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

GLUCOSAZONE

MALTOSAZONE

GALACTOSAZONE

LACTOSAZONE

QUALITATIVE TESTS FOR CARBOHYDRATES

MOORES 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

FEHLINGS 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

FEHLINGS 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

NYLANDERS TEST Nylanders 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

BARFOEDS TEST Barfoeds 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

SELIWANOFFS 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 Seliwanoffs Reagent
Resorcinol and concentrated sulfuric acid

QUALITATIVE TESTS FOR CARBOHYDRATES

SELIWANOFFS TEST Seliwanoffs 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 Seliwanoffs 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

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