A positive Molisch's test indicates the presence of a carbohydrate or any compound that dehydrates to furfural or hydroxymethylfurfural in the presence of H2SO4. A purple- or red-colored ring forming at the interface of the two liquids in the test tube indicates the positive result. Compounds other than carbohydrates cause false positives. However, a negative result is definitive in confirming the absence of carbohydrates.
A Molisch's test requires mixing the test solution with Molisch's reagent. One prepares this reagent by dissolving alpha-naphthol in 95 percent ethyl alcohol. After mixing the test solution and reagent, the individual adds 2 milliliters of the test solution to a test tube and pours 2 milliliters of sulfuric acid down the side of the slanted test tube without mixing. The mineral acid is very heavy, so it forms a bottom layer.
The color change is due to a condensation reaction between the sulfuric acid and the two molecules of the phenol. While directions generally call for alpha-naphthol, other phenols, including thymol and resorcinol, also create the colored ring.
The test works for all carbohydrates, including monosaccharides, disaccharides and polysaccharides. In addition, the test is positive for nucleic acid and glycoprotein, which convert to monosacharides upon exposure to strong mineral acids.