Molisch test

When heated with a strong acid, pentoses and hexoses are dehydrated to form furfural and hydroxymethylfurfural derivatives respectively (Figure 9.20), the aldehyde groups of which will then condense with a phenolic compound to form a coloured product. This reaction forms the basis of some of the oldest qualitative tests for the detection of carbohydrates, e.g. the Molisch test using concentrated sulphuric acid and a-naphthol. 

Molecular extinction coefficients, 1135 Molecular weight, by Rast camphor method, 1037 Molisch test, 453 ... 

There are a number of well-known tests for carbohydrates which depend on color formation. Thus in the Molisch test the substance is treated with mineral acid in the presence of a-naphthol and a characteristic purple color is formed. In Seliwanoff s test only ketoses give an immediate red color when warmed with mineral acid and resorcinol, and similarly ketoses give a blue coloration when warmed with sulfuric acid and diphenylamine in the Ihl-Pechmann reaction. 

In 1933, Fischer and Schmitz first claimed to have isolated a pure heparin preparation. Their lengthy extraction and isolation procedure culminated in the isolation of a microcrystalline brucine salt 32 times as active as the starting material. No evidence other than that of appearance was quoted in support of the claimed crystallinity of the product. It was concluded that heparin was a carbohydrate (Molisch test) and contained a uronic acid. 

The molisch test used for detecting carbohydrate in solution is based on following principle. When cone. H2SO4 is added slowly to a carbohydrate solution containing a-naphthol, a pink colour is produced at the juncture. The heat generated during the reaction hydrolyse and dehydrate it to produce furfural or hydroxymethyl furfural which then react with a-naphthol to produce the pink colour. 

A) Molisch Test for Carbohydrates. Apply the test to small amounts of the following sucrose, starch, and paper fibers. Place in a test tube 2 ml of one per cent carbohydrate solution or dispersion. Add 2 drops of a 10 per cent solution of a-naphthol in alcohol, and mix. Add this carefully down the side of a tube containing 2 ml of concentrated sulfuric acid so that it will form a separate layer. Carbohydrates give a purple color at the junction of the two liquids. 

Test for cellulose esters. Cellulose esters respond to the Molisch test for carbohydrates. The sample is dissolved in acetone and treated with 2-3 drops of 2% ethanolic solution of a-naphthol a volume of 2-2.5 ml of concentrated H2SO4 is so added as to form a lower layer. A red to red-brown ring at the interface of the liquids indicates cellulose (glucose). A green ring at the interface indicates nitrocellulose and differentiates it from other cellulose esters. 

The determination of the presence of carbohydrate in a sample is obtained by performing the Mohsch test [1] (see section 3.4 in Chapter 3 for the chemistry involved in the Molisch test). The test is sensitive down to 10 p,g/mL and is relatively broad but specific for all types of carbohydrates, with the exception of sugar alcohols, 2-deoxy sugars, and 2-amino-2-deoxy sugars or 2-acetamido-2-deoxy sugars. 

General color reactions of this type include the Molisch test with a-naphtfiol and concentrated sulfuric acid and the anthrone reaction with anthrone and sulfuric acid. The latter is used for quantitive colorimetric determinations. Reaction conditions can be chosen so that only ketoses react (HCl - - resorcinol, Seliwanoff s test) or only pentoses (HCl -b orcinol. Rial s test). These reactions help to distinguish the various sugars. 

There are a number of well-known colour tests for monosaccharides, such as condensation of the acid degradation products with phenolic substances. Perhaps the most frequently cited example of this is the Molisch test in which carbohydrates give a purple colour with a-naphthol in the presence of concentrated sulphuric acid. The presence of ketoses and of 3,6-anhydro-hexoses is often detected by the characteristic red colour rapidly developed with acid resorcinol (Seliwanoff test), and the presence of 2-deoxypentoses may be shown by the deep blue colour they yield with diphenylamine after degradation with acid to 5-hydroxy-levulinaldehyde. Before an individual sugar can be identified, however, it must be isolated as a pure substance in crystalline form or converted to a characteristic crystalline derivative. The derivative the author considers the best for the characterisation of the better known monosaccharides is given in Table I (p. 57). 

Slightly more than one half the constituents of fraction (G) were precipitated by basic lead acetate at pH 8, the active principle remaining in the filtrate (0). This preparation (0) was free of carbohydrates as judged by the Molisch test. Thus, proteins, fats, and carbohydrates had been eliminated from the raw beef liver without a noteworthy loss in activity. 

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