Phytochemical analysis for plant’s crude extract By……. Ass. Lec. Farah J. Hashim Ass. Lab. Sumaya S. Hashim Alkaloids Alkaloids generally include alkaline substances that have nitrogen as part of a ring structure. More than 6500 alkaloids are known and are the largest class of secondary compounds. They are very common in certain plant families. Detection of Alkaloids. Mayer’s test: Mayer’s reagent was prepared by: 1.Solution A: 1.358 g of Mercuric chloride was dissolved in 60 ml of distilled water. 2.Solution B: 2.5g of potassium iodide was dissolved in 10 ml of distilled water. Both solution were mixed and the volume was completed to 100 ml with distilled water. The test: Few drops of freshly prepared Mayer’s reagent were added to 5 ml of the sample, a white precipitate will appear if Alkaloids were present. Wagner’s reagent: Tow grams of potassium iodide was dissolved in 5 ml of distilled water ,then 1.27 g of iodide was added and the volume was completed to 100 ml with distilled water. The test: Few drops of freshly prepared Wagner’s reagent were added to 1 ml of the sample, a brown precipitate were appear of alkaloids were present. Terpenoids • Terpenoids are dimers and polymers of 5 carbon precursors called isoprene units (C5 H8). • Terpenoids often evaporate from plants and contribute to the haze we see on hot sunny days. They are expensive to make; they often take 2% of the carbon fixed in photosynthesis; carbon that could otherwise be used for sugars. Detection of Terpenoids and Steroids: Four milliliters of acetic acid anhydride and 1 ml of concentrated sulfuric acid( H2SO4 ) added to 2ml of chloroform then mixed with 1 ml of crude extract. The appearance of pink color indicated the presence of Terpens while formation of blue color after leaving the sample for 1minute indicated the presence of Steroids. Saponins Saponins are a class of chemical compounds, one of many secondary metabolites found in natural sources, with saponins found in particular abundance in various plant species. More specifically, the are amphipathic glycosides grouped, in terms of phenomenology, by the soap-like foaming they produce when shaken in aqueous solutions, and, in terms of structure, by their composition of one or more hydrophilic glycoside moieties combined with a lipophilic triterpene derivative. Detection of Saponins: Five milliliters of the sample were added to three ml of mercuric chloride (1%) solution ,formation of white precipitate indicating presence of saponins. Phenols • Compounds that contain a fully unsaturated six carbon ring linked to an oxygen are called phenolics. • Salicylic acid (basic part of aspirin) is a simple phenol. Detection of Phenols: Mix equal volumes of 1% ferric chloride solution and alcoholic crude extract .The appearances of blue-green color indicated the presence of Phenols. Flavonoids are complex phenolics . They are often sold in health food stores as supplements to vitamin C. The most commonly available flavonoid is rutin from buckwheat. Detection of Flavonoids: Four milliliters of 95% ethanol were added to 1 ml of the sample and then placed in boiling water path for 25-30 minutes. Then few drops of potassium hydroxide 0.5N were added to 5ml of the sample. The presence of flavonoids was indicated by the formation of dark yellow color. Tannin Tannin is a naturally occurring polyphenol found in plants, seeds, bark, wood, leaves and fruit skins. About 50% of the dry weight of plant leaves are tannins. As a characteristic of wine, tannin adds both bitterness and astringency as well as complexity. Wine tannins are most commonly found in red wine, although white wines have tannin from being aged in wooden barrels. Detection of Tannin: Few drops of lead acetate (1%) were added to 1ml of the sample, appearance of white gel precipitate indicating tannins were present. Resin Resin is a natural or synthetic compound that begins in a highly viscous state and hardens with treatment. Typically, it is soluble in alcohol, but not in water. The compound is classified in a number of different ways, depending on its exact chemical composition and potential uses. It also has many applications, ranging from art to polymer production, and many consumers interact with products that contain it on a daily basis. Detection of Resin: Ten milliliters of the sample were added to 20 ml of acidic distilled water with hydrochloric acid 4%,formation of turbidity Indicates to positive test. Glycoside A compound formed by the reaction of a pyranose sugar with a nonsugar molecule (an aliphatic or aromatic hydrocarbon) termed the aglycone. The aglycone replaces the hydrogen in the hydroxyl group of carbon atom one of the sugar ring. Glucose is the sugar component of many glycosides, such compounds being called glucosides. Some rare sugars are only found in glycosides, e.g. digilalose, which has only been detected in certain Digitalis glycosides Detection of Glycosides. Kid's reagent: Solution A: 1g of 3,5 di nitrate benzoic acid was dissolved in 50 ml of 95% methanol. Solution B: 1g of potassium hydroxide was dissolved in 50ml of 95% ethanol. Then mix 0.4 ml from solution A with 0.6 ml from solution B. The test: Add few drops of Kid's reagent to 5 ml of the sample, the presence of Glycosides is the formation of violet ring color.