CHAPTER 3

MATERIALS AND METHODS

3.1

Methods

3.1.1

Preparation of Daphnia sp.

Daphnia magna is obtained from Pusat Perikanan Air Tawar at Felda Titi, Jelebu, Negeri
Sembilan. The Daphnia magna was kept in an environment with bacteria as its food
source. The Daphnia magna is kept at room temperature.

Figure 3.1 Daphnia was placed in aquarium

3.1.2

Extraction of Caffeine from Green Tea

Seventeen tea bags were obtained and the net mass of tea in each bag was recorded. In a
500 ml beaker, 300 ml of distilled water was heated to just below its boiling point. The
tea bags were swirled for several minutes. The tea solution was allowed to cool at room
temperature. The tea solution was poured into a 250 ml of extraction funnel. Twenty
milliliters of methylene chloride (dichloromethane) was added and the swirling
technique was used. When two layers were present, the organic layer at the bottom of
the extraction funnel was carefully stored in a beaker covered with aluminum foil. The
aqueous layer was re-extracted two more times with 20 ml portions of methylene
chloride. All organic layers were collected in the same vessel. The combined layer was
taken and run passed a filter funnel with filter paper that contain 10g of anhydrous
sodium sulphate to dry the solution so that the solution looks transparent and 3 pallets of
sodium hydroxide that helps to deprotonates the caffeine to the organic solution. The
filtrate was collected in air tight bottle and placed in a box until extraction using rotary
evaporator.

3.1.3

Extraction of Caffeine from Coffee

Fifty grams of coffee was obtained and the net mass of coffee was noted. In a 500 ml
beaker, 400 ml of distilled water was heated to just below its boiling point. The coffee
was swirled for several minutes. The coffee solution was allowed to cool at room
temperature. The coffee solution was poured into a 250 ml of extraction funnel. 20 ml of
methylene chloride (dichloromethane) is added and the swirling technique was used.

When two layers were present, the organic layer at the bottom of the extraction funnel
was carefully stored in a beaker covered with aluminum foil. The aqueous layer was reextracted two more times with 20 ml portions of methylene chloride. All organic layers
were collected in the same vessel. The combined organic layer was taken and run passed
a filter funnel with filter paper that contain 10 g of anhydrous sodium sulphate to dry the
solution so that the solution looks transparent and 3 pallets of sodium hydroxide that
helps to deprotonates the caffeine to the organic solution. The filtrate was collected in air
tight bottle and placed in a box until extraction using rotary evaporator.

Figure 3.2 Separation of organic layer

3.1.4

Concentration using Rotary Evaporator

The round bottom flask was rinsed with a small amount of methylene chloride since it is
the organic layer that is used for tea solution and coffee solution. Then, the collected tea
solution was poured into the round bottom flask. The rotary evaporator was run with
small amount of methylene chloride to avoid any contamination of the crude caffeine.
The rotary evaporator was set to 75 revolutions per minute. The temperature of the water
was set to 40

because organic solvent used boils at 38 . Then, the rotary

evaporator was turned on. The rotary evaporator was kept running until the tea solution
was dried up on observation. The rotary evaporator was then turned off. The precipitate
was collected using a spatula into a dish. Then the total weight of collect precipitate was
measured using analytical balance. The precipitate was then transferred into an air tight
bottle labeled caffeine from tea. The extraction using rotary evaporator was then
repeated using coffee solution.

Figure 3.3 Rotary Evaporator

3.1.5

Testing of Crude Caffeine

The crude caffeine obtained from extraction of green tea and coffee was tested using
digital melting point machine. A capillary tube was pushed into the caffeine from green
tea. Only a small amount was needed for the capillary tube. The device was turned on
and the required melting point was set at 238 0C. The capillary tube containing caffeine
from green tea was placed into the device. Then, the start button was pressed. As the
temperature rise, the caffeine was observed through a window in the machine. At 238 0C,
the caffeine was observed and determined. The method was repeated for caffeine from
coffee and the observation was recorded.

3.1.6

Preparation of Caffeine Solution from Crude Caffeine

Crude caffeine of coffee of 0.002 g was weighed using analytical balance. 1 ml of

distilled water was added into a petri dish. The crude caffeine was then added into the
petri dish. The mixture was stirred so that the crude caffeine of coffee dissolved
completely into the water. This was the 0.2% caffeine solution. For 0.4% caffeine
solution, 0.004 g of crude caffeine was added to 1 ml of distilled water. For 0.6%
caffeine solution, 0.006 g of crude caffeine was added to 1 ml of distilled water. For
0.8% caffeine solution, 0.008 g of crude caffeine was added to 1 ml of distilled water.
For 1.0% caffeine solution, 0.010 g of crude caffeine was added to 1 ml of distilled
water. After all of the solution was prepared, it can be used for the Daphnia magna. It
was repeated for the crude caffeine from green tea.

3.1.7

Testing the Caffeine using Daphnia sp.

A light microscope was set up. A low power objective of 40x magnification was used.
One single Daphnia magna was selected from the Daphnia magna culture. By using a
pipette, the selected Daphnia magna was carefully sucked out of the culture and then
transferred to a slide along with an appropriate amount of pond water. By using a filter
paper, the excess water on the slide was absorbed so that the Daphnia magna lies on its
side and has limited movement so that it can be viewed under the microscope. A small
amount of water was left so that the Daphnia sp. can survive. The slide was then placed
on the microscope stage and held in position using stage clips. The microscope was
adjusted by first adjusting the coarse focusing knob and the fine focusing knob until a
fine image of the Daphnia magna was observed. The position of the slide was adjusted
until the heart of the Daphnia magna can be seen clearly.

One member of the group was assigned to observe the Daphnia magna using the
microscope and the number of heart beat made by the Daphnia magna within 30
seconds was counted using a counter. The time was recorded by another member. The
steps were repeated to obtain another 2 set of data from 2 more Daphnia magna. The
average reading was calculated from all of the data that was recorded. The Daphnia
magna was then returned to a separate beaker containing some pond water. Another
Daphnia magna was tested and placed into the same separate beaker. Then a few drops
of 0.2% caffeine solution were added to the Daphnia magna. The Daphnia magna was
left in the caffeine solution for 1 minute. After that, its heart beat was counted by the
same member and same method. These steps were repeated by using solutions of 0.4%,

0.6%, 0.8% and 1.0% respectively. Each average value of heart beat of Daphnia magna
was multiplied by two to obtain the value of heart beat of Daphnia magna in unit beats
per minute. The resulted were tabulated. A bar graph showing the comparisons of
Daphnia magna with treatments and without treatments was plotted. A line graph of the
heartbeat of Daphnia magna per minute against the concentration of caffeine solution
was drawn.

3.2

MATERIALS

3.2.1

Apparatus

Beakers
Thermometer
250ml Extraction funnel
Filter funnel
Small air tight bottle
Round bottom flask
Rotary evaporator
Spatula
Analytical balance

Petri dish
Weighing boat
Light microscope
Pipette
Counter

3.2.2

Chemicals

Tea bags
Coffee
Distilled water
Methylene chloride
Aluminum foil
Anhydrous sodium sulphate
Pallet sodium hydroxide
Filter paper
Lens paper