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BIOCHEMISTRY LABORATORY

Geromil J. Lara, RMT, MSMT

QUALITATIVE TESTS FOR CARBOHYDRATES
MOLISCH TEST
± Carbohydrates are dehydrated by concentrated sulfuric acid to form hydroxymethylfurfural will react with alphanaphthol (Molisch reagent) to yield a purple condensation product ± Not specific for carbohydrates

QUALITATIVE TESTS FOR CARBOHYDRATES ANTHRONE TEST ± Sugars react with the anthrone reagent under acidic conditions to yield a bluegreen color ± The sample is mixed with sulfuric acid and the anthrone reagent and then boiled until the reaction is completed. ± Determines both reducing and nonreducing sugars because of the presence of the strongly oxidizing sulfuric acid .

QUALITATIVE TESTS FOR CARBOHYDRATES OSAZONE FORMATION (Phenylhydrazine Test) ± Reducing sugars form characteristic osazone crystals when heated with an excess phenylhydrazine ± This property is attributed to the presence of aldehyde or ketone group in their molecules ± -OH group immediately adjacent to the keto group is oxidized to a keto group and adds to phenylhydrazine to form the yellow to pale orange osazones .

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GLUCOSAZONE .

MALTOSAZONE .

GALACTOSAZONE .

LACTOSAZONE .

caramel . which becomes more marked upon acidification ± Due to the liberation of aldehyde which subsequently polymerizes to form resinous substance.QUALITATIVE TESTS FOR CARBOHYDRATES MOORE¶S TEST ± When reducing sugar is heated with an alkali it turns yellow to orange ± Finally dark brown liberating the odor of caramel.

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QUALITATIVE TESTS FOR CARBOHYDRATES FEHLING¶S TEST ± Fehling I consists of 7 g of hydrated copper(II) sulfate dissolved in 100 mL of distilled water ± Fehling II is made by dissolving 35 g of potassium sodium tartrate and 10 g of sodium hydroxide in 100 mL of distilled water ± Equal volume ± deep blue solution .

QUALITATIVE TESTS FOR CARBOHYDRATES FEHLING¶S TEST ± Aldolases are easily oxidized to yield carboxylic acids ± Cupric ion complexed with tartrate ion is reduced to cuprous oxide (brick red ppt.) .

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. cupric hydroxide would precipitate from the basic solution.Tartrate ions as a complexing agent to keep the copper ion in solution. Without the tartrate ions. so the reduction of Cu2+ to Cu+ by reducing sugars results in the formation of an orange to red precipitate of Cu2O. The tartrate ion is unable to complex cuprous ion Cu+.

QUALITATIVE TESTS FOR CARBOHYDRATES NYLANDER¶S TEST ± Nylander¶s Reagent ‡ A solution of Rochelle salt (potassium sodium tartrate). potassium or sodium hydroxide. and bismuth subnitrate in water Bi(OH)2NO3 + KOH Bi(OH)3 + KNO3 reducing sugar 2 Bi(OH)3 2 Bi + 3 O + 3 H2O heat (black precipitate) .

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QUALITATIVE TESTS FOR CARBOHYDRATES BARFOED¶S TEST ± Barfoed¶s Reagent ‡ Cupric acetate and acetic acid ‡ Reducing monosaccharides are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide within three minutes. .

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QUALITATIVE TESTS FOR CARBOHYDRATES PICRIC ACID TEST ± Picric acid (yellow) is reduced to picramic acid (mahogany red) in alkaline solution ± Presence of reducing sugars .

g. when heated. ketoses (e.QUALITATIVE TESTS FOR CARBOHYDRATES SELIWANOFF¶S TEST ± a chemical test which distinguishes between aldose and ketose sugars ± is based on the fact that. fructose) are more rapidly dehydrated than aldoses ± Seliwanoff¶s Reagent ‡ Resorcinol and concentrated sulfuric acid .

QUALITATIVE TESTS FOR CARBOHYDRATES SELIWANOFF¶S TEST ± Seliwanoff¶s Reagent ‡ Resorcinol and concentrated sulfuric acid ± Hydrolysis of polysaccharides and oligosaccharides yield simple sugars ± dehydrated ketose then reacts with the resorcinol to produce a deep cherry red color ± aldoses may react slightly to produce a faint pink color ‡ Fructose (fast-reacting) and sucrose .

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QUALITATIVE TESTS FOR CARBOHYDRATES INVERSION OF SUCROSE ± Catalyzed by H ions to form glucose and fructose ‡ Sucrose is dextrorotatory ± Neutralized by barium hydroxide ± Barium sulfate = precipitate ± Glucose and Fructose = filtrate ± Seliwanoff¶s Test .

with simultaneous production of maltose (colorless) .QUALITATIVE TESTS FOR CARBOHYDRATES IODINE TEST ± Iodine in potassium iodide ‡ Reacts with starch to form a deep purple color ± Heating of the colored solution ‡ Lighter in color or diminishing purple color ± Add few drops of sodium thiosulfate ‡ To hydrolyze the starch to split fragments of gradually diminishing molecular size and complexity.

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QUALITATIVE TESTS FOR CARBOHYDRATES MUCIC ACID TEST ± Sugar + Nitric Acid ± Oxidation of galactose and hydrolysis of lactose will yield insoluble mucic acid ± Mucic acid crystals ± also known as galactaric acid ‡ Melts at 213 degrees Celsius ‡ Insoluble in alcohol and nearly insoluble in cold water .

ether. benzene. boiling alcohol and other organic solvents .CHARACERIZATION OF LIPIDS SOLUBILITY ± Insoluble in water ± Insoluble in ordinary solvents ± Readily dissolve in chloroform.

penetrate through it producing a translucent spot ‡ Fats are non-volatile ‡ In RT. the spot of water can absorb enough heat from the air and evaporized ‡ But the spot of grease can never absorb enough heat to evaporized ‡ When the liquid is inside the sheet of paper. it diffracts light ± TRANSLUCENT PHENOMENON .CHARACERIZATION OF LIPIDS FORMATION OF TRANSLUCENT SPOT ± Lipids have a characteristic greasy feel ± When brought in contact with a substance like paper.

5. fatty acids are neutral below pH 4.5 and charged above pH 4.5 ± Fats containing high unsaturated fatty acids are neutral in reaction. Therefore.CHARACERIZATION OF LIPIDS REACTION OF FATS ± Fatty acids are carboxylic acids and are therefore weak acids ± For fatty acids. generally speaking. the value of pKa is around 4. but when exposed to air become acidic due to hydrolysis which results from the liberation of volatile fatty acids .

CHARACERIZATION OF LIPIDS ACROLEIN FORMATION .2H2O Glycerol Acrolein (acrid odor) dehydrating agent (potassium bisulfate) .

CHARACERIZATION OF LIPIDS EMULSIFICATION OF FATS .

CHARACERIZATION OF LIPIDS EMULSION ± Is a mixture of two or more materials that are ordinarily immiscible ± Droplets of the dispersed component rapidly coalesce to form a separate layer ± Emulsifying agent must be present to stabilize the emulsion ± Lecithin in the egg will serve as emulsifier .

CHARACERIZATION OF LIPIDS SAPONIFICATION OF LARD ± Alcoholic Potash ‡ KOH dissolved in ethanol ‡ To neutralized fatty acids in the lard ± A metallic salt of fatty acid is formed ± Hydrophobic tails extend into the greasy droplets whereas the polar heads of the soap molecules face toward the water .

CHARACERIZATION OF LIPIDS SAPONIFICATION OF LARD ± Sodium Carbonate ± to produce hard soap Palmitin Palmitate + Glycerol Soap .

CHARACERIZATION OF LIPIDS SEPARATION OF CHOLESTEROL AND TRIGLYCERIDES ± Precipitate = cholesterol digitonide after digitonin precipitation ± Supernatant = triglycerides ± PRECIPITATE tested for Salkowski Test and Liebermann-Burchard Test ‡ Test for cholesterol .

CHARACERIZATION OF LIPIDS SALKOWSKI TEST ± The presence of a double bond in one cholesterol rings is responsible for its ability to form color products in the presence of concentrated inorganic acids ± Sulfuric acid ‡ Results in dehydration of cholesterol molecule with a formation of a red bicholestadien disulphonate ± Bluish color between the 1st layer (chloroform) and 2nd layer (H2SO4) .

CHARACERIZATION OF LIPIDS LIEBERMAN-BURCHARD TEST ± Deep green color (+) ± due to the hydroxyl group (-OH) of cholesterol reacting with the reagents (acetic anhydride and concentrated sulfuric acid) and increasing the conjugation of the un-saturation in the adjacent fused ring .

TRANSITIONAL PAGE .