Methodology for Carbohydrates

Color reactions of carbohydrate
Carbohydrates are widely prevalent in the plant kingdom, comprising the mono-, di-, oligo-, and polysaccharides. The common monosaccharides are the glucose, fructose, galactose, ribose etc. the disaccharides, i.e., the combination of two monosaccharides include sucrose, lactose and maltose. Starch and cellulose are polysaccharides consisting of many monosaccharide residues. Cellulose is most abundant organic compound on this planet since it forms part of cell wall in plants.

Aldehydes (-CHO) and ketones (=CO) are active groups in carbohydrates. Carbohydrates contain many hydroxyl groups as well. The number of hydroxyl groups varies with the number of carbon atoms. Monosaccharides contain the free aldehyde group (maltose) and some do not have the free ones (sucrose). The polysaccharides, starch and cellulose, are polymers of monosaccharides linked through the active groups.

The chemical properties of the saccharides vary depending upon the number of hydroxyl groups and the presence or absence of –CHO/=CO groups. These variations are the basis in the development of color reactions to identify the saccharides.

Some simple tests used to identify the presence/absence of certain saccharides are listed below:

» Iodine Solution: Add a few crystals of iodine to 2% potassium iodide solution till the color becomes deep yellow.
» Fehling’s Reagent A: Dissolve 34.65g copper sulphate in distilled water and make up to 500mL.
» Fehling’s Reagent B: Dissolve 125g potassium hydroxide and 173g Rochelle salt (potassium sodium tartrate) in distilled water and make up to 500mL.
» Benedict’s Qualitative Reagent: Dissolve 173g sodium citrate and 100g sodium carbonate in about 800mL water. Heat to dissolve the salts and filter, if necessary.
Dissolve 17.3g copper sulphate in about 100mL water and add it to the above solution with stirring and make up to volume to 1L with water.
» Barfoed’s Reagent: Dissolve 24g copper acetate in 450mL boiling water. Immediately add 25mL of 8.5% lactic acid to the hot solution. Mix well. Cool and dilute to 500mL.
» Seliwanoff’s Reagent: Dissolve 0.05g resorcinol in 100mL dilute (1:2) hydrochloric acid.
» Bial’s Reagent: Dissolve 1.5g orcinol in 500mL of concentrated HCl and add 20 to 30 drops of 10% ferric chloride.

The reactions of carbohydrates are given in table as below:

Reactions of Carbohydrates

Molisch’s Test
Add two drops of Molisch’s Reagent (5% 1-naphthol in alcohol) to about 2mL of test solution and mix well.

Incline the tube and add about 1mL of concentrated sulphuric acid along the sides of the tube.
Observe the color at the junction of the two liquids.
A red-cum-violet ring appears at the junction of the two liquids
The color formed is due to the reaction of alpha-naphthol with furfural and/or its derivative formed by the dehydration of sugars by concentrated sulphuric acid. All carbohydrates react positively with this reagent.
Iodine Test
Add a few drops of iodine solution to about 1mL of the test solution
Appearance of deep blue color
This indicates the presence of starch in the solution

The blue color is due to formation of starch-iodine complex.
Fehling’s Test
To 1mL of Fehling’s solution ‘A’, add 1mL of Fehling’s solution ‘B’ and a few drops of the test solution. Boil for a few minutes.
Formation of yellow or brownish-red precipitate
The blue alkaline cupric hydroxide present in  solution, when heated in the presence of reducing sugars, gets reduced to yellow or red cuprous oxide and it gets precipitated. Hence, formation of the colored precipitate indicates the presence of reducing sugars in the test solution.
Benedict’s Test
To 2mL of Benedict’s reagent add five drops of the test solution. Boil for five minutes in a water bath. Cool the solution.
Formation of red, yellow or green color/precipitate.
As in Fehling’s test, the reducing sugar because of having potentially free aldehyde or keto group reduce cupric hydroxide in alkaline solution to red colored cuprous oxide. Depending on the sugar concentration yellow to green color is developed.
Barfoed’s Test
To 1mL of the test solution add about 2mL of Barfoed’s reagent. Biol it for one minute and allow to stand for a few minutes.
Formation of brick-red precipitate.
Only monosaccharides answer this test. Since Barfoed’s reagent is weakly acidic, it is reduced only by monosaccharides.
Seliwanoff’s Test
To 2mL of Seliwanoff’s reagent add two drops of test solution and heat the mixture to just boiling.
Appearance of deep red color
In the concentrated HCL, ketones undergo dehydration to yield furfural derivatives more rapidly than do aldoses. These derivatives form complexes with resorcinol to yield deep red color.
It is a timed color reaction specific for ketones.
Bial’s Test
To 5mL of Bial’s reagent add 2-3mL of solution and warm gently. When bubbles rise to the surface cool under the tap
Appearance of green color or precipitate
It is specific for pentoses, they get converted to furfural. In the presence of ferric ion orcinol and furfural condense to yield a coloured product.
Test for non-reducing sugars such as sucrose:

Do Benedict’s test with the test solution
No characteristic color formation
Indicates the absence of reducing sugars in the given solution
Add 5 drops of concentrated HCl to 5mL of test solution in another test tube. Heat for five minutes on a boiling water bath.
Add 10% sodium hydroxide solution to give a slightly alkaline solution (test with red litmus paper). Now perform Benedict’s test with this hydrolysed solution
Appearance of red or yellow color
Indicated the formation of reducing sugars from non-reducing sugars after hydrolysis with acid.
Mucic Acid Test
Add a few drops of conc. HNO3 to the concentrated test solution or substance directly and evaporate it over a boiling water bath till the acid fumes are expelled. Add a few drops of water and leave it overnight
Formation of crystals
The both end carbon groups are oxidized to carboxylic groups. The resultant saccharic acid of galactose is called mucic acid which is insoluble in water.
Osazone Test
To 0.5g of phenyl hydrazine hydrochloride add 0.1g of sodium acetate and 10 drops of glacial acid. To this mixture add 5mL of test solution and heat on a boiling water bath for about half an hour. Allow the tube to cool slowly and examine the crystals under a microscope.
Glucose, fructose and mannose produce needle-shaped yellow osazone crystals. Whereas lactosazone in mushroom-shaped. Different osazones show crystals of different shapes. Maltose produces flower-shaped crystals.
The ketoses and aldoses react with phenyl-hydrazine to produce a phenylhydrazone which in turn reacts with another two molecules of phenylhydrazine to form the osazone.
1. For osazone test, the reaction mixture should be between pH 5 and 6. Fructose takes 2min to form the osazone whereas for glucose it is 5min. the disaccharides take a longer time to form osazone. Disaccharides form crystals only on cooling.
2. When a mixture of carbohydrates is present in the test sample, chromatographic methods should be employed to identify the individual sugars.

1. Sadasivam S and Theymoli Balasubramanian (1985). Practical Manual (Under Graduate) Tamil Nadu Agricultural University, Coimbatore p 2.