QUALITATIVE TESTS FOR CARBOHYDRATES

CARBOHYDRATES QUALITATIVE EXPERIMENTS

INTRODUCTION:

Carbohydrates are the major part of our diet which play an important role to human bodies. It provides  60-70% of total energy required by our body. Glucose, one of the most important constituents of our diet which are usually obtain from the plant source in the form of starch. The glucose is rapidly utilized by our body or stored in the form of glycogen in the liver. 

Carbohydrates contain carbon, hydrogen and oxygen atoms, they are generally hydrates of the carbon. The name carbohydrates are used because of the presence of hydrogen and oxygen usually in the ratio of 2:1 (H₂O), for example Glucose has the formula C₆H₁₂O₆ which can be written as C₆(H₂O)₆. The general formula for most of the carbohydrates are C_nH_2nO_{n ,} but this is not true for all sugar.

CLASSIFICATION:

Carbohydrates chemically are aldehydes and ketones of polyhydroxy alcohols. Based on the functional group carbohydrates are classified into:-

1. Aldose Sugar / Aldoses (Eg. Glucose)

2. Keto Sugar / Ketoses (Eg. Fructose)

Based on the number of sugar units they are classified into:-

1. Monosaccharides (Eg. Glyceraldehyde, Glucose, Fructose etc)

2. Disacharides (Eg. Maltose, Lactose, Sucrose etc)

3. Ploysaccharides (Eg. Starch, Glycogen, Cellulose etc)

Monosaccharides are classifed based on the number of sugar units as:-

1. Trioses (Eg. Glyceraldehyde, Dihydroyacetone)

2. Tetroses (Eg. Erythrose, Threose)

3. Pentoses (Eg. Ribose, Deoxyribose)

4. Hexoses (Eg. Gluose, Frutose)

5. Heptoses, Octoses and more higher number do occurs in nature

   

TEST FOR CARBOHYDRATES:

1] MOLISCH'S TEST

It is general test for carbohydrates. Molisch's test when it is positive its indicates the presence of carbohydrates in the solution.

Principle

The carbohydrates when treated with conc. sulfuric acid (H₂SO4), its undergoes dehydration to form furfural or its derivatives. The furfural and its derivatives are highly reactive with alpha naphthol and form violet colored complex at the junction of two liquids.

Procedure

In a test tube take 2ml of carbohydrate solution and add 2 drops of Molisch's reagent and mix it. Add approx 2ml of conc. sulfuric acid by the side wall of the tube in the inclined position, so that two distinct layers are produced. The bottom layer is acid. The formation of violet ring between two layers indicates the presence of carbohydrates.

Notes:

1. All carbohydrates except amino sugars give this test.

2. Disaccharides and polysaccharides are easily hydrolysed by conc. sulfuric acid into monosaccharides so they also give positive test.

3. In Anthrone test, in place of alpha naphthol, anthrone is used for the test of carbohydrates.

4. Pentose sugar give rise to furfural where as hexose sugar give rise to derivative of ferfural such as hydroymethyl furfural.

5. Due to contamination of sulfuric acid sometime green or some other colored ring are formed when the test is performed with control (water).

6. Molisch's test is given by sugar with at least 5 carbon atoms because it involves furfural formation which is a 5 carbon compound. So less than 5 carbon Molisch's test is negative.

7. Molisch's reagent: Dissolve 1gm of alpha nephthol in 100ml of ethanol or methanol.

2] IODINE TEST

It is the test for polysaccharides

Principle

The polysaccharides absorb iodine and form colored complex.

The complex is formed under the neutral or acidic conditions. Under alkaline conditions, iodine react with NaOH, so blue color disappears. When alkali is neutralized with acid, iodine is released and blue color reappears.

Procedure

Take 2ml of sugar solution in two test tubes. To one add several drops of NaOH solution. Than add 1 to 2 drops of diluted iodine solution to each tube.

In case of starch, the test tube in which no NaOH has been added blue color will appeared. On heating test tube blue color will disappeared while on cooling its reappears whereas if excess HCl is added in test tube containing NaOH its get neutralized and blue color will now formed in that tube also. 

Similar test with glycogen will give reddish brown color and with dextrin color varying from brownish red to no color.

Notes:

1. The color formed by polysaccharide depends on the length of linear or branched sequence.

2. In linear sequence like amylose give deep blue color.

3. In branched chain like amylopectin give purple color.

4. In highly branched chain like glycogen give reddish brown color.

5. Dextrine formed by partial degradation of starch give brownish red color as chain length become smaller whereas on extensive degradation of starch the corresponding dextrin formed may not yield any color at all. 

6. On heating the molecule get unfolded and releases iodine due to which its become decolourization whereas on cooling molecule get refolded and react with iodine again to give color.

3] BENEDICT'S TEST

Test for reducing sugars.

Principle

Reducing sugar with free alddehyde and ketone groups in alkaline conditions undergoes tautomerise and form enediols. This enediols are strong reducing agents. They reduce cupric ions of Benedict's solution to couprous ions which ultimately form cuporous oxide and form color precipitate depending on the concentration of sugar.

Procedure

Take 2.5ml of Benedict's reagent in a test tube add 4 drops of sugar solution. Keep test tube in hot water bath for 2 to 3 min. Allow to cool spontaneously (do not cool in cold water bath). A color precipitate is form ranging from green, yellow, orange-red or brick red depending on concentration of sugar.

COLOR	Approx. AMOUNT OF REDUCING SUGAR
No change of Blue color	Absent of reducing sugar
Blue changes to Green ppt.	0.1 gm% to 0.5 gm% of reducing sugar
Blue changes to Yellow ppt.	0.5 gm% to 1.0 gm% of reducing sugar
Blue changes to Orange-red ppt.	1.0 gm% to 2.0 gm% of reducing sugar
Blue changes to Brick-red ppt.	More than 2.0 gm% of reducing sugar

Notes:

1. Benedict's reagent prepared by dissolving 173 gm of Sodium citrate and 100 gm of sodium carbonate in about 100 ml of water, by heating. If there is any turbidity, it should be removed by filteration. Copper sulfate solution (17.3 gm opper in 100 ml water) is slowly added with constant stirring to the citrate carbonate solution and the volume is made up 1 liter.

2. Sodium citrate is act as metal chelator, used to keep hydroxide in the solution.

3. Sodium carbonate is a mild alkali which convert cuprous ion to cupric ion. 

4. Benedict's test is the modification of Fehling's test.

4] BARFOED'S TEST

This test is used to differentiate between reducing monosaccharides and disachharides.

Principle

The monosaccharides with free aldehyde or ketone group can cause the reduction of copper ions present in copper acetate to couprous oxide, of red precipitate. This test is reduction test for monosaccharides an the reduction is brought in an aciic solution.

Procedure

take 1 ml of Barfoed's reagent add 4 drops of sugar solution, keep the tube in boiling water bath or 5 min, take out the tube and observe the precipitate. The monosaccharide sugar will give positive test yielding red precipitate of cuprous oxide.

Notes:

1. Barfoed's reagent is prepared by dissolving 13.3 gm of neutral copper acetate in 200 ml of water and then adding 1.0 ml glacial acetic acid.

2. Barfoed's test is also a copper reduction test but this test is carried out in acidic medium instead of alkaline medium.

3. Under acidic conditions, the reduction is takes place less efficiently.

4. Monosaccharide respond quicker to the test its takes 2-5 min only whereas disaccharide boil continues for about 10 min or more disaccharide also hydrolyzed to monosaccharides and give positive test.

5. To distinguish reducing monosaccharide to disaccharide by Barfoed's test by controlled time of heating and pH.

 

5] SELIWANOFF'S TEST

This test distinguishes between aldose and  ketoses. It is positive for keto sugars.

Principle

Principle is same as Molisch test that is sugar on reacting with acid form furfural derivatives by dehydration. Ketohexoses acted on by HCl at 100 degree Celsius to form 5-hydroxymethyl ferfural which on condensation with resorcinol give cherry red colored soluble compound.

Procedure

Take 1 ml of Seliwanoff's reagent add 2 drops of sugar solution. Place the tube in boiling water bath exactly for 30 sec. Note the color change, if any, and then continue the heating for 5 min and periodically observe the  color change.

Notes:

1. Seliwanoff's reagent is 0.05% resorcinol in on. HCl diluted 1:1 with water.

2. Ketoses (naturally fructose) give cherry red color.

3. Aldoses give test weakly and slowly. If boiling continues for more than 2 min. may give rise to false positive test due to hydrolysis of disacharides.

4. This test is also given by sucrose that is hydrolyzed during the course of test yielding fructose as one of the products.

6] OSAZONE TEST

Several reducing sugar form characteristic shaped crystals.

Principle

A solution of reducing sugar when heated with phenyl hydrazine in aidic medium a characteristic yellow crystalline compounds called OSAZONE are formed. Simple sugars like glucose, fructose and mannose produce the same osazone because of the similarities in their molecular structures.

Procedure

Take about 300 mg of phenyl hydrazine mixture then add few drop of glacial acetic acid and then 5 ml of sugar solution. Shake well and heat in a boiling water bath for 30 to 45 min. Take tube out of  water bath and allow it to cool slowly. Yellow crystals of osazone will appear. 

Examine the crystals under the microscope.

Glucosazones (needle shaped crystals)

            Given by Glucose, Fructose and Mannose. 

Maltosazones (sunflower shaped crystals)

            Given by Maltose.

Lactosazones (cotton ball shaped crystals)

            Given by Lactose.

Notes:

1. Phenyl hydraine mixture is prepared by mixing equal weights of phenyl hydrazine hydrochloride and anhydrous sodium acetate. The mixing is to be done throughly in a mortar.

2. Glucose and Fructose give identical osazones because except the first two carbon (which are involved in the formation of osazone) the remaining four carbon atoms have same configuration in both of them. 

3. Sucrose as such does not form any osaone because it has no reducing groups available for reaction with phenylhydrazine. On hydrolysis, however it give rise to osazone.

4. Osazone of disaccharides are soluble in hot water. Therefore, in these case osazone do not participate during heating. They appear only after cooling.

 

GENERAL SCHEME FOR DETECTION OF CARBOHYDRATES

(Except galactose, the  general scheme for detection of unknown carbohydrates are followed)

 

************************************************************************************************