Carbohydrate: Tests and Estimation | Biochemistry

In this article we will discuss about:- 1. Test for Carbohydrates 2. Quantitative Estimation of Redu­cing Sugars of Carbohydrate.

The carbohydrates — most common and one of the constituents of animal body — composed of carbon, hydrogen and oxygen. Chemically carbohydrates are polyhydroxy aldehydes or ketones.

Carbohydrates are of three forms:

Monosaccharide (one molecule C₆H₁₂O₆), Oligosaccharide (two to 10 molecules) and Polysaccharide (above 10 molecules).

Monosaccharide:

Glucose, Galactose, Fruc­tose are reducing sugars.

Disaccharide:

Sucrose, Lactose and Mal­tose are non-reducing sugars.

A. Test for Carbohydrates:

I. General Test for Carbohydrates:

1. Molisch’s Test: Carbohydrate:

Procedure:

Take two test tubes. Mark A and B. Take 5 ml of sample solution in test tube A. Add two drops of Molisch’s reagent. Mix the two thor­oughly. Take 2 ml conc. H₂SO₄ in test tube B and pour it gently in test tube A along its side, so that two do not mix.

Observation:

A purple or pink ring appears at the junction of the two solutions.

Inference:

The given sample is a carbohydrate.

2. Foulger’s test: Fructose:

Procedure:

Take 0.5 ml of the sample solution in a test tube, add 3 ml Foulger’s reagent, boil and shake the tube gently.

Observation:

Blue colour appears.

Inference:

The sample contains Fructose.

3. Seliwanoff’s test: Fructose:

Procedure:

Take 3 ml Seliwanoff’s reagent in a test tube, add 1 ml sample solution to it; boil for 30 seconds.

Observation:

Red to orange colour appears.

Inference:

Fructose present in the sample.

II. Colour Test: Reducing Mono­saccharide:

1. Barfoed’s Test: Reducing Monosaccharide:

Procedure:

Take 5 ml Barfoed’s reagent in a test tube, add 4 ml sample solution, stir and boil for one or two minutes. Allow to stand for at least 15 minutes.

Observation:

Orange or red coloured ppt. of cuprous oxide appears.

Inference:

The sample contains reducing monosaccharide.

2. Benedict’s Test: Reducing Monosaccharide:

Procedure:

Take 5 ml Benedict’s reagent in a test tube, add 0-5 ml sample solution. Mix thoroughly; heat to boil for 2 minutes and cool under tap water.

Observation:

Red, Yellow or green ppt. of cuprous oxide appears.

Inference:

The sample contains reducing monosaccharide.

3. Moor’s Test: Glucose:

Procedure:

Take 4 ml sample solution in a test tube, add an equal volume of 2% sodium hydroxide solution and boil.

Observation:

The solution turns yellow in the beginning and reddish brown later due to formation of Caramel (a condensed product of glucose).

Inference:

The sample contains glucose.

4. Picric Acid Test: Reducing Monosaccharide:

Procedure:

Take 3 ml sample solution in a test tube, add 1 ml saturated picric acid solution and 1 ml 1% or 4% NaOH solution and warm.

Observation:

Red colour appears (picrylic acid).

Inference:

The sample contains reducing monosaccharide.

5. Fehling’s Test: Reducing Monosaccharide:

Procedure:

Take 5 ml Fehling’s solution in a test tube, add 2 ml sample solution and boil.

Observation:

Yellow or brick-red (cuprous oxide) ppt. appears.

Inference:

The sample contains reducing monosaccharide.

6. Tommer’s Test: Reducing Monosaccharide:

Procedure:

Take 2 ml 0.5% copper sulphate solution in a test tube, add 2 ml sample solution and mix. Add 2 ml 40% NaOH solution, boil and cool.

Observation:

Red or yellow ppt. (cuprous oxide) appears.

Inference:

The sample contains reducing monosaccharide.

7. Nylander’s Test: Reducing Monosaccharide:

Procedure:

Take 5 ml sample solution in a test tube, add 0-5 ml Nylander’s reagent, boil for 3 minutes and cool.

Observation:

Black ppt. develops (bismuth sub-nitrate reduced to black bismuth).

Inference:

The sample contains reducing monosaccharide.

8. Methelene Blue Test: Reducing Mono­saccharide:

Procedure:

Take 3 ml distilled water and 0.5 ml 40% NaOH solution in a test tube, add a drop of methylene blue, boil and cool. The colour of solution remains blue. Add 1 ml sample solution, boil and cool.

Observation:

Blue colour of the solution disappears. (Methylene blue reduced to leuco-methylene blue — colourless)

Inference:

The sample contains reducing monosaccharide.

9. Potassium Ferricyanide Test: Reducing Monosaccharide

Procedure:

Take 3 ml 1% potassium ferricyanide solution in a test tube, add 1 ml 40% NaOH solution. Boil and add 10 drops (drop by drop) of the sample solution and keep on boiling.

Observation:

The yellow colour of ferricyanide decolorizes.

Inference:

The test solution contains reducing monosaccharide.

III. Colour Test for Disaccharide:

1. Fearson’s Test: Sucrose:

Procedure:

Take 4 ml sample solution in a test tube, add 4 drops of 10% methylamine hydrochloride solution, boil for 30 seconds, add 5 drops of 20% NaOH solution.

Observation:

Yellow colour appears, which turns red.

Inference:

The sample contains sucrose.

IV. Colour Test for Glycogen: (Poly­saccharide):

Iodine test: Glycogen:

Procedure:

Put 2 ml sample solution in a test tube, boil and cool at room temperature. Add 1 or 2 drops of iodine solution.

Observation:

Red or wine red colour appears.

Inference:

The sample contains glycogen.

V. Colour Test for Starch:

Starch is a polysaccharide. Rice, Wheat, Maize, Potato are chief sources of starch.

Iodine solution:

Prepare a 2% potassium iodide (Kl) solu­tion in alcohol. Add sufficient amount of iodine crystals to colour it deep yellow.

In depression of a porcelain test plate, place a small amount of starch. Add 1 to 3 drops of dilute iodine solution. Iodine complexes are formed, resulting a blue colour.

VI. Detection of Salivary Amylase:

Activity of ptyalin or amylase present in saliva is determined by digestion of starch.

Reagents:

1. Starch Solution 1 %:

Soluble starch 1 g

Distilled water 100 ml

Preparation:

Put 1 g starch to 5 ml distilled water in a 250 ml conical flask and mix thoroughly. Add 95 ml distilled water and heat till boiling point— stir constantly. Cool and add a few drops of toluene or chloroform as a preservative.

2. Standard Iodine Solution:

Collection of saliva:

Clean the teeth, gargle with a mild antisep­tic and rinse thoroughly with water. Hold a piece of sour foodstuff, like tamarind or a piece of green mango or some such material — in front of the tongue. A profuse secretion of saliva starts immediately. Collect the saliva from under the tongue with a pipette or medicine dropper.

Technique:

Control:

To one drop of 1% starch solution in a white porcelain plate add one drop iodine solution. The colour of the mixture turns deep blue, showing presence of starch.

Test:

Place 5 ml 1% starch solution in a test tube and add 5 drops of saliva. Mix and place the tube in a water bath at 37°C and incubate for 15 minutes.

Test at intervals by mixing one drop of the mixture with one drop of iodine solution in a white porcelain plate. Note the colour.

With time, the colour changes to purple, red and finally yellow. This indicates presence of ptyalin amylase in the saliva which has digested (hydrolysed) starch.

B. Quantitative Estimation of Redu­cing Sugars (Benedict’s Method):

Reducing sugars, viz., glucose, fructose, lactose, galactose, pentose and others reduce, curpric sulphate (blue) to cuprous sulphate (yellow precipitate) and the blue colour disappears.

Reducing sugar solution:

Dissolve 0.5 g glucose in about 20 ml distilled water. Make up the volume to 100 ml. The concentration of sugar solution is 0.5%, i.e., 5 mg per ml.

Benedict’s solution:

Reagents:

Copper sulphate (crystalline) 18 g

Sodium carbonate (crystalline) 200 g

Sodium potassium citrate 200 g

Potassium ferrocyanide solution 5% 5 ml

Potassium sulpho-cyanate 125 g

Distilled water

Preparation:

Dissolve the given amount of carbonate, citrate and sulpho-cyanate in about 700 ml distilled water by heating and filter.

Dissolve the copper sulphate in about 10 ml distilled water with constant stirring. Cool and mix the two solutions. Add the ferrocyanide solution. Cool and add distilled water to make the volume 1 litre.

The strength of the solution: 2gm reduc­ing sugar is required to reduce 1 ml Benedict solution.

Technique:

Take 25 ml Benedict’s solution in a 150 ml conical flask. Add 3 to 4 g anhydrous sodium carbonate. Add a few pieces of small glass beads to prevent bumping of the solution during heating. Heat the solution to boil. Add the sugar solution (unknown concentration) to be tested, little at a time but quickly from a burette. Repeat addition of solution till the blue colour begins to disappear.

A chalk-white precipitate is formed. From this point add the sugar solution in drops till the last trace of blue colour just disappears. The reagent should be kept in boil­ing during titration. Loss due to evaporation can be made up by adding distilled water.

Calculation:

Read from the burette the amount of sugar solution required to reduce 25 ml Benedict’s solution. 25 ml Benedict’s solution requires 25 x 2 = 50 mg reducing sugar. Therefore 50 mg sugar was present in the total amount of solution of unknown concentration used. Calculate the concentration of sugar in the unknown solution.