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Carbohydrate Biochemistry Tests

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GENERAL REACTIONS OF CARBOHYDRATES

MOLISCH'S REACTION:

. PRINCIPLE: .

Sugars on reaction with dehydrating agents like concentrated strong acids (concentrated H2S04) yield furfural and furfural derivatives, such as hydroxyrnethyl furfural, Which condense with a-naphthol and give a reddish violet ring. .

REAGENTS:

1. Molisch's Reagent:

i- a-naphthol,

ii- Ethyl alcohol

2. 'Concentrated H2S04

/ .

3. Original solution (O.S.) - containing. a carbohydrate.

PROCEDURE:

To 2ml of sugar solution (original solution) add 2 to 3 drops of Molisch's

reagent Mix thoroughly. Carefully pore 5 ml concentrated H2S04 along the side of the test

,

tub.e. Acid being heavier will form a layer beneath the sugar solution. The formation of' a reddish violet ring at the junction of the two liquids indicates the presence' of carbohydrates. This test is very sensitive and is given by all the carbohydrates.

REACTIONS GIVEN BY MONOSACCHARIDES AND DISACCHARIDES

1. COPPER REDUCTION TESTS:

. Carbohydrates which give reduction tests have free aldehyde or ketonic groups, and

are called "reducing sugars".

PRINCIPLE: .

Alkaline copper reagents (Benedict's and Fehling's reagents) are reduced by

the reducing sugars with the' formation of yellow, orange or red precipitate. .

. The reaction of acid copper reagents (Barfoed's reagent) with reducing sugars

is slow and 'can be used to distinguish monosaccharides from disaccharides.

a. FEHLING'S TEST:

1. Fehling's reagent:

i. Solution A - Copper sulphate solution, and

ii. Solution B - Alkaline tartrate solution.

These solutions are preserved in separate bottles. Fehling's reagent is

freshly prepared by mixing equal volumes of so~ution-A w!th solution-B.

2. Original solution (O.S.) - containing a carbohydrate.

PROCEDURE:

. To.1ml of sugar solution (original solution) in a test tube, add 1ml of Fehling's reagent. Mix and boil .carefully. The production of yellow 'or brownish-red precipitate of cuprous oxide indicates the presence of reducing sugars in the sample.

b. BENEDICT'S TEST:

'1. Benedict's reagent: .

i. Copper sulphate,

ii. Sodium Citrate, and

iii. Sodium Carbonate.

2. Original solution (O.S.) - containing a carbohydrate.

PROCEDURE:

To 5ml of Benedict's reagent in a test tube add 8 drops of sugar solution (original solution). Mix thoroughly and heat to boil for 2 minutes. Allow the tube to cool. The solution, in addition to formation of a precipitate, will change colour from blue to green, yellow, orange or red depending upon the amount of reducing sugar present. This test can be used as a rough quantitative test for the clinical evaluation as shown in the following table:

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