Applied Science BTEC Nationals/Chemical Laboratory Techniques/Caffeine Extract

Isolation Of Caffeine From An Impure Mixture (Tea).

This method is adapted from that of Eric Paul, Bergen Academies, 2000 [1]

The experimental details given here are given in good faith and are believed to be safe and workable methods. However, the authors cannot take responsibility for the consequences of performing these experiments.

The experiments are written for experienced science teaching staff to use as instructions for a supervised class of students. The experiments are not designed for students or inexperienced members of the public to perform without supervision. If you wish to attempt the experiments, ensure that you have completed a legally adequate risk assessment beforehand and that you work within the constraints of the risk assessment.

Introduction

Natural products are substances produced in living systems. They may be useful to humankind, especially in the field of medicine. For example, taxol is a substance derived from Pacific yew trees that has been shown to be effective against certain types of cancer. The extraction of such substances is necessary before their value can be evaluated.

The extraction of a natural product, caffeine, from tea leaves, has been chosen since the starting ingredients are relatively easy to come by, and we will still find a reasonable level of challenge. Caffeine is a natural product found in coffee and tea. It is chosen here because it is relatively easy to extract compared to some of the other substances we might be interested in. Efficient Extraction of caffeine from tea leaves relies heavily on the properties of caffeine and the other components present in the tea bag. Tea leaves constitute of cellulose, tannins, proteins and pigments, saponins and other minor components.

Caffeine

Objective

To obtain the greatest mass of caffeine from four bags of tea.

Reagents

• Dichloromethane (methylene dichloride):

The compound is highly volatile and the vapour irritates eyes, skin and lungs; depresses the central nervous system and may cause liver damage. Prolonged action may cause cardiac arrest. The liquid can be absorbed through the skin and is toxic if ingested. Use only in a fume cupboard. Wear gloves as well as usual protective clothing (labcoat and safety glasses).

• Sodium carbonate
• Anhydrous magnesium sulphate

Murexide test

• Potassium chlorate (I)
• Concentrated hydrochloric acid
• 2 mol dm⁻³ ammonia solution

Precautions

Wear eye protection and laboratory coat at all times.

Use dichloromethane and concentrated hydrochloric acid in fume cupboard, wearing gloves

Materials and Equipment

rotary evaporator machine	three 400-cm3 beakers
1000-cm3 beaker	basin for ice
ice (cubes or crushed)	tea (any tea containing caffeine)
petri dish	100-cm3 graduated cylinder
glass stirring rod	NaHCO3
500-cm3 separating funnel	ring stand with ring
expendable rubber tubing	dichloromethane
regular funnel	hood
goggles	250-cm3 conical flask
filter paper	Na2SO4 (anhydrous)
rubber tubing	aspirator or vacuum setup
round-bottom flasks	gloves
organic waste can	kitchen mitts
stirring rod	Bunsen burner, mat and tripod

Perform this activity in groups of three or four. Every member has a lab book in which procedures, observations, and data must be recorded. The members of each group have specific tasks to do.

Task #1 is safety engineer, the person who directs the others and reminds them to wear goggles and to follow other safety procedures.

Task #2 is balance technician, responsible for recording masses and other significant data.

Task #3 is “setups technician,” who sets up various pieces of equipment, such as the separating funnel.

For four-member groups, there is a director distinct from the safety engineer.

Method

1. Put on goggles.
2. Set up the separating funnel in advance. Place the ring stand in the hood. It is desirable to place rubber bumpers on the iron ring so as to protect the glass from breakage. The rubber for this will come from expendable rubber tubing. Cut a short section of tubing, then cut it open lengthwise, so that it can be wrapped around the ring. Place these bumpers approximately 120o from each other on the iron ring, and this will protect the funnel against damage when it is placed in the iron ring.
3. Prepare a basin of ice for cooling the beaker of tea later.
4. In a hood start heating the water in the rotary evaporator.
5. The objective is to produce the greatest mass of caffeine per tea bag. Students are given this objective in advance, so they can do research in the library, on the Internet, and by talking to experts, to identify promising teas for extraction of the most caffeine. Start with four tea bags.
6. Four tea bags are to be massed. It is important that tea leaves not leave the bags, so the bags should not be punctured, but strings and tags should be cut off and paper wrappers removed before massing.
7. Using the graduated cylinder, measure 100 cm³ of distilled water into a 400 cm³ beaker.
8. Mass 2.0 g of NaHCO₃ and use glass rod to stir into beaker of water.
9. Submerge tea bags in the solution in the beaker.
10. Place a clean, dry petri dish on the top of the beaker to prevent splattering. Thus covered, the beaker should be placed on a tripod and heated with a Bunsen burner. Continue heating until the water boils.
11. Decant the liquid to a 400 cm³ beaker and examine the liquid to see if any tea leaves have escaped the bags. If the liquid is free of tea leaves, then proceed to the next step. Otherwise, repeat steps 6 - 11.
12. Cool the tea by setting the beaker in a basin of ice.
13. Ensure separating funnel valve shut. Using an ordinary funnel, pour the tea into the separating funnel.
14. Put on gloves. (The subsequent steps, which involve dichloromethane, should be performed in a fume cupboard while wearing gloves.)
15. The separating funnel should be placed in the hood if it has not been already.
16. Using a graduated cylinder, measure 25 cm³ of dichloromethane. Dichloromethane and the tea mixture can form an emulsion difficult to separate if allowed to be agitated, so slowly pour the dichloromethane into the separating funnel.
17. Avoid shaking the separating funnel too vigorously. Stopper the separating funnel and gently tilt the mixture back and forth. Vigorous shaking will cause a bothersome emulsion to form.
18. After the bottom layer is removed from the separating funnel, it must pass through a drying agent (such as anhydrous Na₂SO₄). The purpose of this is to remove any H₂O from the bottom layer. A second filtration setup is used. Set up the conical flask close by the separating funnel, and place a funnel in the flask. Line the funnel with filter paper. Place a small amount of dichloromethane on the filter paper so that it will adhere to the sides of the funnel. This setup will be used in the next step to remove Na₂SO₄ after it has removed the last traces of water.
19. Place a conical flask under the separating funnel to catch the bottom layer. Unstopper the flask and gently open the valve to allow only the bottom layer to pass through. Turn the valve to the shut position before any of the top layer passes through. Add anhydrous Na₂SO₄ to the liquid and stir. Pour the contents of this flask through the paper-lined conical funnel so as to remove the Na₂SO₄.
20. Set up the rotary evaporator. Ensure tubing is hooked up for cooling water, including a secure hose leading to a drain. Turn on the cooling water.
21. Record the mass of a round-bottom flask. Transfer the filtrate to the round-bottom flask, and attach this to the appropriate end of the rotary evaporator. Ensure a receiving flask is also in place. Start the rotation of the sample flask. Gently lower the sample flask into the water, but if the solvent boils too violently, elevate the sample flask and lower the temperature of the water, then try again. Eventually, the solvent will evaporate and is collected in the receiving flask. The caffeine sample will encrust the first flask.
22. Weigh your flask with its contents to determine the yield, in grams, of caffeine.⁵ Remove your caffeine to a sample tube and label it.

Test small samples as follows

Measure the melting point and compare it to the expected value

Perform the murexide test:

(In the fume cupboard, wearing gloves)

1. In a watch glass, mix a small amount of your sample with 2-3 drops of concentrated hydrochloric acid. Use a glass rod for mixing.
2. Add a few small crystals of potassium chlorate (I) and mix well.
3. Heat the watch glass over a boiling water bath until the sample is dry.
4. Allow to cool.
5. Moisten with a drop of ‘bench’ (2 mol dm⁻³) ammonia solution. The sample should turn purple.

The test is for purines in general and gives murexide, a violet compound:

Murexide is a useful indicator for metals complexes such as nickel. The nickel complex is yellow.

Questions

1 Polyphenols are a potential contaminant. The addition of carbonate prevents them from contaminating the caffeine because the weakly acidic polyphenols undergo the following equilibrium:

Low pH phenol	High pH ‘phenoxide’ ion

Explain why this prevents polyphenols becoming an impurity.

2 Which layer is dichloromethane and which is aqueous? Give the densities of water and dichloromethane in your explanation.

3 What effect does this have? Record your observations and their explanation.

4 Why use this piece of equipment?

5 What is the amount of caffeine in tea? What is your percentage yield?

6 Identify the ‘amide’ parts of caffeine.

7 Phenols are aromatic molecules. Explain what this means. What is its main functional group?

References

Selinger, B (1978) Chemistry in the Market Place (2nd ed), John Murray (Publishers) Ltd, London p415-7

Mann, FG & Saunders, BC (1960) Practical Organic Chemistry (4th ed), Longman, London, p387

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Illustrated method