Date: 21 September 2017

Isolation of Caffeine from Tea Leaves

Pichayathida Siriwechdaruk, Salisa Simaroj, Pattranith Pattanaanunsuk, Nakreetipe

Sudhisanronakorn, Lalida Suriyanonrin

Abstract

An extraction for caffeine from tea leaves by the use of solid-liquid and liquid-liquid

extractions was performed in order to determine whether if caffeine exist within tea leaves or

not, and from the extraction, is the extraction pure or not. The following techniques has been

described: thin-layer chromatography (TLC), rotary evaporation, crystallization, and liquid-

liquid extraction. Moreover, the chemicals used within all of the process was identified and

described. At the end of the experiment, the existence of caffeine within tea leaves could not be

confirmed due to the error of the experiment.

Objective

This experiment was conducted in order to isolate caffeine from the tea sample prepared

with water to determine whether if there is caffeine in tea leaves or not and whether if the crude

sample obtained from the process of extraction is pure or not.

Introduction

This experiment includes the usage of various chemicals which are: dichloromethane,

ethanol, sodium sulfate, hexane, ethyl acetate, and iodine in order to extract caffeine from tea

leaves. The techniques used within this experiment are TLC (Thin Layer Chromatography),

liquid-liquid extraction, recrystallization, and rotary evaporation.

 Caffeine (8 10 4 2 ) is an natural occurring and existing organic compound. Caffeine

have the melting point of 238 degree Celsius. Caffeine could be found in seeds, nuts, or leaves of

plants [)] . The appearance of caffeine is a soft, white crystal or powder. It is odorless and have a

slightly bitter taste. It is classified as a central nervous system stimulant. In 150 ml of tea, the

approximated amount of caffeine is 2-5 mg [+] . Caffeine is polar, hence, it is water soluble [,] .

Dichloromethane (+ + ) is a clear, colorless, volatile, and non-flammable liquid

commonly used as solvent. The density of dichloromethane is 1.33 g/3 .If exposed to high

temperature, dichloromethane could emit harmful, toxic fumes. It is also commonly used in the

process of decaffeinating coffee [0] due to the greater solubility of caffeine in dichloromethane

than in water. The solubility of caffeine in dichloromethane is 140 mg/ml when the solubility of

caffeine in water is 22mg/ml [1] .

Ethanol (, + ) is a clear, colorless, highly flammable and volatile liquid with

pungent, bitter taste commonly used as disinfectant. It is also used as solvent. The density of

ethanol is 0.79 g/3 [2] .

Sodium Sulfate (+ 0 ) is an anhydrous salt, meaning that it doesnt contain water,

used as drying agent. It dissociates itself in water in order to form sodium ions (5 ) and sulfate

ions (4 26 ). It usually come in the form of colorless crystals or fine white powder. It is odorless

and tastes bitter and is insoluble in alcohol and is hardly dissolved in water [7] .

Hexane (6 14 ) is a naturally occurring acyclic hydrocarbon commonly used as

extraction solvent. Hexane could be found in citrus, scallops, and some species of plants. It is a

clear, colorless liquid with petroleum-like odor. It is non polar and, hence, is insoluble in

water [8] .
 Ethyl Acetate (, + , ) is a compound found in alcoholic beverages, radishes,

fruit juices, and cereal crops. It is commonly used as a solvent and typically used in the

manufacturing of decaffeinated coffee and tea. The physical characteristic of ethyl acetate is that

it is a clear, colorless liquid with fruity odor. It is a slightly polar compound due to the ester

group and hence is soluble in water [9] .

Iodine (2 ) is a dark violet to black solid crystals with slightly metallic luster to it. The

odor of iodine is very sharp and pungent. It is very volatile [);] and could be used in thin layer

chromatography (TLC) to react with the spots to make the spots visible [))] .

Thin Layer Chromatography (TLC) is a method used to analyze mixtures by separating

the composing compound, helping with identifying the compound and the number of compounds

composing the mixture. TLC consists of three steps, spotting, development, and visualization.

The sample that would be analyzed would be dissolved in volatile solvent to produce dilute

solution to be spotted onto the TLC plate. The step of spotting consist of using a capillary to

transfer the diluted sample onto one end of the TLC plate. Then, the step of development

follows. The step of development includes soaking the TLC plate into the solvent and let the

solvent, with the sample to move along the TLC plate with capillary action. The outcome

depends on the polarity of each of the compound present. The compound with more polarity

would be closer to the starting line when compared to the compound with less polarity. Lastly,

visualization is the step that the difficulty depends on the substance. If the substance is colored,

then visualization is just looking at the plate. However, as some of the compound is clear and

colorless, visualization process is needed. The visualization process could be done by exposing

the TLC plate to iodine vapor and letting it react with the spots on the TLC plate. From the TLC

process, retention factor (= ) could be determined by dividing the distance travelled by the
solvent over the distance from the starting line to the center of the spot. By using the retention

factor, the compound could be determined [))] .

Rotary evaporation technique is a technique used to separate volatile solvent and non-

volatile sample with most efficiency. It works by lowering the boiling point of the solvent

through the reduction of pressure. Then, the sample is rotated by the rotor vap in order to

increase the surface area of the sample, enabling the solvent to be evaporated while the sample is

heated. After that, the vapor of the solvent would be passing the coil filled with cold distilled

water in order to precipitate the solvent. After the process is done, the non-volatile sample would

be left within the rotating round bottom flask (evaporating flask) while the solvent would be

within the receiving flask [)+] .

Recrystallization is a process used to purify nonvolatile solids. It involves dissolving the

solid in a solvent at a temperature in which its almost boiling. Then, let the solvent cool down

and wait until the solution crystallizes. After the crystals developed, the crystals could be

collected, washed, and dried. This process do not involve any breakage of chemical bonds [),] .

Liquid-liquid extraction is a technique used in environmental, clinical, and industrial

laboratories. During this process, the solute separates into two immiscible (cannot be mixed)

phases, one is usually organic while another usually is aqueous [)+] .

Materials

1. Tea

2. Boiling water

3. Beakers

4. Graduated cylinder

5. Separatory funnel & cork

6. Erlenmeyer flask

7. Stand and clamp

8. Watch glass

9. Dichloromethane (+ + )

10. Tea sample

11. Ethanol (, + )

12. Sodium Sulfate (+ 0 )

13. Suction flask

14. Buchner funnel

15. Filter paper

16. Pump

17. Rotary Evaporator

18. Round bottom flask

19. Capillary tube

20. TLC plate

21. Ethyl Acetate (, + , )

22. Hexane (6 14 )
 23. TLC chamber

24. Iodine (2 )

25. Thermometer

26. Petri dish

27. Forcep

28. Melting capillary tube

29. Melting point apparatus

Procedure

Part 1: Caffeine Extraction

1. Prepare tea sample by letting the tea bags steep in boiling water. Leave the tea bag within

the water for a while in order to obtain the tea sample. This process is the solid- liquid

extraction. It is the extracting process of caffeine from tea leaves (solid) with hot water

(liquid).

2. Strain the tea leaves out of the tea solution by either picking out the tea bags or filter the

tea leaves out.

3. Retrieved 15 ml of the tea sample by using a graduated cylinder. Pour the obtained

sample into the separatory funnel. Making sure that the stopcock is closed before the

substance was pour into the funnel.

4. Obtain dichloromethane and pour it into a beaker. Cover the beaker with a watch glass in

order to keep the volatile substance within the beaker.

5. Measure 15 ml of dichloromethane by using graduated cylinder. Pour the measured

dichloromethane into the separatory funnel. Close the beaker that contains

dichloromethane in order to prevent the volatile substance to evaporate.

6. Hold the separatory funnel horizontally with two hands. Turn the stopcock open in order

to release the gas that had been produced within the separatory funnel. Turn the stopcock

back to the horizontal position in order to close the funnel again and avoid spillage.

7. Slightly mix the solution in the flask by moving in C-shape. Then, carefully release the

gas created inside the flask (raise the end in order and point. Repeat this step two to three

times.

8. After letting the solution rest still for a while, the organic part of the mixture will be

sinken at the bottom of the flask due to the higher density of dichloromethane compared

to water. Separate the dichloromethane and caffeine mixture from the aqueous solution

by gradually open the stopcock of the separatory flask to let the solution flow and then

close the stopcock when it almost reach the layer of the tea solution.

9. Collect the dichloromethane and caffeine mixture (the organic part) in the Erlenmeyer

flask.

10. Repeat step 5 to 10 for three times in order to extract as much caffeine from the sample as

possible.

11. Dispose the leftover aqueous solution and clean the separatory funnel.

12. Pour the organic mixture back into the separatory funnel and add 15 ml of ethanol into

the funnel in order to get rid of any water residue within the mixture. Wait for the

separation of the layers. If there is any separations present, proceed to gradually open the

stopcock of the separatory flask in order to transfer the dichloromethane, which sinks due

to the greater density, to an erlenmeyer flask

13. Add sodium sulfate to the dichloromethane. If the sodium sulfate clump together, add

more sodium sulfate to the dichloromethane until some of the sodium sulfate appears as

fine powder and does not clump together.

14. Prepare Buchner funnel with filter paper and place them on the suction flask which is

connected to the pump.

15. Open the pump, then carefully pour the solution into the Buchner funnel to filter out the

solid sodium sulfate from the mixture. Discard the filtered sodium sulfate.

16. Wash the funnel with dichloromethane, ensuring that all of the caffeine were transferred

into the flask and non were stuck onto the funnel and to recover some of the adsorbed

product.

17. Collect the organic solution, a mixture of caffeine and dichloromethane and transfer it

into a round bottomed flask.

18. Perform rotary evaporation by using a rotary evaporator in order to obtain pure

dichloromethane in a beaker and the solid caffeine extract would be left within the round

bottom flask.

19. Transfer the caffeine extract from the round bottom flask onto a watch glass.

20. Leave the crude caffeine extract to dry overnight.

21. Collect some of the crude caffeine extract to be purified through the process of

recrystallization.
Part 2: Thin Layer Chromatography

1. Prepare TLC plate and TLC chamber

2. Mark a line and two dots on the line, which is about one centimeter from the edge of the

plate, on one end of the TLC plate by using a pencil to indicate the starting line with the

points for the sample of crude caffeine obtained by the processes in the previous part and

the recrystallized caffeine. Be careful not to touch the matte side of the TLC plate.

3. Add some ethanol to the caffeine in order to make it easier to be transferred onto the TLC

plate.

4. Use a capillary tube to transfer the caffeine sample onto the TLC plate. Use separate

capillary tube for the crude caffeine and the purified caffeine.

5. Repeat step 4 for about 15-20 times, dotting the sample onto the TLC plate at the same

spot repeatedly in order to ensure that there is enough sample on the plate. Spot until a

small, white spot was made on the line.

6. Combine hexane and ethyl acetate by a 1:1 ratio to be used as the solvent (mobile phase)

of the TLC process.

7. Add the hexane and ethyl acetate mixture into the TLC chamber, trying to pour the

mixture into the chamber until it reaches about 1 cm over the bottom of the chamber so

that when the plate is placed, the solvent would be at the starting line.

8. Place the TLC plate into the chamber. Close the lid and let the process run until the

solvent reaches 1 cm under the top edge.

9. While TLC is running, prepare a petri dish with Iodine crystals. Close the lid of the petri

dish when not in use.

 10. Using a forcep, take the TLC plate out of the chamber. Place the plate into the petri dish

with the iodine crystals. Close the lid of the petri dish after the TLC plate was placed into

the dish.

11. Wait until dots appears on the plate.

12. Compare the distance of the dot from the starting line of both crude and purified caffeine.

Also, observe the amount of dots/spots present in each of the sample.

Part 3: Determining the melting point of the extracted caffeine

1. Insert one thermometer into the melting point apparatus.

2. Fill the crude caffeine sample into the melting capillary tube by using the open end of the

capillary tube to scoop the crystals into the tube. Tap the closed end of the capillary tube

against the table gently several times in order to force the sample to fall down into the

bottom of the tube. Repeat this step until there is 1-2 mm of packed sample within the

tube.

3. Repeat step 2 but replace the crude caffeine sample with the purified caffeine crystals

acquired from recrystallization.

4. Insert both capillary tubes into the block of the melting point apparatus.

5. Set the heating controller to 20@ /min

6. Reduce to 10@ /min once the thermometer reaches 60% of the expected melting point

7. Observe the melting of the substance through the eyepiece while recording the melting

point by observing the temperature shown by the thermometer.

Results

Figure 1: Caffeine extract before being dried overnight

Figure 2: Crude caffeine after dried overnight Figure 3: Purified caffeine acquired from recrystallization
 Figure 4: The TLC plate with the crude sample on the right (the one with dots) and the purified caffeine sample on

the left (right plate) and the TLC plate with purified caffeine prepared by the instructor (left plate)

The resulting substance from the process of extraction appears to be a fine powder

(Figure 1). The coloring of the fine powder is yellow to white before being dried overnight. The

white powder is in the center of the watch glass while the powder that appears more yellow in

color is on the outer edge. After being dried overnight and mixed, the crude caffeine (Figure 2)

powder appears creamy in color. The color is not white nor yellow. On the other hand, the

sample that has been purified by the process of crystallization (Figure 3) was lessen in amount,

but appears white in color. The size of the recrystallized caffeine and the crude caffeine is

relatively the same.

Through the TLC process, it could be observed that the crude caffeine is a mixture of at

least two to three substances due to the two to three spots showing on the TLC plate (Figure 4).

(Note: the spot is not very distinguishable, hence the approximated amount of spot is 2-3)

However, the substance within the recrystallized caffeine couldnt be distinguished since there is

no spot showing.
 From the measurement of melting point by using the melting point apparatus, it could be

seen that the melting point of the crude caffeine measured is within the range of 225 227 .

However, the melting point of the recrystallized caffeine couldnt be measured since the crystal

didnt melt.

Discussion

From the process of TLC, it was illustrated that the crude caffeine obtained from the

extraction process wasnt pure and was composed with at least two to three substances. On

contrast, the information regarding the purified caffeine couldnt be obtained from the

experiment, and hence, the purity of the recrystallized caffeine couldnt be concluded through

the process of TLC.

In terms of melting point, while the melting point acquired from researches is238@ , the

melting point of the crude caffeine extracted is in the range of 225 227@ , only 9 13@

different from the reference, hence, there is a possibility that the crude caffeine extracted is a

mixture of caffeine and compounds with lower melting point. From the observation and the

comparison of the melting point of the samples, it was shown that neither the recrystallized

caffeine nor the crude caffeine extracted was pure due to the difference in melting point.

From the experiment, it could be determined that the strongest intermolecular force

present in caffeine is dipole-dipole. There are two intermolecular forces present in caffeine:

dipole-dipole and london force. Since caffeine is a polar compound due to the presence of

Oxygen and the absence of the overall cancelling of electronegativity within the compound, the

dipole-dipole intermolecular force is present. Moreover, since london force is present in every

compounds, london force which is weaker than dipole-dipole intermolecular force is also present

in caffeine
 During the experiment, emulsion could be observed; especially during the liquid-liquid

extraction process. The emulsion could be a result of having a large amount of compounds with

low surface tension (which could be resulting from the weak intermolecular forces such as

london force), preventing the distinct separation of the two compounds. Another reason that

could be used to explain the presence of the emulsion is that the sample could contain

compound(s) with emulsifier-like properties and allow some of the polar and nonpolar

substances to interact. Formation of emulsion is common in liquid-liquid extraction due to the

need to properly mix the two substances (in this case, the tea solution and the dichloromethane)

together. In order to break the emulsion, the separatory funnel could be gently shaken, tapped, or

swirled. Another easy way to break the emulsion is to just wait for the separation of the two

layers. Moreover, the addition of the drying agent into the organic solution could also help with

the breakage of the emulsion between the two layers. The drying agent, in this case the

anhydrous sodium sulfate, will pull the residue of the unwanted aqueous mixture out from the

wanted mixture of caffeine and dichloromethane. If the drying agent wasnt put into the

extracted organic mixture before evaporation, the evaporated product might be impure since

some of the aqueous mixture might be present.

In this experiment, it couldnt be confirmed that caffeine is obtained from tea leaves due

to the error with the purified caffeine crystals obtained from recrystallizing the extraction.

However, it is likely that caffeine is obtained from the tea leaves due to its similar physical

characteristics and the similar boiling point. The crude sample obtained from extraction is

impure. The impurity of the sample could be confirmed by using TLC, where about two spots are

present on the TLC plate. Compared to the extract product with the others, it could be seen that
the extracted product is similar and both are impure. Two to three spots are present both the

product done by extraction by my group and the product produced by others.

The possible errors that lead to the inability to confirm whether if the crude sample

acquired through the process of extraction is caffeine or not are the lack of polarity of the

solvent. The reason that proves that the lack of polarity of the solvent could lead to the inability

of determining whether if the sample is caffeine or not is the clumping of the spots on the TLC

plate. This shows that the solvent (the mobile phase) doesnt have enough polarity to bring/repel

the components of the sample along the TLC plate. Moreover, the sample that was transferred

onto the TLC plate might not be enough for determining the components within the sample.

There are many possible errors that might lead to the impurity of the crude sample as

observed during the TLC process. Firstly, there might be other compound within tea leaves that

have similar properties as caffeine, meaning that it could also be extracted with dichloromethane

while the caffeine is being extracted. Secondly, there might be some impurities during the liquid-

liquid extraction, meaning that there might be some unwanted aqueous layer mixed with the

extracted organic layer. Also, during the liquid-liquid extraction process, the separation of layers

might not be completed due to the lack of time. During the TLC process, there might be some

contamination of the TLC plate due to touches and other compound that is preexisting on the

plate. Lastly, the waiting time during the process of transferring the sample onto the TLC plate

might not be enough and resulted the dispersion of the sample.

In order to avoid errors, a new solvent system that has more polarity might be used within

the lab and a new mobile phase that is more suitable to the sample might be used in the future.

Moreover, the time used in liquid-liquid extraction process might be added in order to let the

layers separate completely and the extraction could be done more carefully in order to lessen the
unwanted aqueous that is mixed with the wanted organic layer. Also, more sample could be used

during the TLC process in order to make sure that there would be some sample that could be

tested for sure.

Conclusion

To conclude, the experiment was unsuccessful since it couldnt be determined whether if

caffeine was extracted from tea leaves or not. However, the probability of the presence of

caffeine within the extraction from tea leaves is high due to the similar characteristic and boiling

point of the crude sample and the reference information on caffeine and it could be determined

that the extraction obtained from the tea leaves is impure due to the presence of more than one

spot during the TLC process.

References

[1] National Center for Biotechnology Information. (n.d). Caffeine. Retrieved September 21,

2017 from https://pubchem.ncbi.nlm.nih.gov/compound/caffeine#section=Top

[2] The Chemistry of Caffeine. (n.d). Retrieved September 23, 2017 from

http://www1.udel.edu/chem/C465/senior/fall00/Caffeine/Chemistry.htm

[3] Case Study: Removing caffeine from Coffee. (August, 2015). Retrieved September 23, 2017

from https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/

Physical_Properties_of_Matter/States_of_Matter/Supercritical_Fluids/Case_Study%3A_

Removing_caffeine_from_Coffee

[4] National Center for Biotechnology Information. (n.d). Dichloromethane. Retrieved

September 23, 2017 from https://pubchem.ncbi.nlm.nih.gov/compound/dichloromethane

#section=Top

[5] Extraction of Caffeine from Tea.(2011). Retrieved September 23, 2017 from

vlab.amrita.edu/?sub=3&brch=64&sim=169&cnt=1

[6] National Center for Biotechnology Information. (n.d). Ethanol. Retrieved September 23,

2017 from https://pubchem.ncbi.nlm.nih.gov/compound/702#section=Top

[7] National Center for Biotechnology Information. (n.d). Sodium Sulfate. Retrieved September

23, 2017 from https://pubchem.ncbi.nlm.nih.gov/compound/sodium_sulfate#section=Top

[8] National Center for Biotechnology Information. (n.d). Hexane. Retrieved September

24, 2017 from https://pubchem.ncbi.nlm.nih.gov/compound/hexane#section=Top

[9] National Center for Biotechnology Information. (n.d). Ethyl Acetate. Retrieved September

24, 2017 from https://pubchem.ncbi.nlm.nih.gov/compound/8857#section=Top

[10] National Center for Biotechnology Information. (n.d). Iodine. Retrieved September
 24, 2017 from https://pubchem.ncbi.nlm.nih.gov/compound/iodine#section=Top

[11] Thin Layer Chromatography.(n.d). .Retrieved September 24, 2017 from

http://www.chem.umass.edu/~samal/269/tlc.pdf

[12] ROTARY EVAPORATION: THE ROTOVAP. (n.d). Retrieved September 24, 2017 from

http://www.chem.ucalgary.ca/courses/351/laboratory/rotavap.pdf

[13] Recrystallization. (1999). Retrieved September 24, 2017 from

http://www.pitt.edu/~bandik/organicweb/recrystallization.html

[14] Harvey, D. (2017). Liquid-Liquid Extractions. Retrieved September 27, 2017 from

https://chem.libretexts.org/Textbook_Maps/Analytical_Chemistry_Textbook_Maps/Map

%3A_Analytical_Chemistry_2.0_(Harvey)/07%3A_Collecting_and_Preparing_Samples/

7.7%3A_Liquid%E2%80%93Liquid_Extractions