Qualitative test for Carbohydrates

The qualitative tests for carbohydrates such as (Molish, Benedict, Fehling, Osazone, and Barfoed tests) are used to detect the presence of reducing and non-reducing, mono and disaccharides in a sample. Here are common qualitative tests for carbohydrates:

EXPERIMENTAL ANALYSIS

1. Molish Test

The molish test is a simple, rapid, and chemical-sensitive test to detect the presence of carbohydrates (sugars, and starches) in a sample.

Principle

Dehydration of carbohydrates from the purple compound with α-naphthol.

Requirements

• α-naphthol
• conc. sulfuric acid
• sample solution
• conical flask
• tripod stand
• iron stand
• beakers
• stirrer
• test tubes
• thermometer

Procedure

1. Prepare a sample solution
2. Add 2 to 3 drops of α-naphthol to the sample
3. Carefully add 1 to 2 ml of con. sulfuric acid
4. Mix it well
5. Observe the purple ring or violet color.

Results

• The purple color indicates a positive test
• no color change means a negative test (no carbohydrates)

Application

• clinical diagnosis (urine glucose testing)
• food analysis (presence of sugar)
• different biochemical research works

Limitations

• The non-specific test detects all types of carbohydrates.
• False positive with non-carbohydrate compounds.

2. Fehling test

Fehling test is a chemical test that is used to detect the presence of reducing sugars such as monosaccharides and disaccharides in a sample.

Principle

Reducing sugars reduces copper || ions to copper| ions, forming red precipitates.

Requirements

• Fehling solution A copper|| sulphate
• Fehling solution B sodium hydroxide and Rochelle salt
• sample solution
• titrating apparatus
• water bath

Procedure

• Make a sample solution in a clean and dry beaker.
• Mix 01ml of Fehling A and 01 ml of fehling B, stirr it well.
• Add 1 to 2 ml of sample.
• Boil the solution for 05 to 10 minutes.
• Observe for red precipitates.

Result

• red precipitates for a positive test
• no precipitates mean a negative test

3. Benedict test

Benedict test is a test that indicates the strength of reducing sugars such as strong, weak, and moderate.

Principle

Reducing sugars reduces copper|| to copper| ions that form a colored compound.

Requirements

• Benedict reagent
• sample
• titrating instruments
• boiling apparatus

Procedure

• Make a sample solution in water
• mix 1ml of benedict reagent with 10ml of sample.
• boil it for 05 minutes.
• observe for color changes.

Results

• Blue color for negative test
• green for reducing sugars
• yellow for moderate reducing sugars
• orange red shows the presence of strong reducing sugars

4. Barfoed test

Barfoed test is a chemical test used to detect the presence of monosaccharides and disaccharides ( classification of lipids). It’s a useful test in biochemistry and food science. Here’s how it works:

Principle of Barfoed test

• Reducing sugar such as mono and disaccharides reduced copper acetate present in the reagent into copper oxide in an acidic medium.
• sugars with hemiacetal are in equilibrium with an open chain form in a solution and behave as a reducing agent towards containing metal salts
• This test relies on the ability of monosaccharides to reduce copper ll ions to copper l ions, forming a red precipitate of copper l oxide, disaccharides, and polysaccharides do not react with the reagent under these conditions.

Barfoed Reagent

• 0.33M copper acetate in dist. water
• Acetic Acid ( 1% solution )
• Dissolve 1.1g of copper acetate in 20ml distilled water and filter out the solution
• Add 0.18ml acetic acid infiltrate
• Copper acetate with acetic acid decreases the Ph and makes the medium acidic

Apparatus

• Beakers
• stirrer
• thermometer
• tripod stand
• conical flask
• water bath
• heating apparatus like spirit lamp

Procedure

1. Take three separate test tubes, clean them, and label as monosaccharides, disaccharides, and control respectively.
2. Add 1ml sample in both mono and di saccharides labeled tubes
3. Add 1ml sample dist. water in the control tube.
4. Add 3 ml of Barfoed reagent in all the test tubes.
5. Place the tubes in a water bath.
6. Observe the color change.

Limitations

• The test is not specific to a particular monosaccharide.
• Some disaccharides may give a false positive result if they are partially hydrolyzed to monosaccharides during the tests.

Result of this qualitative test 

1. Glucose (mono) gives precipitates of brick reddish brown color in 2- 3 mins.
2. Disaccharides (maltose) give ppt in 10 mins.
3. The control tube remains unchanged, which means the absence of monosaccharides (glucose, fructose) and the presence of disaccharides (e.g. sucrose) or polysaccharides.

The Barfoed test is a simple and useful tool for detecting monosaccharides, but it should be used in conjunction with other tests for more specific identification.

5. Salliwanoff’s test

Salliwanoffs test is a chemical qualitative test used to detect the presence of cholesterol, aldose, and ketose sugar in a sample. This test is simple, rapid, and sensitive, and should be used with other tests for confirmation.

Applications

• Food analysis( e.g. cholesterol in oils)
• Pharmaceutical analysis
• Clinical diagnosis (e.g. cholesterol levels in blood)

Principle

A dehydration reaction due to hydrolyzed groups of sugar. Salliwanoffs reagent is resorcinol in dilute hydrochloric acid. Ketones ( e.g. fructose) are more readily dehydrated by HCl than the aldose to form hydroxyl methyl furfural which then condenses with resorcinol to form a red complex.

Requirements

• The apparatus is the same as we use in the Barfoed test
• Salliwanoffs reagent
• sample
• Salliwanoffs Reagent
• Dissolve 0.01gresorcinol in 6.6ml conc. HCl.
• Add distilled water up to 13.4ml

Procedure

• Take a test tube, and wash it with clean water.
• Add 01 ml sample (fructose) solution in it.
• Now, add 2ml reagents and mix them.
• Place the test tube in a boiling water bath.
• Focus on color changing.

Result

A reddish color appears in 1min if keto sugar is present. Faint pink appears for aldose sugar.

6. Osazone test

The osazone test is a chemical qualitative test used to detect and identify reducing sugars (monosaccharides and disaccharides)in a sample.

What is Osazone?

Carbohydrates derivative which is formed when any carbohydrate reacts with phenyl hydrazine to form osazone.

• Osazone is also known as 1,2- phenylhydrazine.
• Osazone is a different shape crystal for every monosaccharide.
• The differentiation is based on time for different reducing sugars.

Crystal color is because of phenylhydrazine.

Osazone reaction

Osazone Composition

• 2g phenyl hydrazine HCl+ 2g sodium acetate
• 9 -10 drops of glacial acetic acid

Procedure

• Take a test tube and add a 5ml sample into it.
• Now add freshly prepared phenylhydrazine mixture and boil it.
• Heat the mixture gently to 60- 80°C for 5- 10 minutes.
• Then, the formation of crystals.
• Observe the crystals under a microscope.

Result

• Positive result: Yellow, orange, or crystalline precipitate forms.
• Negative test: No precipitate forms.

Uses of Osazone test

It is used for the identification of reducing sugar

• Clinical diagnoses (urine glucose testing)
• food analysis( sugar detection)
• Biochemical research

Limitations

• Do not distinguish between specific reducing sugars.
• False positives with non-carbohydrate-reducing sugar.

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qualitative tests for carbohydrates