Extraction and isolation of Caffeine from tea leaves Reading assignment: Techniques in Organic Chemistry 2nd ed pages 158-159. 3rd ed pages 198-199. General background and overview of the experiment: Humankind has historically used advantageous compounds derived from plants and animals. For instance, various extracts from plants have been used as teas, potions, medicines and poisons. Though these extracts can contain a mixture of many different chemicals, often only one or few are responsible for the activity of the extract. For decades such extracts were used directly from their natural sources. This practice has many disadvantages, as the composition of the extract can vary from time to time and it is highly dependent on the availability of the natural source. At the beginning of the 19th century chemists made the first attempts to isolate the active components within these natural mixtures. The first compound to be isolated and purified was morphine from opium. Sertürner accomplished this by extracting opium with hot water and precipitating morphine with ammonia. He obtained colorless crystals that were poorly soluble in water but soluble in acids and alcohol. To make sure that the effect of the compound was identical to that of raw opium he tested the crystals on himself… Following this initial experimentation, many natural products were isolated and their structures were determined. Once a structure was elucidated, chemists were able to devise synthetic methods to synthesize these compounds. This was the beginning of modern pharmaceutical chemistry. Today, researches are still looking for new compounds from natural sources. Potential drugs are often isolated from sea creatures, like sponges or slugs, parts of plants that were used in traditional medicine, or new a species discovered in the rain forest. In this lab we will extract and purify caffeine from tea leaves. First water soluble compounds will be extracted from dry tea leaves with boiling water. Then, caffeine will be preferentially extracted from the water into organic solvent. The solvent will be removed and the crude material will be purified by sublimation. Caffeine extraction Caffeine (Figure 1) belongs to a group of compounds known as alkaloids. Alkaloids are a diverse group of compounds that are found primarily in plants and contain basic nitrogen atom(s). The basic nature of these compounds makes them exists mostly as salts. Other well-known alkaloids include morphine, strychnine, quinine, ephedrine, and nicotine. NNOOCH3NNH3CHOHOONCH3H Figure 1. Structure of caffeine (left) and morphineThe major sources for caffeine are the seeds of the coffee plant (Coffea Arabica), cola nuts, Mate which is used as tea in Paraguay and Tea leaves (Camellia Sinensis). Three types of tea are commercially produced from tea leaves (green, oolong and black) which differ only in their processing methods. To obtain green tea, fresh leaves are steamed to destroy the natural enzymes that cause fermentation. If however, the leaves are allowed to ferment an enzymatic oxidation process occurs which gives rise to oolong tea. Longer fermentation times yields black tea. Choosing extraction conditions for isolation of a product from its natural source depends on both the properties of the compound and the composition of the source. In our case caffeine is soluble in both water and organic solvents. It is possible to extract caffeine from leaves by solid/liquid extraction to hot water. The medium polarity of caffeine implies that it can be further separated from water soluble compounds by extraction to a polar non-protic solvent like methylene chloride (KCH2Cl2/H2O~10). Designing an efficient extraction scheme requires analyzing the major components of tea leaves. These include cellulose, proteins and amino acids, tannins, pigments and saponins. Cellulose The major component of leaves is cellulose, which serves as a rigid and insoluble structural element. Cellulose is a linear polymer composed of D-glucopyranose units connected through carbons 1 and 4. The fully equatorial conformation of β-linked glucopyranose residues stabilize the chair structure of the ring and minimize its flexibility. Although cellulose carries many hydroxyl groups, it is not water soluble due to its high molecular weight. OHOHOOHOHOOHOOHOHOOOOHOHHOOHOOHOHHOn123451234β (1-4) Figure 2. Structure of cellulose. Tannins Tannins are polyphenolic compounds (having OH on aromatic ring…) with molecular weights of 50-20,000. Tea tannins are soluble in water and therefore extracted from the leaf and responsible for the typical bitter taste of tea. Tea tannins belong to a subgroup named hydrolysable tannins. The core structure is D-glucose, to which several units of gallic acid are attached, via ester bonds. (Figure 3) It is important to note the gallic acid is poly functional, and able to form multiple ester bonds.HOHOHOgallic acidOHOHOOHOHHOOHOOOOOGGGOOOOGD- glucoseOOGG=ester bondOOH Figure 3. Typical structure of hydrolysable tannins. Note that multiple units of gallic acid are attached by ester bonds. The presence of soluble tannins in tea leaves complicates the isolation of caffeine, as low molecular weight tannins are also soluble in methylene chloride. However, we can take advantage of the chemical reactivity of the ester bonds in hydrolyzble tannins. When tea leaves are boiled in the presence of weak bases, such as CaCO3, the ester bond is cleaved. This cleavage produces glucose and a calcium salt of gallic acid. These very polar compounds will stay in water and will not be extracted into the methylene chloride. Additionally, the base also converts caffeine molecules the may be present as salts to the free base, increasing its solubility in methylene chloride. Proteins and pigments Proteins and pigments are very soluble in water and therefore do not present problems to the separation of caffeine by extraction. The content of pigments varies between different kind of teas and the level of oxidation that the leaves were exposed to. Saponins Saponins are compounds that have amphiphilic structure, i.e.