Organic Chemistry

Module 7. Carbohydrates

Lesson 24
DETECTION OF CARBOHYDRATES

Carbohydrates are polyhydroxy aldehydic or ketonic compounds or anhydrides of such compounds or their derivatives.

Carbohydrates have reactive groups which are responsible for their chemical behavior. There are

1. The glycosidic hydroxyl group
2. The alcoholic hydroxyl group and,
3. The free aldehyde or ketone group

Most of the reactions of Carbohydrates are carried out in aqueous solution, which means that all three types of groups will be present and available for reaction in mutarotating mono- and di- saccharide. When chemical reagents react specifically with 1 or 3 in mono and di saccharides, the equilibrium,
alpha form ⇐ = ⇒ aldehyde form ⇐ = ⇒ beta form (referred to previous structures mention)
or
alpha form ⇐ = ⇒ ketone form ⇐ = ⇒ beta form (referred to previous structures mention) is shifted in favour of the group which is being used up. Some reagents react with more than one group. Such, reactions are the basis for detection of different types or classes of carbohydrates.

24.1 Alpha-Napthol Reaction (Molisch test)

24.1.1 Principle

Concentrated sulphuric acid hydrolyses glycosidic bonds to give the mono- saccharides which are then dehydrated to furfural and its derivatives such as hydroxyl methyl furfural. These products then combine with sulphonated alpha-napthol to give a purple complex (conc. solution of organic compounds may give a red or violet color due to the charring action of the sulphuric acid).

This reaction is a general one for the presence of carbohydrate and other organic compound that give furfural with conc. sulphuric acid.

24.1.2 Materials

1. Conc. H₂SO₄
2. Alpha-napthol (5% in ethanol)
3. 1% sugar solution

24.1.3 Method

Add two drops of the alpha napthol solution to 2 ml of test solution,then carefully pour about 1 ml conc. H₂SO₄ down the side of the tube so as to form two layers. A redish-violet zone appears at the junction between the two liquids. Repeat the test using water.

24.2 The Anthrone Reaction

24.2.1 Principle

The anthrone reaction is generally test for carbohydrates. The principle is the same as outlined above except that the furfural reacts with anthrone to give blue-green complex.

24.2.2 Materials

1. Conc. H₂SO₄
2. Anthrone solution (0.2% in conc. H₂SO₄)
3. 1% sugar solution

24.2.3 Method

Add 5 drops of the test solution to about 2 ml of the anthrone reagent, mix thoroughly and observe the color change. Blue green color indicates presence of carbohydrate.

24.3 Fehling’s Test (for Reducing Sugar)

24.3.1 Principle

Carbohydrates with a free or pontentially free carbonyl group have the ability to reduce solution of various metallic ions such as Fehling’s solution in which case rust-brown cuprous oxide is precipitated. Fehling’s test is too sensitive for the routine detection of glucose in urine since it can give a false positive test due to the action of urates in urine. Excess ammonia and ammonium salts interfere with the test.

24.3.2 Materials

1. Fehling’s solution A
2. Fehling’s solution B
3. Sugar solution

24.3.2 Method

Mix equal volumes of Fehling’s solution A and B and a few drops of the test solution to 1 ml of the mixed Fehling’s solution and boil. Brick red precipitates show the presence of reducing sugars. Test the Fehling’s solution with water.

24.4 Benedict’s Test (for reducing sugar)

24.4.1 Principle

Benedict modified the Fehling’s test to product single solution which is more convenient for tests as well as being more stable than Fehling’s reagents. This is also a copper reduction test in which sugars are oxidized to their corresponding acids and cupric hydroxide is reduced to cuprous oxide.

24.4.2 Materials

Add 5 drops of the solution to 2 ml of Benedict’s reagent and place in a boiling water bath for 5 mins. Compare the sensitivity of Benedict’s and Fehling’s test, using increasing dilutions of 1% glucose.

24.5 Picric Acid Test (for Reducing Sugar)

24.5.1 Principle

Reduction of picric acid to picramic acid by the reducing sugar produces mahogany-red color (dark brown-red).

24.5.2 Materials

1. Sugar solution
2. Saturated picric acid
3. 10% Na₂CO₃

24.5.3 Method

To 5 ml of the sugar solution add 2 to 3 ml of saturated picric acid solution, add about 1 ml of 10% Na₂CO₃. Warm. Note the development of mahogany-red color is the presence of reducing sugar due to the reduction of picramic acid.

24.6 Barfoed’s Test (for Monosaccharide)

24.6.1 Principle

Barfoed’s reagent is weakly acidic and is only reduced by monsaccharides. Prolonged boiling may hydrolyse di-saccharide to give a false positive reaction. The precipitate of cuprous oxide is less dense than with the previous two tests and it is best to leave the tube to stand to allow the precipitate to settle. The color of the cuprous oxide is also different, being more a brick-red rather than orange-brown obtained in the previous tests.

24.6.2 Materials

1. Sugar solution
2. Barfoed’s reagent

24.6.3 Method

Add 1 ml of the test solution to 2 ml of Barfoed’s reagent, boil for one minute and allow to stand. Reduction indicated glucose, fructose, mannose, galactose, pentose, possibly along with non-reacting carbohydrates. No reduction indicates lactose, or maltose or both.

24.7 Bial’s Test (for Pentose)

24.7.1 Principle

When pentoses are heated with conc. HCl, furfural is formed which condenses with orcinol in the presence of ferric ions to give a blue-green color. The reaction is not absolutely specific for pentose since prolonged heating of some hexoses yields hydroxymethyl furfural which also reacts with orcinol to give colored complexes.

24.7.2 Materials

1. Sugar solution
2. Amyl alcohol
3. Bial’s orcinol reagent

24.7.3 Method

Add about 2 ml of the test solution to 5 ml of reagent in a test tube and heat until boiling commences. A blue green color indicates positive result. Cool the tube, then add 2-3 ml of amyl alcohol and shake.

24.8 Selwanoff’s Test (for Ketose)

24.8.1 Principle

Ketoses are dehydrated more rapidly than aldoses to give furfural derivatives which then condenses with resorcinol to form a red complex, prolonged heating of the solution under investigation must, therefore, be avoided. Furfural-hydrochloric acid reacts with resorcinol with formation of orange to red color.

24.8.2 Materials

1. Sugar solution
2. Seliwanoff’s reagent

24.8.3 Method

Add two drops of carbohydrate solution to 2 ml of Seliwanoff’s reagent and warm in a boiling water bath for 1 min. Note the appearance of a red color.

24.9 Iodine Test (for Polysaccharide)

24.9.1 Principle

Iodine forms colored adsorption complexes with polysaccharides, starch gives a blue color with iodine, while glycogen and partially hydrolyzed starch react to form red-brown colors and no color with sugars (only color of iodine).

24.9.2 Materials

1. Cellulose, glycogen, starch, inulin and sugars (1% solution)
2. Iodine solution

24.9.3 Method

Acidify the test solution with dilute HCl, then add 2 drops of iodine and compare the colors obtained with that of water and iodine.

24.10 Osazone Formation Test

The ‘osazones’ are yellow solids with characteristic crystalline forms and rates of formations. Osazone formation is, therefore, very useful in the identification of carbohydrates which are usually difficult to crystallize.

24.10.1 Materials

1. Phenylhydrazine mixture
2. 5% sugar solution
3. Alcohol

24.10.2 Principle

When a solution of reducing sugar is heated with phenylhydrazine yellow crystalline compound called osazone are formed. In general, each individual sugar will give rise to an osazone of a definite crystalline form which is typical for that sugar.

The reaction of phenylhydrazine with free aldehyde and ketone groups of sugars may be considered to be an example of the formation of a special types of Schiff’s base, a hydtazone, followed by the formation of a double Schiff’s base, the osazone.

24.10.3 Procedure

To 300 mg phenylhydrazine mixture add 5 ml of the sugar solution, shake well and heat on a boiling water bath for 30 to 45 mins. Allow the tube to cool slowly. Note the time required for crystal formation. Also examine crystals for size, shape, melting point etc. The crystals can be purified by recrystalization from alcohol.

24.10.4 Note

Determination of the time required for formation of the insoluble yellow osazone is a valuable means of identifying the various sugars. The time required for osazone formation (mins.) is given in table below:

Table 24.1 Time required for osazone formation