PART 2 BIOCHEMICAL PHARMACEUTICALSUnit 6 Isolation of Caffeine from TeaIn this experiment，Caffeine will be isolated from tea leaves. The major problem of theisolation is that caffeine does not occur alone in tea leaves，but is accompanied by other natural substances from which it must be separated. The major component of tea leaves is cellulose，which is the major structural material of all plant cells. Cellulose is a polymer of glucose. Since cellulose is virtually insoluble in water，it presents no problems in the isolation procedure. Caffeine，on the other hand，is water soluble and is one of the major substances extracted into the solution called tea.”Caffeine comprises as much as 5 percent by weight of the leaf material in tea plants. Tannins also dissolve in the hot water used to extract tea leaves. The term tannin does not refer to a single homogeneous compound，or even to substances which have similar chemical structure. It refers to a class of compounds which have certain properties in common. Tannins are phenolic compounds having molecular weights between 500 and 3000. They are widely used to tan”leather. They precipitate alkaloidsz and proteins from aqueous solutions. Tannins are usually divided into two classes: those which can be hydrolyzed and those which cannot. Tannins of the first type which are found in tea generally yield glucose and gallic acid when they are hydrolyzed. These tannins are esters of gallic acid and glucose. They represent structures in which some of the hydroxyl groups in glucose have been esterified by digalloyl groups. The non-hydrolyzable tannins found in tea are condensation polymers of catechin. These polymers are not uniform in structure，but catechin molecules are usually linked together at ring positions 4 and 8. When tannins are extracted into hot water，the hydrolyzable ones are partially hydrolyzed，meaning that free gallic acid is also found in tea. The tannins，by virtue of their phenolic groups，and gallic acid by virtue of its carboxyl groups，are both acidic. If calcium carbonate，a base，is added to tea water，the calcium salts of these acids are formed. Caffeine can be extracted from the basic tea solution with chloroform，but the calcium salts of gallic acid and the tannins are not chloroform soluble and remain behind in the aqueous solution.The brown color of a tea solution is due to flavonoid pigments and chlorophylls，as well as their respective oxidation products. Although chlorophylls are somewhat chloroform soluble，most of the other substances in tea are not. Thus，the chloroform extraction of the basic tea solution removes nearly pure caffeine. The chloroform is easily removed by distillation(by 61C)to leave the crude caffeine. The caffeine may be purified by recrystallization or by sublimation. 68 Catechin Gallic Acid In a second part of this experiment，Caffeine will be converted to a derivative. A derivative of a compound is a second compound，of known melting point，formed from the original compound by a simple chemical reaction. In trying to make a positive identification of an organic compound，it is often customary to convert it into a derivative. If the first compound，Caffeine in this case，and its derivative both have melting points which match those reported in the chemical literature (e. g.，a handbook)，it is assumed that there is no coincidence and that the identity of the first compound，Caffeine，has been definitely established.Caffeine is a base and will react with an acid to give a salt. Using salicylic acid，a derivative salt of Caffeine，Caffeine salicylate，will be made in order to establish the identity of the Caffeine isolated from tea leaves. Special Instructions Be careful when handling chloroform. It is a toxic solvent，and you should not breathe it excessively or spill it on yourself. When discarding spent tea leaves，do not put them in the sink because they will clog the drain. Dispose of them in a waste container. Procedure Place 25g of dry tea leaves，25g of calcium carbonate powder，and 250ml of water in a 500ml three neck round bottom flask equipped with a condenser for reflux. Stopper the unused openings in the flask and heat the mixture under reflux for about 20 minutes. Use a Bunsen burner to heat. While the solution is still hot，filter it by gravity through a fluted filter using a fast filter paper such as E&D No. 617 or S&S No. 595. You may need to change the filter paper if it clogs.Cool the filtrate (filtered liquid)to room temperature and，using a separatory funnel， extract it twice with 25ml portions of chloroform. Combine the two portions of chloroform in a 100ml round bottom flask，Assemble an apparatus for simple distillation and remove the chloroform by distillation. Use a steam bath to heat. The residue in the distillation flask contains the caffeine and is purified as described below (crystallization). Save the chloroform that was distilled. You will use