Objective:

• to detect carbohydrate in the given solution

Principle:

Anthrone test is also another general test for all carbohydrates. In this test also, carbohydrate gets dehydrated when react with conc. H2SO4 to form furfural. This furfural reacts with anthrone to give bluish green colored complex.

Reagents:

• test solution: 5 % Glucose, 5 % Sucrose, 5 % Starch
• Anthrone reagent: 0.2 % anthrone in conc. H2SO4

Procedures

1. Take 1ml of sample in test tube.
2. Take 1ml of distilled water in another tube as control.
3. Add 2ml of anthrone reagent to all the tubes.
4. Mix thoroughly all the content of the tube.
5. Observe for color change in bluish green.

Result interpretation:

• positive test: all carbohydrate give test positive

Anthrone Test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect reducing sugar ( carbohydrate having free aldehyde or ketone functional group)

Principle:

The Reducing sugar under alkaline condition form enediols. Benedict’s solution contains milder alkali Na2CO3. Enediols are powerful reducing agents. They can reduce cupric ions to cuprous ions which is the basis for Benedict’s reaction. The cuprous hydroxide during the process of heating is converted to red cuprous oxide.

Reagents:

• test solutions: 5 % Glucose, 5 % Sucrose
• Benedict’s reagent: CuSO4.5H2O solution with Sodium carbonate and sodium citrate
• Water bath
• Dry test tubes
• Pipettes

Procedures

1. Take 1ml of test sample in dry test tube.
2. Take 1ml of distilled water in another tube as control.
3. Add 2ml of Benedict’s reagent to all the tubes.
4. Keep in water bath for 5 minutes.
5. Look for the development of brick red precipitate.

Result interpretation:

• positive benedict’s test: color change from blue to brick red ppt ( glucose)
• Negative Benedict’s test: no change in color( sucrose)

Benedict’s Test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect reducing sugar
• to distinguish monosaccharides from reducing disaccharides

Principle of Barfoed’s test:

Barfoed’s test is used for distinguishing monosaccharides from reducing disaccharides. Monosaccharides usually react in about 1-2 minute while the reducing disaccharides take much longer time between 7-12 minutes to react with the reagent. Brick red color is obtained in this test which is due to formation of cuprous oxide.

Reagents for Barfoed’s test:

• test solution: 5 % Glucose, 5 % Sucrose, 5 % Maltose, 5 % Lactose, 5 % Starch
• Barfoed’s reagent: cupric acetate in 1% acetic acid
• Water bath
• Dry test tubes
• Pipettes

Procedure of Barfoed’s test:

1. Take 1ml of test sample in dry test tube.
2. Take 1ml of distilled water in another tube as control.
3. Add 2ml of Barfoed’s reagent to all the tubes.
4. Keep in boiling water bath.
5. Look for the development of brick red precipitate.
6. Note the time taken to develop the color.

Result interpretation of Barfoed’s test:

• Positive Barfoed’s test: development of brick red color ppt within 3-5 minutes
• Negative Barfoed’s test: absence of red color

** reducing disaccharides also give positive barfoed test on prolong heating

Barfoed’s Test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to identify carbohydrate from other biomolecules

Principle of Molisch’s test:

Molisch’s test is a general test for all carbohydrates. In this test, carbohydrates when reacted with conc. H2SO4 get dehydrated to form furfural and its derivatives.

When monosaccharide are treated with conc H2SO4 or conc HCl, -OH group of sugar are removed in the form of water and furfural is formed from pentose sugar and hydroxymethyl furfural is formed from hexose sugar. These products reacts with sulphonated α- naphthol to give a purple (violet red) colored complex.

Reagents for Molisch’s test:

• test solution: 5 % Glucose, 5 % Sucrose, 5 % Starch
• Molisch’s reagent (5 % α naphthol in ethanol)
• H2SO4
• Dry test tubes
• pipettes

Procedure of Molisch’s test:

1. Take 2ml of sample in dry test tube.
2. Take 2ml of distilled water in another tube as control.
3. Add 2-3 drops of Molisch’s reagent to the solution.
4. Gently pipette 1ml conc. H2SO4 along the side of the tube so that two distinct layers are formed.
5. Observe color change at the junction of two layers.
6. Appearance of purple color indicates the presence of carbohydrates.

Result Interpretation of Molisch’s test:

Positive Molisch’s test: purple color complex

Negative Molisch’s test: no purple color

Molisch’s Test: Objectives, Principle, Reagents, Procedure and Result

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Objective:

• to detect reducing sugar in a given solution

Principle of Fehling’s test:

Fehling’s test is one of the sensitive test for detection of reducing sugars. Fehling’s reagents comprises of two solution Fehling’s solution A and solution B. Fehling’s solution A is aqueous copper sulphate and Fehling’s solution B is alkaline sodium potassium tartarate ( Rochelle salt). Rochelle salts (sodium potassium tartarate) present in the reagent acts as the chelating agent in this reaction.These two solution are mixed in equal amount before test.

On heating an aldehyde or reducing sugar with Fehling’s solution give reddish brown prepitate. Formation of red precipitate of cuprous oxide denotes the presence of reducing sugar.

Reagents:

• test solution: 5 % Glucose, 5 % Sucrose, 5 % fructose, 5 % Lactose, 5 % Starch
• Fehling’s reagent (solution A: CuSO4.5H2O
• Fehling’s reagent ( solution B: Sodium potassium tartrate)
• Water bath
• Pipettes
• Dry test tubes

Procedure of Fehling’s test:

1. Take 1ml of sample in dry test tube.
2. Take 1ml of distilled water in another tube as control.
3. Add 1ml of Fehling’s reagent (A and B) to all the tubes.
4. Keep in boiling water bath.
5. Look for the development of red precipitate.

Result interpretation:

• Positive Fehling’s test: reddish brown ppt ( glucose, fructose, lactose)
• Negative Fehling’s test: No red ppt (sucrose, starch)

Fehling’s  Test: Objective, Principle, Reagents, Procedure and Result

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1. Seliwanoff’s test: Principle, reagents, procedure and result

Principle of Seliwanoff’s test:

Seliwanoff’s test is used to distinguish aldoses from ketoses. On treatment with conc. Acid, ketoses are dehydrated more rapidly to give furfural derivatives and on condensation with resorcinol give cherry red complex. The test will be answered by fructose, sucrose and other keto containing carbohydrates.

Reagents:

• test solution: 5 % Glucose, 5 % Sucrose, 5 % Fructose.
• Seliwanoff’s reagent (0.5% resorcinol in 3N HCl)
• Water bath
• Pipettes
• Dry test tubes

Procedures

1. Take 1ml of sample in test tube and take 1ml of distilled water in another tube as control.
2. Add 3ml of Seliwanoff’s reagents in both test tube
3. Keep the test tubes in water bath for 1-2 minutes.
4. Look for the development of red color.

**If the reaction is allowed for longer time, aldoses also produce positive results.

Result Interpretation of Seliwanoff’s test:

• Positive seliwanoff’s test: Fructose and sucrose
• Negative seliwanoff’s test: glucose, distilled water

2. Bial’s Test: Principle, reagents, Procedure and result

Principle of Bial’s test:

Bial’s test is useful in distinguishing pentoses sugar from hexoses sugars. Pentosses ( such as ribose sugar) form furfural in acidic medium which condense with orcinol in presence of ferric ion to give blue green colored complex which is soluble in butyl alcohol.

Reagents:

• test reagent: 5 % Glucose, 5 % Ribose, 5 % Fructose
• Bial’s reagent
• Water bath
• Dry test tubes
• Pipettes

Procedures of Bial’s test:

1. Take 2ml of Bial’s reagent in test tube.
2. Add 4-5 drops of test solution to this reagent.
3. Keep in water bath for 30 seconds.
4. Look for the development of bluish green color.

Result interpretation for Bial’s test:

• Positive Bial’s test: formation of blue color ( eg. Ribose sugar)
• Negative Bial’s test: formation of any other color indicates negative test. Hexose sugar ( glucose, fructose) generally gives green, red or brown color product.

3. Iodine  Test: Principle, reagents, Procedure and Result

Principle of Iodine test for carbohydrate:

Starch when reacted with I2 forms absorbed compound that gives blue color. On heating or on addition of alkali like NaOH or KIH, color disappears. This reaction is only physically association where I2 traps in the coiled structure of polysaccharide. On heating or on addition of alkali; the coiled structure becomes linear and the I2 molecules are released and the color disappears. The test will be answered by fructose, sucrose and other keto containing carbohydrates.

Reagents

• 5 % Glucose, 5 % Sucrose, 5 % Fructose, 5 % Starch
• Iodine solution
• Water bath
• Dry test tubes
• Pipettes

Procedure for Iodine test for carbohydrate

1. Take 2ml of sample in test tube and take 2ml of distilled water in another tube as control.
2. Add 5 drops of iodine solution to all the tubes.
3. Look for the development of blue color.
4. Heat the solution, the blue color will disappear and on cooling the color reappears.

Result interpretation of Iodine test:

• Positive iodine test: dark blue color (starch)
• Negative iodine test: glucose, fructose and sucrose

Tests for specific carbohydrates: Seliwanoff’s  test, Bial’s test and Iodine test

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