Biochemistry Lab - 06 Tests for Carbohydrates

BIOCHEMISTRY LABORATORY • MODULE 6 - TESTS FOR CARBOHYDRATES GENERAL TESTS FOR CARBOHYDRATES • • Molisch test o carbohydrates when treated with concentrated sulphuric acid undergo dehydration to give furfural derivatives o these compounds condense with α-naphthol to form colored products o this is a sensitive but a non-specific test and is given positive by all types of carbohydrates o reagent: α-naphthol in 95% ethanol o (+) result: an appearance of reddish violet or purple colored ring at the junction of two liquids Anthrone test o furfural derivatives from the reaction of the monosaccharides with concentrated sulfuric acid reacts w/ anthrone yielding green to blue green color o very sensitive test for carbohydrates that it will give a positive result with a filter paper (cellulose paper) o reagent: anthrone in diluted H2SO4 solution o (+) result: green to blue-green color (may vary on the quantity of sugar present) • • TEST FOR REDUCING SUGARS • • • Moore’s test o when a solution of reducing sugar is heated with an alkali (NaOH), it turns yellow to orange and finally dark brown, liberating the odor of caramel o reagent: alkali solution (NaOH) o (+) result: yellow-orange to dark brown solution with caramel odor Tollen’s test o test for reducing aldose from ketose o it is common known as the silver-mirror test, due to its positive result with aldoses o reagent: AgNO3 in aqueous NaOH (ammonia is used to dissolved the precipitate) o (+) result: silver mirror Fehling’s test o carbohydrates w/ free aldehyde/ketone groups have ability to reduce solutions of various metallic ions o the presence of aldehydes o reagent: Fehling’s A [blue aqueous solution of copper (II) sulfate pentahydrate] & Fehling’s B [clear solution of aqueous potassium sodium tartrate (Rochelle salt) and a strong alkali (NaOH or KOH)] o (+) result: brick red precipitate Nylander’s test o depends on the reduction of bismuth subnitrate in a weakly alkaline solution into metallic bismuth o it is also utilized as a medical test for the detection of glucose in the urine o reagent: bismuth subnitrate in alkaline (using NaOH or KOH) potassium sodium tartrate solution o (+) result: formation of black precipitate Benedict’s test o reducing sugars under alkaline conditions tautomerise and form enediols (powerful reducing agents) o they reduce cupric ions to cuprous form and are themselves converted to sugar acids o it is a semi-quantitative test; the color of the precipitate gives a rough estimate of a reducing sugar present in the sample Barfoed’s test o this test is used to distinguish reducing monosaccharides from disaccharides by controlling pH and time of heating o monosaccharides react very fast whereas disaccharides react very slowly o (+) result: scanty brick red precipitate SPECIFIC TEST FOR CARBOHYDRATES • Bial Orcinol test o test for pentoses o the test reagent dehydrates pentoses to form furfural o furfural further reacts with orcinol and the iron ion present in the test reagent o reagent: ferric chloride in orcinol-HCl solution o (+) result: formation of bluish product (all other colors indicate a negative result for pentoses) o hexoses generally react to form green, red or brown products o o • Mucic acid test o test for the presence of galactose o when galactose is treated with strong oxidizing agents such as HNO3, it yields a saccharic acid o this test allows for the differentiation of galactose (and lactose since it will be hydrolysed by the HNO3 to its monosaccharides) to other monosaccharides since the latter would yield soluble dicarboxylic acid while galactose produces insoluble mucic acid o reagent: concentrated nitric acid o (+) result: formation of insoluble crystals of mucic acid • Seliwanoff’s test o test for ketohexoses o this test is given positive by ketohexoses so it is answered by fructose, sucrose and other fructosecontaining carbohydrates o this test distinguishes between glucose and fructose o (+) result: cherry red color • Phenylhydrazine test o osazone crystal formation o a solution of reducing sugar when heated with phenylhydrazine, characteristic yellow crystalline compounds called osazones are formed o these crystals have definite crystalline structure, precipitation time and melting point for different reducing sugars o reagent: phenylhydrazine-HCl o (+) results: yellow to pale orange crystals with specific shape and formation • Iodine test o test for polysaccharides iodine forms a coordinate complex between the helically coiled polysaccharide chain and iodine centrally located within the helix due to adsorption the color obtained depends upon the length of the unbranched or linear chain available for complex formation