygen-based elements of mammalian bodies.

Carbohydrates are polyhydroxy aldehydes or ketones chemically. Monosaccharide (one molecule C6H12O6), Oligosaccharide (two to ten molecules), and Polysaccharide (more than ten molecules) are the three types of sugars (above 10 molecules).

Monosaccharide: Glucose, Galactose, Fruc­tose are reducing sugars. Disaccharide: Sucrose, Lactose and Mal­tose are non-reducing sugars. Polysaccharide: Starch, Cellulose, Amylopectin, Chitosan.

Qualitative Tests for Carbohydrates (Biochemistry)

Materials Required

  1. Test Tubes
  2. Test Tube Stand
  3. Pipettes
  4. Dropper
  5. Glass Rod
  6. Microscope
  7. Glass Slides
  8. Test Tube Holder
  9. Spatula
  10. Blotting Paper

Chemicals Required

  1. Distilled water
  2. Concentrated hydrochloric acid
  3. Sodium hydroxide
  4. Concentrated Sulphuric acid
  5. Iodine solution
  6. Phenylhydrazine hydrochloride
  7. Sodium acetate
  8. Glacial acetic acid
  9. Concentrated nitric acid
  10. Phenylhydrazine hydrochloride
  11. Sodium acetate
  12. Glacial acetic acid.

Identification

Molisch’s test

Molisch’s test is a sensitive qualitative tests for carbohydrates, named after Austrian botanist Hans Molisch, for the presence of carbohydrates, based on the dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to produce an aldehyde, which condenses with two molecules of a phenol, resulting in a violet ring.

Principle

In the presence of sulphuric acid, the sugars undergo dehydration to furfural or hydroxymethyl furfural, which condenses with α-naphthol (1-hydroxy naphthalene), resulting in the formation of a purple coloured ring. The sulphuric acid also brings about the hydrolysis of glycosidic bonds of oligo and polysaccharides.

Reagent

Molisch’s [α-naphthol reagent (5% w/v in ethyl alcohol)]: Dissolve 5 g of α-naphthol crystals in 100 mL ethyl alcohol.

Procedure

2-3 drops of Molisch’s reagent must be added to a small amount of the analytes in a test tube and mixed well. Now, a few drops of concentrated sulphuric acid must be added drop-wise along the walls of the test tube to facilitate the formation of a layer and avoid mixing. The development of a purple ring at the layer formed by the concentrated acid is a positive indicator for Molisch’s test.

Result

A purple ring appears at the interphase of sugar solution and the conc. sulphuric acid. If no purple or reddish-purple colour arises, the given analytes does not contain any carbohydrate.

Iodine test

Iodine test is a qualitative tests for carbohydrates, it used to distinguish mono or disaccharides from certain polysaccharides like amylase, dextrin, and glycogen.

Principle

Molecular iodine reacts with polysaccharides forming coloured adsorption complexes. Variations in the colour of the polysaccharide-iodine complex is observed (starch gives blue colour, while glycogen forms a red brown colours).

Reagent

Prepare a 3% (w/v) solution of potassium iodide in distilled water. Add few crystals of iodine until the solution becomes deep yellow in colour.

Procedure

1 mL of a given sample should be placed in a clean, dry test tube. In a separate tube, control 1 mL of pure water. Add a few drops of iodine solution to both tubes and vortex them together. Examine how the colour appears in the test tubes. In a water bath, heat the test tubes until the colour fades. Remove the test tubes from the oven to cool.

Result

If the colour of the solution changes when you add iodine, it means the solution contains polysaccharide. The colour formed is used to determine the polysaccharide’s nature. The presence of amylase or starch in the solution is indicated by the appearance of a blue tint. Dextrin is present if a reddish-purple tint emerges. Glycogen is present if a reddish-brown tint emerges.

Tests for Reducing Sugar

Fehling’s test

Fehling’s solution, also known as Fehling’s reagent, is a chemical reagent used to differentiate between aldehydes and ketones other than α-hydroxy ketone. In practise, it is used to determine the presence of reducing and non-reducing sugars in carbs. Fehling’s test is the test that is used for this purpose. For aromatic aldehydes, Fehling’s solution is ineffective in qualitative tests for carbohydrates.

Principle

Fehling’s test is based on the same idea as Benedict’s test. When aldehydes are introduced to Fehling’s solution, the bistartratocuprate (II) complex quickly oxidises them. Copper (II) ions are reduced to copper (I) ions during this process, resulting in a red copper (I) oxide precipitate (Cu2O). A good result is indicated by the presence of red precipitate.

Reagent

Fehling’s reagent A:  Dissolve 6.92 grams of cupric sulphate in distilled water and make up the volume to 100 mL in a volumetric flask. Store this reagent in a reagent bottle.

Fehling’s reagent B: Dissolve 34.6 grams of sodium potassium tartrate and 25 grams of potassium hydroxide in distilled water and make up the volume to 100 mL with distilled water and store in a reagent bottle.

Procedure

Take 1ml of sample in dry test tube. Take 1ml of distilled water in another tube as control. Add 1ml of Fehling’s reagent (A and B) to all the tubes. Keep in boiling water bath for 1-2 minutes. Make observations and record if there is any development of red precipitate.

Result

The appearance of a reddish-brown precipitate indicates a positive result and the presence of reducing sugars (glucose, fructose, lactose). The absence of the reddish precipitate or the appearance of deep blue color indicates a negative result and lack of reducing sugars (sucrose, starch).

qualitative tests for carbohydrates

Qualitative Tests for Carbohydrates

Benedict’s test

Benedict’s test is a straightforward chemical procedure for detecting reducing sugars (qualitative tests for carbohydrates). Carbohydrates with a free aldehyde or ketone functional group in their chemical structure are known as reducing sugars.

Principle

Benedict’s test is carried out by heating the reducing sugar in the presence of Benedict’s reagent. The presence of alkaline sodium carbonate transforms sugar to enediols, a powerful reducing agent. Due to the development of cuprous oxide, the mixture will change colour from blue to brick-red precipitate during the reduction phase (Cu2O). Cupric (Cu2+) or copper (I) copper is converted to cuprous (Cu+) or copper (I) copper (II). Cuprous oxide, which is red in colour, is insoluble in water and must be separated. When the sugar content is high, the colour turns crimson and the amount of the precipitate grows.

Reagent

One litre of Benedict’s Solution can be prepared from 100 g of anhydrous sodium carbonate, 173 g of sodium citrate and 17.3 g of copper (II) sulfate pentahydrate.

Procedure

2 mL (10 drops) Benedict’s reagent, pipette into three clean and dry test tubes. In each test tube containing Benedict’s reagent, add approximately 1ml of each of the test solutions and water. Heat the mixture directly over the flame or in the test tubes in a boiling water bath for 3-5 minutes. Look for colour changes in the solution or the production of precipitate in the test tubes.

Result

Positive Benedict’s test: color change from blue to brick red precipitate (glucose), Negative Benedict’s test: no change in color (sucrose) and water.

Barfoed test

The barfoed test is a qualitative tests for carbohydrates used to determine whether or not monosaccharide is present. It is determined via the reduction of copper (II) acetic acid to copper (I) oxide (Cu2O), which results in a brick red mixture.

Principle

The basis of Barfoed’s test reaction is the reduction of cupric acetate by reducing monosaccharides and disaccharides. When cupric acetate is converted to cuprous oxide, a brick red precipitate forms.

Reagent

Barfoed’s reagent: 0.33M solution of copper acetate is added to 1% acetic acid. The freshly prepared reagent should be used for the assay.

Procedure

1 mL of a given sample should be placed in a clean, dry test tube. If disaccharides are employed, their concentration should not exceed 1% (w/v). In a separate tube, control 1 mL of pure water. Add a few drops of Barfoed’s reagent to both tubes and vortex them together. Allow 1-2 minutes for the test tubes to soak in the water bath. Boiling should not last longer than 2 minutes, or the disaccharides may hydrolyze into monosaccharides, resulting in a positive result.

Result

Brick Red precipitation within 5min boiling in case of a monosaccharide, while 7-12 min for disaccharide. This delay in the development of the color of in case of disaccharides is due to slow reduction of disaccharide during the reaction.

Seliwanoff’s Test

Seliwanoff’s test is a qualitative tests for carbohydrates that distinguishes between the sugars aldose and ketose. The ketone/aldehyde usefulness of ketoses distinguishes them from aldoses.

Principle

Ketosugars (ex. Fructose) when exposed to acid medium undergo dehydration forming hydroxymethyl furfural more rapidly than aldohexoses. This furfural condenses with resorcinol (m-dihydroxy benzene) to produce a deep pink colour molecule. This test is helpful to distinguish between aldoses and ketoses.

Reagent

Resorcinol Solution (0.5%): Weigh 0.5 g of resorcinol in about 40 ml of distilled water. Add 35 ml of concentrated HCl (12 N) to it and make up the volume to 100 ml using distilled water.

Procedure

To begin the test, fill a test tube halfway with Seliwanoff’s reagent (5 mL). Make sure the amount of reagent you’re pouring into the test tube doesn’t exceed 5 ml by measuring it first. Pick up the material that will be tested, measure 1 ml of it, and pour it into the test tube. The solution should be heated using hot water in the third stage. Boil the solution for 5 minutes in boiling water, and then wait for the results. After some time has passed, you will see that the colour of the test tube has changed.

Result

Positive Seliwanoff’s Test: If the colour changes to red, the Seliwanoff’s Test is affirmative, indicating keto sugar (Fructose and Sucrose) is present in the solution.

Negative Seliwanoff’s Test: If no red colour or a faint pink tint appears, your result is negative, indicating that Aldose sugar (Glucose) is present in the solution.

Bial’s test

Bial’s test is a qualitative tests for carbohydrates for the presence of pentoses. It is named after Manfred Bial, a German physician. The components include orcinol, hydrochloric acid, and ferric chloride. A pentose, if present, will be dehydrated to form furfural which then reacts with the orcinol to generate a coloured substance.

Principle

This test is used to distinguish between pentose monosaccharide and hexose monosaccharide. Bial’s reagent contains concentrated HCl as a dehydrating acid, orcinol and ferric chloride as condensation reagent. The test reagent dehydrates pentoses to form fufural and dehydrates hexoses to form 5-hydroxymethyl fufural, fufural reacts with orcinol and ferric chloride to produce blue-green complex, while 5-hydroxymethyl fufural produce muddy-brown color complex.

Reagent

300 mg orcinol is dissolved in 5 mL ethanol. Add 3.5 ml of this mixture to 100ml of 0.1% solution of FeCL3.6H2O. The resulting reagent should be stored in a dark bottle and utilised within a few hours.

Procedure

In clean dry test tube add 1 ml of 5% ribose solution (pentose). In the second test tube add 1 ml of 5% glucose solution (hexose). For each tube add 2.5 ml of Bial’s reagent and mix well. Keep both tubes in boiling water bath 10 minutes and allow the tubes to cool down to room temperature and measure the optical density of the solutions at 620 nm against a blank.

Result

The presence of a blue-green complex in the sample shows the presence of pentoses. The concentration of ribose sugar in the sample can be determined using the graph. RNA detection can also be interpreted in the same way.

Mucic Test

This is a galactose-specific qualitative tests for carbohydrates. A sugar is oxidised to carboxylic acid by hot nitric acid. To produce dicarboxylic acids, aldoses are oxidised at both ends of the ring-opened form.

Principle

Monosaccharides like galactose, (galactose containing sugars such as lactose gives positive response to this reaction). The presence of strong acids like nitric acid, produce saccharic acids. The saccharic acid formed is insoluble and form clear crystals. This acid derivative is known as galactaric or meso-galactaric acid (mucic acid), thus the name for the test.

Procedure

Mix 3 drops of the carbohydrate solution (galactose and lactose) and 3 drops of the concentrated Nitric Acid on a glass slide. Pass the mixture over a small flame until it is almost dry. Cool the mixture at room temperature; examine the crystals under the microscope.

Result

The results obtained were insoluble precipitate or crystals which indicate the presence of a galactose. The results viewed under the microscope with high power objective were rectangular in shape and only a few in numbers. The result of the experiment yield to insoluble precipitate which is a positive result for Mucic Acid Test

Test for sucrose

Hydrolysis Test

Sucrose gives a positive test as it is a disaccharide consisting of fructose and glucose. Generally, 6M HCl is used to run this test.

Principle

Sucrose does not reduce Benedict’s reagent but hydrolysed and neutralized product of sucrose will answer for Benedict’s reagent. This indicating the presence of reducing sugar in the hydrolysis of sucrose.

Procedure

To 5ml of sample solution add 3 drops of conc. hydrochloric acid and boil for 1 minute. Cool and neutralize with 20% sodium carbonate solution till the effervescence ceases to the neutralized solution. Add 5ml of Benedict’s reagent and boil for 2 minutes and cool.

Result

The results obtained were Red colour precipitate  which indicate the presence of a Sucrose.

Confirmatory test for Carbohydrates

Osazone test

A chemical qualitative tests for carbohydrates for detecting decreasing sugars is the osazone test. This test can even distinguish between different reducing sugars based on the appearance time of the complex.

Principle

Reducing sugars upon reaction with Phenylhydrazine produces osazones, which are the characteristic derivatives of carbohydrates. These osazone derivatives have definite crystalline shape. These crystals made it easy and possible to confirm the type of carbohydrate. Sugars lacking free anomeric hydroxyl group (non-reducing sugars) do not respond to this test. Whereas glucose, fructose and mannose produces similar type of osazone, i.e., glucosazone. An osazone crystal differs from the other with respect to time of crystallization, crystal shape and melting point.

Procedure

Fill a clean, dry test tube with 5 mL of test solution. To the test tube, add 0.3 g of osazone mixture and five drops of glacial acetic acid. To dissolve all of the ingredients, thoroughly mix everything together and gently warm the test tube in the water bath if necessary. Keep the test tube in boiling water and watch the crystals form at different times. Examine the crystal’s form under a microscope at a modest magnification.

Result

Qualitative tests for carbohydrates: Confirmatory test for Carbohydrates

Different sugars can be identified based on the shape and structure of the crystals as well as their appearance time. Maltose crystallises into distinct petal-shaped crystals in this test. Lactose, on the other hand, forms puff-shaped crystals. Galactose, another material, will produce phombi-plate shaped crystals.

Further Readings

Reference

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