International University, Vietnam National University - HCMC

School of Biotechnology
Department of Biochemistry

ORGANIC CHEMISTRY LABORATORY

REPORT 7: LIQUID- LIQUID EXTRACTION
INSTRUCTOR: PhD. Hoàng Lê Sơn

TEACHING ASSISTANT: Nguyễn Thanh Phong

DATE OF SUBMISSION: 16-07-2019

Group member:

1. Trương Thị Ngọc Hằng - BTBCIU16041

2. Võ Minh Hạnh - BTBCIU16083

3. Nguyễn Hồng Anh Phương - BTBCIU16015

4. Nguyễn Tấn Phú – BTBCIU16057

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 International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry

I. Abstract
The experiment was about using a liquid-liquid extraction technique to remove
tea-consisting caffeine and recrystallization to reform caffeine crystals. The main
principle is the compound's polarity and solubility. The solution is separated into two
layers and the layer required either top or bottom layer is collected. After this
experiment, about 0.03 g of caffeine out of 30.00 g of tea was acquired, and the
percentage yield of crystallization was 60.00%.

II. Introduction
Tea is one of the world's most widely used caffeinated drinks. The caffeine
(C8H10N4O2) discovered in tea is a bitter, white, crystalline methylxanthine and a
member of a compound class known as alkaloids. Caffeine also triggers increased
respiration and heart rate, as well as nervousness and insomnia. Although caffeine
has demonstrated physical dependence, it is also capable of enhancing alertness,
learning ability, and exercise efficiency (NCBI, 2013). Tea leaves, where caffeine is
discovered, also contain acidic tannins, undecomposed chlorophyll, cellulose, and
pigments. To remove caffeine from tea leaves, caffeine must be present as the free
foundation (Amrita, 2013). To do so, the acidic substances mentioned above must
stay water-soluble.

The extraction of liquid-liquid is the method for separating compounds in

different solvents based on their relative solubility. It is an extraction of a substance
from one liquid to another liquid phase. The easy-to-use technique of static
extraction is commonly used and very efficient in the field of extraction analysis.

A separate funnel, also known as a separation funnel that operates on the

notion of "like dissolves like" that we learned in chemistry engineering. This implies
that if a compound is polar, it can only be dissolved in a polar solvent. In accordance
with this logic, a non-polar compound can only be dissolved in a non-polar solvent.
There are two phases in a separate funnel, the aqueous layer is polar, and the organic
layer is non-polar. Since these two layers share a surface, any polar substances that
were originally in the solution will be taken into the polar (aqueous) layer and any
non-polar substances will be drawn into the non-polar (organic) layer. The greatest
danger when using a separate funnel is pressure build-up. Pressure accumulates
during blending when gas responses happen. This issue can be addressed readily by
regularly opening the stopper at the top of the funnel while blending. This should be
performed with the top of the funnel pointing away from the body. Hold the stopper
in position when shaking, or it may dislodge causing the fluids to spill. Simply keep
the stopper in place with one side to account for this. The solvent is the key to
effective liquid-liquid extraction separation. Less toxic (non-toxic), not to mix water,
solubility of component in solvent, proportion of component capable of extraction,
density, vapor pressure, viscosity, interfacial tension, etc. Liquid-liquid extraction has

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 International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry

several applications in bioprocess technology: fermentation and algae broths,

removal of elevated boiling organics from sewage, removal of carboxylic acid,
protein separation and removal.

The advantages of this process include its effectiveness towards compounds

with high boiling points even at low concentrations, azeotropic mixtures (mixes with
points that, when boiled, have the same composition in vapor as initially, therefore
it cannot be modified by easy distillation) and heat-sensitive compounds such as
big organic molecules. The disadvantages are the high consumption of potentially
poisonous solvent, which also needs expensive machinery when reused in the
scheme. Thus, a proper choice of the solvent is needed in order to achieve a
sustainable process and generates difficulties for the large-scale sector to implement
the process.

III. Materials and chemicals

• Materials:

Some equipments were prepared, including one Erlenmeyer flask 250 mL, a
heating plate, a vacuum filter, one cylinder 10 mL and a cylinder 100 mL, a beaker
10 mL, a balance, and one separatory funnel 250 mL. Besides, the chemical reagent
was also prepared with the sample was dry tea leaf, methylene chloride, benzene,
and petroleum ether.

• Method:

The first step was the preparation of sample, tea solution was prepared by
boiling thirty grams of dry tea and 100 mL of water in ten minutes, after the tea
solution was filtered the binder by suction filtration. The liquid of caffeine in tea
was saved and kept it cool down, then the solution was transferred to the
separatory funnel 250 mL and then extracted three times with methylene chloride.
Methylene chloride allowed caffeine could be dissolved. When using the separatory
funnel containing solution did not shake the layers vigorously and use a swirling
motion. Moreover, the combination of methylene chloride extracts and evaporation
the extract to dryness on the steam bath made the crude caffeine, after transferring
the crude caffeine to a 50 mL beaker and added 5 mL of benzene, the heating it in
water bath. Benzene could dissolve chlorophyll in crude caffeine and also dissolve
caffeine. The important step to recrystallization was adding 10 ml of petroleum
ether, the product of recrystallization could be collected by suction filtration and
washed with 1 mL of petroleum ether which allowed to air-dry. Finally, the final
products were used for determining the melting boil.

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 International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry

IV. Result

1. Extraction process:

Figure 1: Two separate solvent layers Figure 2: Methylene chloride extract

Figure 3: Crude caffeine after extraction process

2. Purification process:

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International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry

Figure 4: Caffeine crystal

Figure 5: Recovered crystal weight

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 International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry

Figure 6: Caffeine’s melting point

V. Discussion
In the case of Caffeine extraction from tea powder, the organic solvent
dichloromethane is used because caffeine is more soluble in dichloromethane (140
mg/ml) than it is in water (22 mg/ml). The dichloromethane - caffeine mixture can
then be separated on the basis of the different densities of dichloromethane and
water because dichloromethane is much denser than water and insoluble in it. The
lower layer contained caffeine that diluted in methylene chloride.

Residual water is separated from dichloromethane by drain out the

dichloromethane through separating funnel, thus dichloromethane passed through
the funnel while polar solvents such as water is still remained in the funnel. Water
and dichloromethane is slightly soluble in each other. So, after separating the
solvents, residual water will remain the organic layer. Therefore, drying out the
solution to collect crude caffeine is necessity.

Recrystallize and determination melting point were performed to purify the

collected sample. In the recrystallize process, be careful to wash with petroleum

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 International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry
ether so as not to dissolve any caffeine. When putting petroleum ether, crystal was
formed. After filtrating and dryness was completed, caffeine was done in
crystallization. The percentage of recovery caffeine crystal from crude caffeine was
60%, and melting point of caffeine crystal was 223.8 degree Celsius, which was a bit
lower compare to 225 Celsius degree, official melting point of caffeine. It was
because of the impurity crystal, the crystal might be contaminated, or the paper
powder of the filter paper was mixed in.

VI. Conclusion

Caffeine is the world's most popular drug and can be found in many beverages
including tea. Caffeine was isolated from black tea leaves by using solid-liquid
extraction followed by liquid-liquid extraction. Therefore, solute in organic solvent,
caffeine was mix with methylene chloride which was the good organic solvent.

VII. Reference

• Organic Chemistry Laboratory Manual of the experiment 2. Ho Chi Minh:

International University, HCMC, 2018.
• vlab.amrita.edu,. (2011). Extraction of Caffeine from Tea. Retrieved 20 July 2019,
from vlab.amrita.edu

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