Laboratory Experiments in Biochemistry for the Health Sciences

EXPERIMENT 3

Extraction of Caffeine from Tea

OBJECTIVES
 To isolate caffeine from different teas using
microscale extraction
 To assess the caffeine content of tea

DISCUSSION

Extraction is the general term for the recovery of a

substance from a mixture by bringing it into contact with a
solvent which preferentially dissolves the desired material.
The initial mixture may be a solid or liquid, and various
techniques and apparatus are required for different
situations. The reaction product is frequently obtained as a
solution or a suspension in water along with inorganic and
other organic byproducts and reagents. By shaking the
aqueous mixture with a water-immiscible organic solvent,
the product is transferred to it and may be recovered from
it by evaporation of the solvent.

The extraction of a compound from one liquid phase into

another is an equilibrium process governed by the
solubility of the substance in the two solvents. The ratio
of the solubilities is called distribution coefficient, K
= C1/C2, and is an equilibrium constant with a
characteristic value for any compound and pair of solvents
at a given temperature.

In this experiment you shall extract caffeine from tea

leaves. Caffeine is a member of a class of nitrogen-
containing compounds called alkaloids. Caffeine acts as a
stimulant and a diuretic. The compound is found in coffee,
cocoa and tea. Tea is a drink produced by boiling the leaves
of the tea plant Camellia sinensis. The leaves may be
fermented (black tea), non-fermented (green tea) and
Experiment 3  Extraction of Caffeine from Tea
Laboratory Experiments in Biochemistry for the Health Sciences

partially fermented (oolong tea).

Tea leaves contains cellulose (a water insoluble material),

tannins (phenolic compounds), caffeine and a small
amount of chlorophyll. To extract the water soluble
compounds, tea leaves are boiled in water. The cooled
solution is then extracted with an immiscible nonpolar
solvent like dichloromethane (DCM). Caffeine is more
soluble in DCM (140 mg/mL) than in water (22 mg/mL).
However, tannins are also soluble in DCM. To decrease the
solubility of tannins in DCM, the tea leaves can be boiled in
water with sodium carbonate. Sodium carbonate acts as a
base to convert tannins into their conjugate bases,
phenolic anions. Since these are salts, they are more
soluble in water than in DCM. The problem of converting
tannins into salts is that they act like detergents
(surfactants). In extraction we want to have immiscibility
between the organic and aqueous layers. The presence of
surfactants may produce an emulsion between the two
layers. To minimize the formation of emulsions, the
separatory funnel must not be shaken vigorously.

MATERIALS

Bring the following materials

 2 tea bags (green, black, oolong or white teas)
 Sufficient distilled water

Borrow the following materials

 Spatula  separatory funnel
 water bath  Stirring rod
 150 mL beaker  graduated cylinder
 Hot plate  (2) 50 mL Erlenmeyer
 watch glass flask
 centigram balance  Iron ring
 10 mL pipet  iron stand
 Aspirator  50 mL beaker
 Thermometer
REAGENTS

Substance Description
 dichloromethane Liquid
(DCM)
 sodium carbonate powder

Experiment 3  Extraction of Caffeine from Tea

Laboratory Experiments in Biochemistry for the Health Sciences

 sodium sulfate Anhydrous powder

SAFETY PRECAUTIONS
 Thoroughly clean your work area before and after
experimentation.
 Avoid inhaling the vapors of dichloromethane.
Perform the experiment in a well ventilated area.
 Wear your lab coat, safety goggles, safety masks and
lab gloves.
 Be careful handling heating equipment and hot
glasswares.

PROCEDURE

1. Open two tea bags by removing the staple. Weigh the

total contents using an analytical balance. Return the
contents to the bag and staple to secure the contents.

2. Place the bags into a 150-mL beaker, add 30 mL distilled

water and 2.0 g sodium carbonate. Place a watch glass
over the beaker and boil the solution for 10 minutes.

3. Using a thick cloth to protect your hands, decant the hot

liquid into an Erlenmeyer flask. Add 20 mL distilled water to
the beaker and again bring the contents to a boil. Decant
the hot solution to the Erlenmeyer flask also. Extract the
remaining liquid from the bags and transfer to the same
flask. Cool the combined tea extract to room temperature.
The tea bags can now be discarded.

4. Transfer the tea extract to a separatory funnel. Add 5 mL

DCM to the funnel. CAUTION: Avoid inhaling the vapors of
dichloromethane. The instructor shall demonstrate how to
use the separatory funnel. Agitate the funnel to mix the
contents but do not shake vigorously.

5. Allow the contents in the separatory funnel to settle.

There should be two layers. If there is cloudy layer
(emulsion), it is possible to break it by swirling the contents
of the funnel or stirring the middle layer with a stirring rod.

6. Drain the lower DCM layer into a beaker but be careful

not to include any of the aqueous (top layer). If there is a
Experiment 3  Extraction of Caffeine from Tea
Laboratory Experiments in Biochemistry for the Health Sciences

lot of emulsion, include it in the beaker.

7. Repeat steps 4 to 6 using a second 5 mL portion of DCM.

8. Add 0.5 g anhydrous sodium sulfate to the combined

DCM extracts in the flask. The sodium sulfate will absorb
the small amount water from the emulsion or from the
aqueous layer that accidentally drained into the flask.

9. Obtain the exact weight of a small beaker using a

centigram or analytical balance. Decant the liquid from the
flask into this beaker. Add a boiling chip and heat the
beaker in a water bath at a temperature not more than 35
C. You can also use an lectric fan to evaporate the solvent.
The solid that will remain is crude caffeine. Remove the
boiling chip and use the same balance to obtain the exact
weight of substance.

10. Calculate the percent caffeine in your tea sample

DISPOSAL AND CLEAN-UP:

All aqueous solutions can be discarded into the sink. Flush
with generous amounts of tap water. Place solids and paper
into the solid wastes container. Inform your instructor
before placing waste dichloromethane into the halogenated
organic waste container.

Experiment 3  Extraction of Caffeine from Tea

Laboratory Experiments in Biochemistry for the Health Sciences

NAME: ____________________________

GROUP: _________RATING:____________

LAB REPORT DATE PERFORMED: __________________

FOR DATE SUBMITTED: ___________________

EXPERIMENT 3
________________________________________________________________________

Extraction of Caffeine from Tea

RESULTS AND OBSERVATIONS

Tea sample used

Total weight of dried tea leaves (mg)

Weight of crude caffeine (mg)

Color of crude caffeine

Experiment 3  Extraction of Caffeine from Tea
Laboratory Experiments in Biochemistry for the Health Sciences

Percent caffeine in tea leaves

CALCULATIONS

LAB REPORT FOR EXPERIMENT 3 (continued) NAME:

POST-LAB QUESTIONS

1. Give other organic solvents which are often used for extraction.

2. State the characteristics of a good solvent for extraction.

Experiment 3  Extraction of Caffeine from Tea

Laboratory Experiments in Biochemistry for the Health Sciences

SUMMARY AND CONCLUSIONS

Experiment 3  Extraction of Caffeine from Tea