a. Shinoda Test(Magnesium Hydrochloride reduction test): To the test solution add few fragments of Magnesium ribbon add concentrated hydrochloric acid drop wise, pink scarlet, crimson red or occasionally green to blue colour appears after few minutes.
b. Zinc Hydrochloride reduction Test: To the test solution add a mixture of zinc dust and concentrated hydrochloric acid. It gives red colour after few minutes.
c. Alkaline Reagent Test: To the test solution add a few drops of sodium hydroxide solution; formation of an intense yellow colour which turns to colourless on addition of few drops of diluted acid indicates presence of flavonoids.
3.6.4 Test for Proteins and Amino acids
→ Millon’s Test: Test with 2ml of millons reagent (Mercuric nitrate in nitric acid containing traces of nitrous acid), White precipitate appears, which turns red upon gentle heating.
→ Ninhydrin Test: Amino acids and Proteins when boiled with 0.2% solution of Ninhydrin (Indane-1, 2, 3-trione hydrate), Violet colour appears.
3.6.5 Test for sterols and triterpenoids
§ Libermann-Butchard Test: Extract treated with few drops of acetic anhydride, boil and cool, concentrated sulphuric acid is added from the sides of the test tube, shows a brown ring at the junction of two layers and upper layer turns green which shows the presence of triterpenoids.
§ Salkowski Test: Treat extract in chloroform with a few drops of concentrated sulphuric acid shake well and allow to stand for some time, red colour appears at the lower layer indicates the presence of Steroids and the formation of yellow coloured lower layer indicates the presence of triterpenoids.
3.6.6 Test for carbohydrates:
Molisch Test: Treat the test solution with few drops of alcoholic alpha naphthol. Add 0.2ml of concentrated sulphuric acid slowly through the sides of the test tube, a purple to violet colour ring appears at the junction.
Benedict’s Test: Treat the test solution with few drops of Benedict’s reagent (Alkaline solution containing cupric citrate complex) and upon boiling on water bath, reddish brown forms if reducing sugars are present.
Barfoed’s Test: It is a general test for monosaccharides. Heat the test tube containing 1ml of reagent and 1ml of solution of compound in a beaker of boiling water; if red cuprous oxide is formed within 2min, monosaccharide is present, whereas disaccharides on prolonged heating (About 10min) may also cause reduction, owing to partial hydrolysis to monosaccharide.
Caramelisation: Carbohydrate when treated with strong sulphuric acid, they undergo charring with the dehydration along with burning sugar smell.
Selwinoff’s Test: Hydrochloric acid reacts with ketose sugar to form derivatives of furfuraldehyde, which gives red coloured compound when linked with resorcinol. Add compound solution to about 5ml of reagent and boil.
Tollen’s Test: To 100mg of compound add 2ml of Tollen’s reagent and heat gently, a silver mirror is obtained inside the walls of test tube, indicates the presence of aldose sugar.
Bromine water Test: Bromine water gets decolourized by aldose but not by ketose.
Fehling’s Test: Equal volume of Fehling’s A (Copper sulphate in distilled water) and Fehling’s B (Potassium tartarate and sodium hydroxide in distilled water) reagents are mixed and few drops of sample is added and boiled, A brick red precipitate of cuprous oxide forms, if reducing sugars are present.
3.6.7 Test for fats and fixed oils
¤ Stain Test: Press the small quantity of extract between two filter paper, the stain on the paper indicates the presence of fixed oils.
¤ Saponification Test: Add a few drops of 0.5N alcoholic potassium hydroxide to small quantities of various extracts along with a drop of phenolphthalein separately and heat on a water bath for 1-2hrs. The formation of soap or partial neutralization of alkali indicates the presence of Fixed oils and Fats.
3.6.8 Test for Vitamin C
Sodium nitroprusside test: When the test solution is treated with Sodium nitroprusside solution, a blue colour is produced.
Sodium bicarbonate Test: when test solution is treated with sodium bicarbonate solution, a violet colour is produced.
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