Molisch Test

Molisch test is the most common test for carbohydrates. To detect the presence of carbohydrates, samples is first treated with a strong acid. In the presence of concentrated sulphuric acid (H2SO4), the acid acts to hydrolyze the carbohydrates to monosaccharides. Then, the water is removed from monosaccharides, dehydrate monosaccharides to form furfural (for pentose) or 5-hydroxymethyl furfural (for hexoses). Rearrangement of pentoses and pentosans will form furfural while hydroxymethyl is produced from hexoses and hexosans. This compound in turn reacted with 2 -naphtol molecules present in Molisch reagent to form a condensed purple product, at which the rings formed between interface of carbohydrates and sulfuric acid. This is considered as

Tollen’s test and other similar tests, eg Benedict's and Fehling's, will test for aldehydes but will not identify individual compounds. They all rely on aldehydes being susceptible to oxidation whereas ketones are not readily oxidised.

Tollen’s reagent consists of a solution of ammonia and Argentum salt. It is an alkaline solution of ammoniacal silver nitrate and is used to test for aldehydes. Basic conditions are necessary because glucose is oxidised more easily under basic conditions:

RCHO + H2O RCOOH + 2H+ + 2e-

When some silver nitrate (AgNO3) is added to 1 ml of 1% NH4OH, a brown precipitate of silver oxide is formed which dissolves when a second measure of NH4OH solution is added. This precipitate dissolves in aqueous ammonia, forming the diamminesilver(I) ion, [Ag(NH3)2]+. Adding the ammonia to the silver nitrate solution makes the silver ion less susceptible to reduction, which produces silver in a more controlled manner. Ammonia forms a complex with the silver ion, which is more difficult to reduce than the silver ion. This is because silver ions form more stable complexes with NH3 than with water. Aldehydes will be oxidised to silver metal carboxylate ion and precipitated as "silver mirror" on the wall of the test tube. The diamminesilver ion was reduced to metal silver while the aldehyde in turn was oxidized to a carboxylic acid (gluconic acid). Ketones do not react with Tollens'

The solution is then added with NaOH to neutralize the acidity of the solution. Sodium hydroxide is necessary for neutralization as the reduction of the copper(II) ions will not take place in acidic conditions (with present of excess acids). After that, Benedict’s test is carried out by using Benedict’s reagent and heated. At this state, the solution is dark blue in color. Benedict's reagent is an alkaline solution containing Cu(II) ions, which oxidize the aldehyde to a carboxylic acid. In turn, the cupric ions are reduced to cuprous oxide, which forms a red precipitate.

Chemical reaction:

RCHO + 2Cu2+ + 4OH- → RCOOH + Cu2O + 2H2O

Again, Benedict’s solution contains copper sulphate. Reducing sugars, glucose and fructose, reduce soluble blue copper sulphate, containing copper(II) ions to insoluble red-brown copper oxide containing copper(I). This accounts for the precipitate formed.

The iodine test is used to detect the presence of starch. Iodine is insoluble in water. Therefore iodine dissolved in potassium iodide solution to form potassium triiodide solution, then used as a reagent in the test.Iodine form a colored complex with polysaccharide; starch gives blue color when reacted with iodine, while glycogen and partially hydrolyzed starch will react to form a red-brown