Practical Experiments on Carbohydrate Tests, Lecture notes of Organic Chemistry

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Qualitative Tests of Carbohydrate-I

BCH 202 [Practical] 1

➢ Carbohydrates are defined as the polyhydroxy aldehydes or polyhydroxy ketones.

➢ Most , but not all carbohydrate have a formula (CH 2 O)n (hence the name hydrate of

carbon).

➢ In human body, the D-glucose is used.

➢ Simple sugars ends with – ose.

➢ Biological role:

• Carbohydrates are the key source of energy used by living things.
• Also serve as extracellular structural elements as in cell wall of bacteria and plant.

1. Simple sugar (one unit) :

Monosaccharides contain one monosaccharide unit.

2. Complex sugar (more than one) :

1. Disaccharides contain two monosaccharide units.

2. Oligosaccharides contain 3 - 9 monosaccharide units.

3. Polysaccharides can contain more than 9 monosaccharide units.

➢ Complex carbohydrates can be broken down into smaller sugar units through a process

known as hydrolysis.

Carbohydrates Simple Monosaccharides (^) Disaccharides Complex Oligosaccharides Polysaccharides One unite (^) Tow unite 3 -^9 unites^ More than 9 unites

• All monosaccharides are reducing sugars; they all have a free reactive carbonyl group.
• Some disaccharides have exposed carbonyl groups and are also reducing sugars like

lactose. While other disaccharides such as sucrose are non-reducing sugars and will not

react with Benedict's solution.

• Large polymers of glucose, such as starch, are not reducing sugars, since the concentration

of hemiacetal groups is very low.

• Monosaccharide and disaccharid e can be dissolved freely in water because water is a

polar substance.

• Polysaccharide cannot be dissolved easily in water, because, it has high molecular weight ,

which give colloidal solutions in water.

Molicsh test: To identify the carbohydrate from other macromolecules.

Benedict test: For the presence of reducing sugars. Barfoed’s Test: for to distinguish between reducing monosaccharides, reducing disaccharides and non reducing di-polysaccharides (detect reducing monosaccharides). Bial’s Test: To distinguish between pentose monosaccharide and hexose monosaccharide (to detect pentoses). Seliwanoff's Test: To distinguish between aldoses and ketoses (to detect ketoses). 1 2 3 4 5

➢ Objective:

To identify the carbohydrate from other macromolecules lipids and proteins (this test is specific for all

carbohydrates).

➢ Principle:

Two solutions are added : H 2 SO 4 and α-naphthol

1. The test reagent (H 2 SO 4 ) dehydrates pentose to form furfural and dehydrates hexoses to form 5 -

hydroxymethyl furfural.

2. The furfural and 5 - hydroxymethyl furfural further react with α-naphthol present in the test reagent

to produce a purple product.

➢ Method:

1. Two ml of a sample solution is placed in a test tube.

2. 0.5 ml of the Molisch reagent (which α-napthol in

95 % ethanol) is added.

3. The solution is then poured slowly into a tube containing two

ml of concentrated sulfuric acid so that two layers form, producing violet ring appear as

liaison between the surface separations.

➢ Results:

Tube Observation Glucose Lactose Starch Concentrated sulfuric acid is extremely corrosive and can cause serious burns when not handled properly.

➢ Objective:

To distinguish between the reducing and non-reducing sugars (to detect the presence of reducing sugar).

➢ Principle:

• The copper sulfate (CuSO 4 ) present in Benedict's solution reacts with electrons from the aldehyde or

ketone group of the reducing sugar in alkaline medium.

• Reducing sugars are oxidized by the copper ion in solution to form a carboxylic acid and a reddish

precipitate of copper oxide.

• The non-reducing sugars give negative result. reducing sugar carboxylic acid reddish precipitate

16 ➢ Objective: This test is performed to distinguish between reducing monosaccharides, reducing disaccharides and non reducing di- and polysaccharides. ➢ Principle:

• Barfoed’s test used copper (II) ions in a slightly acidic medium.
• Reducing saccharides are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide.
• Different types of reducing sugars react at different rates ➔ Reducing monosaccharides react quickly with Barfoed’s reagent (acidic condition), but reducing disaccharides react very slowly or not at all.
• The non-reducing sugars give negative result. reducing sugar carboxylic acid reddish precipitate

➢ Method:

1. Place one ml of a sample solution in a test tube.

2. Add 3 ml of Barfoed's reagent (a solution of cupric acetate and acetic acid.

3. Heat the solution in a boiling water bath for 6 minutes (after the 3 min check the tubes).

➢ Results:

Tube Observation Glucose Lactose Starch Glucose (+)

➢ Method:

1. Put 2 ml of a sample solution in a test tube.

2. Add 2 ml of Bial's reagent to each tube.

3. Heat the tubes gently in hot water bath.

4. If the color is not obvious, more water can be added to the tube.

➢ Results:

Tube Observation Glucose Ribose

Glucose

Ribose

➢ Objective: To distinguish between aldoses and ketoses (to detect ketoses). ➢ Principle:

• Seliwanoff’s test uses 6 M HCl as dehydrating agent and resoncinol as condensation reagent.

1. The test reagent dehydrates ketohexoses to form 5 - hydroxymethylfurfural ➔ 5 - hydroxymethylfurfural further condenses with resorcinol present in the test reagent to produce a cherry red product within two minutes.
2. Aldohexoses react to form the same product, but do so more slowly giving yellow to faint pink color.