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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:

CONCENTRATION OF  

OBSERV A TIONS SUGAR IN THE CLINICAL 

ORIGINAL SOLUTION EVALUATION 

(%)  

No colour change (Blue) 0.0% Nil 

Green co loured solution with 0.1% Traces 

no precipitate.  

Green coloured solution with 0.1 - 0.5% + 

yellow precipitate.  

Olive green coloured .0.5 - 1% ++ 

. solution with yellow  

precipitate. ..  

Yellow orange coloured 1- 2% +++ 

precipitate.  

Brick red coloured 2% or more. ++++ 

precipitate.

C. BARFOED'S TEST:

1. Barfoed' s Reagent: 

i. Copper acetate, and 

ii. Acetic acid. 

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

. PROCEDURE: 

To 5ml of Barfoed's reagent in a test tube add 0.5ml of sugar solution (original solution). Mix thoroughly and place it in the boiling water bath. Note; the time when 

signs of reduction i.e., formation of a red precipitate of cuprous oxide first appears in the test 

tube. . 

The monosaccharides start forming precipitates in less than 7 minutes where as the precipitates appearing after 7 minutes indicate the presence of disaccharides in the solution.

05 OSAZONE TEST (PREPARATION OF OSAZONES)

REAGENTS: 

1. Phenylhydrazine mixture consisting of: 

i, Phenylhydrazine, and 

ii, Anhydrous sodium acetate. 

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

PROCEDURE: 

Take phenylhydrazine mixture and fill about half an inch of test tube with it. 

o Add 3-Sml of sugar solution (original solution) to the test tube. Mix thoroughly and place it in a boiling water bath. Note the appearance of yellow crystalline precipitate in the test tube. Remove and allow the test tube to cool slowly. Do not cool the test tube under tap water. Examine the crystals under the microscope and make a drawing. Draw only what you see. ' 

Osazones of glucose are formed in 10-15 minutes whereas those of disaccharides take upto 45 minutes. Sucrose does not react with phenylhydrazine to form crystalline compounds called osazones.

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