Introduction to Tests for Carbohydrates

Carbohydrates are biological molecules that are made up of carbon, oxygen, and hydrogen. They are the hydrated carbons having either an aldehydic or a ketonic functional group. They are found in all living organisms. 

Carbohydrates provide energy to the cells upon oxidation. They are also used as energy storage compounds in both animals and plants. They act as monomers or building blocks for making complex biological molecules. Carbohydrates also have some important uses in the industry for the welfare of human beings. Examples of carbohydrates include glucose, ribose, fructose, sucrose, cellulose, starch, etc. 

Carbohydrates present in a solution can be easily identified by performing certain tests in the laboratory. The important tests for carbohydrate detection are as follows. 

Molisch’s Test

It is a screening test for confirming the presence or absence of carbohydrates in a given solution. It is a highly sensitive test for carbohydrates. Monosaccharides, oligosaccharides, and polysaccharides all give positive Molisch’s test. 

Principle

This test is based on the reaction of alpha-naphthol with carbohydrates in the presence of sulfuric acid. The sugars react with alpha-naphthol in an acidic environment to form purple-colored furfural or hydroxymethylfurfural derivatives. The intensity of the color is directly proportional to the amount of carbohydrate present in the solution. 

Apparatus

• Test tube
• Dropper or pipette
• Solution to be tested

Reagent

• Molisch’s reagent: 5% alpha-naphthol solution in ethyl alcohol

Procedure

1. Take 2 ml of the given solution in a test tube
2. Add 1-2 drops of Molisch’s reagent to the above solution
3. Mix the solutions
4. Incline the test tube
5. Add 2 ml sulfuric acid along the side of the test tube

Observations

A violet-colored ring is formed at the junction of the two liquids i.e. solution with Molisch’s reagent and the sulfuric acid. 

Result

The violet ring indicates the presence of carbohydrates in the solution. 

Points to Remember

• To give a positive test, the carbohydrate must have at least five carbons. (It is so because it involves the formation of furfural derivatives that contain five carbon atoms.)
• Impurities in the reagent give green color indicating a false-negative test.
• Oligosaccharides and polysaccharides are first broken down to monosaccharides by acid which then gives the Molisch’s test positive. 
• Proteins and lipids having an attached carbohydrate can also give this test positive.  

Precautions

• Don’t add too much Molisch’s reagent. 
• Don’t pour sulfuric acid directly into the solution. Otherwise, charring of carbohydrates will occur and a black ring will be formed, giving a false negative test. 

Iodine Test

This test is specific for polysaccharides. This test is used to differentiate polysaccharides from the rest of carbohydrates. It is given positive by starch and glycogen. It can also be used to differentiate between glycogen, starch, and cellulose. 

Principle

The iodine test is based on the absorptive properties possessed by large polysaccharide molecules. The glucose chains in most of the polysaccharides are organized to form helices. The space between the turns of the helix can hold small iodine molecules. This is seen with amylase chains found in starch. Glycogen and amylopectin can also absorb these iodine molecules on their surface. The absorptive property of polysaccharides decreases upon heating. 

Blue or red-colored iodine complexes are formed in this test having ill-defined chemical nature.

Apparatus

• Test tube
• Dropper or pipette
• Solution to be tested

Reagent

• Iodine Reagent: 0.5 ml iodine diluted in 5ml distilled water 

Procedure

1. Take 2 ml of the given solution in a test tube
2. Add 2-3 drops of iodine reagent in the above test tube
3. Wait for some time

Observations

When the iodine is added to the solution, the color of the solution changes. It may give the following colors;

• Blue
• Reddish-purple
• Reddish-brown

Result

If the color of the solution changes upon adding iodine, it represents that polysaccharide is present in the solution. The nature of polysaccharides is detected based on the color formed. 

• If blue color appears, amylase or starch is present in the solution
• If reddish-purple color appears, dextrin is present 
• If reddish-brown color appears, glycogen is present

Points to Remember

• The test is highly specific for polysaccharides in the solution
• Cellulose does not give this test, but you will not find cellulose in the solution
• The color disappears on heating and reappears when the solution is cooled
• If the color does not appear upon cooling, it indicates that iodine has vaporized during heating

Precautions

• Don’t add too much iodine to prevent false results

Read more about the Properties of Monosaccharides

Benedict’s Test

It is a test for reducing sugars. Carbohydrates having a free functional group, that is not involved in a glycosidic bond, give this test positive. All monosaccharides and disaccharides except sucrose give positive Benedict’s test. This test is negative for polysaccharides.

Principle

This test is based on the ability of reducing sugars to undergo oxidation in alkaline solutions. In the presence of an alkali, reducing sugars undergo tautomerization to form enediols. These enediols reduce the cupric ions (Cu⁺²) to form cuprous ions (Cu⁺). The cuprous ions from cuprous hydroxide. Upon heating, it is converted to cuprous oxide that forms precipitates. 

The citrate ions present in the reagent release the cuprous ions slowly for reduction and prevent the formation of Cu(OH)₂ until the oxidation-reduction process is completed.

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Solution to be tested

Reagents

Benedict’s Qualitative Reagent is used that contains;

• Copper Sulfate (to provide cupric ions)
• Sodium Carbonate (to make solution alkaline)
• Sodium Citrate (to provide citrate ions)

Procedure

1. Take 5 ml of Benedict’s qualitative reagent in a test tube
2. Add 8 drops of a given solution to the above test tube
3. Mix the solutions
4. Hold the test tube on flame and boil for 2 minutes
5. Allow the solution to cool
6. Look for the precipitates

Observations

The brick-red precipitate is formed at the bottom of the test tube. 

Results

The precipitates of cuprous oxide indicate the presence of reducing sugar in the test tube. 

Points to Remember

• It is also a semi-quantitative test as the color of the precipitate is proportional to the concentration of reducing sugar in the test tube. Maximum concentration that can be tested in 2% at which brick-red precipitates are formed.
• This test is frequently used as a screening test for diabetes mellitus.  
• The test is false positive for ascorbic acid, glutathione, uric acid, etc. 

Precautions

• Adding too much Benedict’s reagent or test solution may give false results. 

Bial’s Test

It is a general test for carbohydrates and is sensitive only to pentoses. Any compound that contains a pentose sugar will give a positive Bial’s test. 

Principle

Pentoses form furfural compounds in the presence of concentrated acid. The furfural compounds formed by pentoses condense with orcinol to form blue-colored compounds.

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Water bath
• Solution to be tested

Reagents

Bial’s reagent is used that is made by dissolving 300 mg of orcinol in 100 ml of concentrated HCL and 0.25 mL ferric chloride solution. 

Procedure

1. Add 3 ml of Bial’s reagent to an empty test tube
2. Add 3 ml of the test solution to the above test tube
3. Heat the test tube in a boiling water bath
4. Allow the solution to cool at room temperature

Observations

A bluish color appears in the test tube upon heating. 

Results

The blue color indicates the presence of pentose sugar in the test solution. 

Points to Remember

• The test is positive for all the compounds that contain pentose sugar like DNA, RNA, etc. 
• Hexoses form a green color with Bial’s reagent.

Precautions

• Don’t allow rapid cooling otherwise, the results may be affected. 

Barfoed’s Test

It is a differentiating test to distinguish between monosaccharides and disaccharides. Barfoed’s test is also based on the reduced ability of sugar. However, sucrose also gives this test positive as it undergoes hydrolysis in the presence of an acid. Monosaccharides give an early positive test while disaccharides give a late positive. 

Principle

Reducing sugar undergo tautomerization in a mildly acidic medium to form enediols. These enediols reduce cupric ions to cuprous ions that form cuprous hydroxide. This cuprous hydroxide is converted to cuprous oxide on heating and precipitates are formed.  

Its principle is similar to Benedict’s test except for the acidic environment. Monosaccharides being strong reducing agents give this test much early.

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Solution to be tested

Reagents

Barfoed’s reagent is used that contains:

• Copper Acetate in Glacial Acetic Acid

Procedure

1. Take 2 ml of Barfoed’s reagent in a test tube
2. Add 2 ml of the test solution to the above test tube
3. Mix the solutions
4. Hold the test tube on flame and boil for minutes
5. Allow to cool at room temperature
6. Look for the precipitates
7. If no precipitates are formed, boil for an additional 10 minutes
8. Allow to cool and look for the precipitates

Observations

1. Red precipitates are formed after the first 5 minutes
2. Red precipitates are formed after additional boiling

Results

1. The formation of red precipitates after the initial first 5 minutes indicates the presence of a monosaccharide
2. If precipitates are formed after 15 minutes, a disaccharide is present in the test solution

Points to Remember

• This test helps to differentiate between monosaccharides and disaccharides
• When the heating period is increased, the disaccharides are hydrolyzed to monosaccharides that give the positive test  

Precautions

• Keep proper track of the boiling time
• Allow gradual cooling at room temperature
• If reheating is necessary, do it after the solution has become cold

Seliwanoff’s Test

This test is used to detect monosaccharides with a ketonic functional group. It is widely used to differentiate fructose, a keto sugar, from glucose and galactose

Principle

This test involves the formation of furfural derivatives by monosaccharides with hydrochloric acid. The furfural derivatives formed by sugar with a ketonic functional group condense with resorcinol to form a chromogen with having cherry-red color. 

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Solution to be tested

Reagents

Seliwanoff’s reagent is used that contains 100 ml of water;

• 50  mg resorcinol
• 33 ml of concentrated HCL

Procedure

1. Take 3 ml of Seliwanoff’s reagent in a test tube
2. Add 1 ml of test solution to the above test tube 
3. Hold the test tube on flame and allow it to boil for 30 seconds
4. Allow to cool at room temperature

Observations

A cherry red color forms in the test tube upon cooling.

Results

The given solution contains a keto-sugar. 

Points to Remember

• Seliwanoff’s test is specific only for hexoses having a ketonic functional group 
• Sucrose also gives a positive test because it is hydrolyzed to glucose and fructose
• It is highly sensitive to sucrose even at a .1% concentration

Precautions

• Prolonged boiling will lead to the conversion of glucose to fructose resulting in a false positive test
• Cooling must be gradual at room temperature

Phloroglucinol test

This test is specifically to detect galactose and lactose in a solution.

Principle

This test also involves the formation of furfural derivates in the presence of concentrated HCL. The furfural derivatives formed by galactose then condense with the phloroglucinol to form a red-colored compound. 

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Solution to be tested

Reagents

Tollen’s reagent is used that contains;

• 10 ml concentrated HCl mixed with 8 ml of 0.5% phloroglucinol

Procedure

1. Take 2 ml of test solution in a test tube
2. Add 2 ml of Tollen’s reagent to the above test tube
3. Mix the two solutions thoroughly
4. Hold the test tube on flame and boil for some time
5. Allow to cool at room temperature

Observations

When the solution is boiled and allowed to cool, it turns yellow to red. 

Results

The change of color to red indicates the presence of galactose in the solution.

Points to Remember

• Lactose also gives this test positive as it is hydrolyzed by acid to yield glucose and galactose. To differentiate between the two, perform Barfoed’s test. 
• The furfural compounds formed by pentoses having a keto group also form a similar red-colored compound with phloroglucinol.  

Precautions

• Boiling for a prolonged time will give a false-positive test due to the conversion of glucose to galactose

Osazone Test

It is a confirmatory test for carbohydrates. It gives you the final inference about the type of carbohydrate present in the solution. Osazone derivative of carbohydrate forms specific crystals that are characteristic of it. The shape of the crystal tells us about the nature of the carbohydrate present.

Principle

Phenyl hydrazine reacts with reducing sugar in an acidic environment at high temperatures to form phenylhydrazone. Phenyl hydrazine further reacts with the sugar on heating and forms crystals of osazone specific to that carbohydrate. 

Apparatus

• Test tube
• Test tube holder
• Dropper
• Pipette
• Stand 
• Spirit or gas lamp
• Light Microscope
• Slides
• Coverslip
• Solution to be tested

Reagents

Osazone mixture is used in this process. It is made by mixing;

• 1 part Phenyl Hydrazine
• 2 parts Sodium acetate
• Few drops of glacial Acetic acid

Procedure

1. Take 5 ml of the given solution in a test tube
2. Add 3 pinches of osazone mixture to the above test tube
3. Mic thoroughly
4. Hold the test  on flame and boil for 5 minutes
5. Check for yellow crystals. If not formed boil further
6. Keep checking after every 5 minutes for yellow crystals
7. Once the crystals are formed, allow the solution to cool at room temperature 
8. Take the crystals out and prepare the slide
9. Observe the slide under the microscope

Observations

Osazone crystals formed when viewed under the microscope have different shapes depending on the type of carbohydrate present.

Results

The following inferences are drawn from the shapes of osazone crystals.

• Needle-shaped/ broom-stick crystals are formed in 5 minutes indicating glucose
• Needle-shaped/ broom-stick crystals are formed in 2 minutes indicating fructose
• Cotton ball-shaped crystals are formed by lactose in 30 minutes
• Sunflower-shaped crystals are formed by maltose in 30 to 40 minutes

Points to Remember

• Osazones are crystalline substances that have a specific structure when viewed under the microscope. 
• They are formed by all reducing sugars.
• Non-reducing sugars do not form osazone crystals unless they are hydrolyzed. 
• Boiling the nonreducing sugars for a longer time releases individual monosaccharides that give the test positive.

Summary

Carbohydrates are the biological molecules that act as the main source of energy for most of the cells. They also perform structural roles. 

The presence of carbohydrates in a solution can be detected by performing the following tests in the laboratory. 

• Molisch’s test, it is positive for all carbohydrates
• Iodine test, it is specific for starch and glycogen
• Benedict’s test, it is positive for reducing sugars
• Bial’s test, it is positive for pentose sugars
• Barfoed’s test, it differentiates between monosaccharides and disaccharides
• Seliwanof’s test, it is positive for monosaccharides with ketonic group
• Phosphoglucirol test, it is positive for galactose or lactose
• Osazone test, it is the confirmatory test in which shape of crystals tell the type of sugar present in the solution

Frequently Asked Questions

How are the carbohydrates in a solution detected?

Carbohydrates are detected in a solution with the help of Molisch’s test. It is a highly sensitive test for carbohydrate detection. Monosaccharides, disaccharides, oligosaccharides and polysaccharides all give a positive Molish test.

How to differentiate between oligosaccharides and polysaccharides in the laboratory?

Oligosaccharides and polysaccharides are differentiated with the help of the Iodine test. The iodine test is positive for starch and glycogen, while negative for oligosaccharides.

Why is Benedict’s test performed?

Benedict’s test is specific for reducing sugars. It is used to differentiate between reducing and non-reducing sugars.

How to differentiate between monosaccharides and disaccharides in the laboratory?

Monosaccharides and disaccharides are distinguished by Barfoed’s test. It is based on the reducing property of sugars. Monosaccharides are confirmed by early positive reactions, while disaccharides give a late positive test.

References

1. Vogel’s Textbook of Quantitative Chemical Analysis, 5th edition
2. Brilliant Biology Student Master Biology Labs
3. https://commons.wikimedia.org/wiki/File:Benedict%27s_test_for_Sugars.png