Chapter 3 Isolation, Purification, Identification Techniques

The objectives of this experiment are; to isolate caffeine from tea leaves through extractionto purify the crude material by sublimation; to identify the purified substance by measuring its melting point.

Background

Caffeine has the molecular formula C8H10N4O2with a molecular weight of 194.19 g/mol. Its chemical name is 3,7-dihydro-l,3,7-trimethyl-l H-purine-2,6-dione. Caffeine belongs to a group of compounds called alkaloids, more specifically, a member of the methylxanthines. The caffeine molecule has base characteristics (alkali-like) and the purine ring system, which is an important framework in living systems.

Caffeine is a chemical with a variety of uses. From medicines to beverages to foods, caffeine is one of the most popular natural products used today. It is the most widely used of all the stimulants and acts to stimulate the heart, central nervous system, and the respiratory system. Its usage can increase blood pressure, contraction force, and volume output by increasing heart rate. A small dose of this compound at an amount of 50 to 200 mg increases alertness and reduces drowsiness and fatigue. Caffeine is the main ingredient of many “stay-awake” pills. It is a smooth muscle relaxant and a diuretic. Caffeine is also a food additive. It can be found in popular soft drinks. However, it needs to be pointed out that caffeine has side effects. Large doses in excess of 200 mg can cause insomnia, restlessness, headaches, and muscle tremors. In addition, continued, heavy use of this chemical may lead to addictiveness. Furthermore, some research connects high caffeine consumption in pregnant women with the malformation of their children.

As a natural product, caffeine constitutes as much as 5% by weight of tea and coffee leaves, and is also present in cola nuts and cacao beans. It can be isolated from these natural sources through a process known as extraction, which is a chemical method of separating a specific component of a solution from the rest of the solution. This method is done by taking advantage of the solubility characteristics of a particular chemical with a given solvent. Caffeine is easily soluble in organic solvents such as chloroform or dichloromethane (CH2Cl2), but only partially soluble in water. In this experiment, caffeine is first separated from tea leaves using hot water since tea leaves consist primarily of cellulose which is insoluble in water. Hot water swells the tea leaves to release caffeine and other water soluble compounds such as tannins, complex substances which are colored phenolic compounds, of high molecular weight. Since tannins are acidic, they can react with a basic salt such as Na2CO3to form salts. These salts are soluble in water, but insoluble in organic solvents. Although caffeine is somewhat soluble in water, it is more soluble in the organic solvent dichloromethane. Therefore, dichloromethane can be used to selectively extract caffeine from its water solution. The sodium salts of the tannins remain behind in the aqueous solution. The dichloromethane solution is then passed through some solid Na2SO4to remove trace water. Evaporation of dichloromethane yields crude material of caffeine, which can be further purified by sublimation.

Sublimation is a phase transformation process in which a solid converts to a gas directly without going through the liquid state. Relatively few solids possess this kind of behavior at atmospheric pressure. Caffeine is one of the few examples. Other examples are solid compounds naphthalene (mothballs), iodine, and solid carbon dioxide (dry ice).

Chemicals possess characteristic physical properties which facilitate their identification. In many cases, a thorough determination of the physical properties of a given substance can be used for its identification. The physical properties of an unknown compound can be compared to properties of known substances that are tabulated in the chemical literature, and identification can be assumed if a match can be made. The physical properties most commonly listed in handbooks of chemical data include color, density, solubility in various solvents, melting point, sublimation characteristics, and so on. The melting point of a compound refers to the temperature at which the solid and liquid states are in equilibrium. A pure substance usually has a quite sharp melting transition and a very narrow range of melting point. Impurities lower the melting point and cause a broadening of the range. The criteria for purity of a solid are the correspondence to the value in the literature and the narrowness of the melting-point range. Thus the purity of caffeine after sublimation can be verified by its melting point. Pure caffeine forms white, hexagonal crystals, which can be ground into soft powder. Its melting point in chemical handbook is 238℃.

Procedures

Commercial tea bags are used as samples for the extraction of caffeine.

Isolation of Caffeine by Extraction

Without tearing the paper, open two tea bags with care. Weigh the contents to the nearest 1 mg and record this weight. Put the tea leaves back into the bags. Then close and securely seal the bags with staples. Place the tea bags in a 150-m L beaker and let the bags lie flat at the bottom. Add 30 m L of distilled water and 2.0 g of anhydrous Na2CO3into the beaker. Heat the water on a hot plate to a gentle boiling. Cover the beaker with a watch glass and continue heating for approximately 15 minutes. Keep the tea bags under water by occasionally pushing them back down with a glass rod, making sure that the tea leaves are covered with as much hot water as possible. Watch for loss of water, additional water may be needed.

Pour the hot, concentrated tea extract into a 50-m L Erlenmeyer flask. Add 10 m L of hot water and carefully press the tea bags with a glass rod. Be careful not to break the tea bags, since the presence of tea leaves in the solution will lead to additional difficulties in the separation process. Add the wash water to the tea extract in the flask. Filter any solids present in the tea extract by gravity. Discard the tea bags. Cool the combined tea solution using an ice-water bath.

Transfer the cool tea extract from the flask to a 125-m L separatory funnel supported on a ring stand with a ring clamp. Into the funnel, add 5.0 m L of dichloromethane. Stopper the funnel and lift it with two hands. Hold the stopper in place with one hand and invert the funnel to gently mix the contents three to four times. Be sure that the liquid is not in contact with the stopcock. When the funnel is inverted for mixing, open the stopcock to release any pressure built up by the volatile solvent. Always point the opening away from any person.

Put the separatory funnel back to the ring clamp. Remove the stopper and let the aqueous layer settle and separate from the dichloromethane layer, resulting in two distinct layers after a few minutes. Carefully manipulate the stopcock and drain the dichloromethane layer at the bottom into a 25-m L Erlenmeyer flask. Try not to transfer any of the aqueous solution along with the organic layer. Add a fresh 5.0 m L of dichloromethane and repeat the extraction. Combine the separated bottom dichloromethane layers. Dry the combined extract by adding 0.5 g of anhydrous Na2SO4. Swirl the flask for better performance.

Using a gravity filtration, filter the dichloromethane-salt mixture to a clean pre-weighed 25-m L side-arm filter flask containing one or two boiling stones. Rinse the salt on the filter paper with an additional 2.0 m L of dichloromethane. Gently heat the flask in a hot water bath to remove the dichloromethane by evaporation. The solid residue is crude caffeine. Weigh the flask to get its weight and determine the percentage yield.

Purification of Caffeine with Sublimation

The caffeine is now purified by sublimation, which is done with a cold finger condenser as the sublimation setup. Using some glycerin as a lubricant, carefully insert the cold finger condenser into a suitable neoprene adapter. Adjust the position of the cold finger so that its tip is 1 cm from the bottom of the side-arm filter flask containing the crude caffeine. Clean any remaining glycerin on the cold finger and dry the cold finger surface.

Connect the cold finger to a faucet with latex tubing and also connect the side-arm filter flask to a vacuum pump by vacuum tubing. Install a trap between the pump and the sublimation setup. When turning on the water, press the cold finger into the filter flask to ensure a good seal. Heat the sample gently and carefully with a microburner to sublime the caffeine. Hold the base of the microburner and move the flame around the flask. Avoid melting the sample. If the caffeine happens to melt, stop heating and allow it to cool before continuing the sublimation. When the sublimation is complete, discontinue heating and allow the system to cool while the vacuum is still on. After the system has cooled, remove the vacuum and carefully collect the purified caffeine from the cold finger.

Weigh the purified solid and calculate the percentage of caffeine in the tea sample.

Identification of Caffeine by Its Melting Point

Determine the melting point of the purified solid using a Thiele tube. First, collect a sample of the caffeine in a capillary tube and seal the tube with a torch afterwards. Attach the sealed melting-point capillary tube to the thermometer using a rubber ring, making certain that the tip of the melting-point capillary containing the solid is next to the mercury bulb of the thermometer. Support the Thiele tube on a ring stand with an extension clamp. Into the Thiele tube, fill silicone oil to a level which is above the top of the side arm. Using a thermometer clamp, support the thermometer with the attached melting-point capillary tube in the oil. Immerse the bulb and capillary tube in the oil, but keep the rubber ring and top of the capillary tube out of the oil.

Very slowly, heat the arm of the Thiele tube with a burner by using a small flame and gently moving the burner along the arm of the Thiele tube. Record the temperature when the solid begins to liquefy and the temperature when the solid is completely a liquid. These two temperatures define the melting-point range.

Compare the melting point obtained with the literature value.