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BIOCHEMISTRY LABORATORYGeromil J. Lara, RMT, MSMT
QUALITATIVE TESTS FOR CARBOHYDRATES
MOLISCH TEST
– Carbohydrates are dehydrated by concentrated sulfuric acid to form hydroxymethylfurfural will react with alpha-naphthol (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 blue-green color
– The sample is mixed with sulfuric acid and the anthrone reagent and then boiled until the reaction is completed.
– Determines both reducing and non-reducing 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
GLUCOSAZONE
MALTOSAZONE
GALACTOSAZONE
LACTOSAZONE
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, which becomes more marked upon acidification
– Due to the liberation of aldehyde which subsequently polymerizes to form resinous substance, caramel
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.)
Tartrate ions as a complexing agent to keep the copper ion in solution.
Without the tartrate ions, cupric hydroxide would precipitate from the basic solution.
The tartrate ion is unable to complex cuprous ion Cu+, so the reduction of Cu2+ to Cu+ by reducing sugars results in the formation of an orange to red precipitate of Cu2O.
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)
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.
QUALITATIVE TESTS FOR CARBOHYDRATES
PICRIC ACID TEST
– Picric acid (yellow) is reduced to picramic acid (mahogany red) in alkaline solution
– Presence of reducing sugars
QUALITATIVE TESTS FOR CARBOHYDRATES
SELIWANOFF’S TEST– a chemical test which distinguishes
between aldose and ketose sugars– is based on the fact that, when heated,
ketoses (e.g. 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
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
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, with simultaneous production of maltose (colorless)
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
CHARACERIZATION OF LIPIDS
SOLUBILITY– Insoluble in water
– Insoluble in ordinary solvents
– Readily dissolve in chloroform, benzene, ether, boiling alcohol and other organic solvents
CHARACERIZATION OF LIPIDS
FORMATION OF TRANSLUCENT SPOT– Lipids have a characteristic greasy feel– When brought in contact with a substance
like paper, 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
REACTION OF FATS– Fatty acids are carboxylic acids and are
therefore weak acids– For fatty acids, the value of pKa is around
4.5. Therefore, generally speaking, fatty acids are neutral below pH 4.5 and charged above pH 4.5
– Fats containing high unsaturated fatty acids are neutral in reaction, 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