CHAPTER 3

MATERIALS AND METHODS

3.1Methods3.1.1Preparation 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

3.1.2Extraction 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.

3.1.3Extraction of Caffeine from CoffeeFifty 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 re-extracted 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

3.1.4Concentration using Rotary EvaporatorThe 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

3.1.5Testing of Crude CaffeineThe 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 2380C, the caffeine was observed and determined. The method was repeated for caffeine from coffee and the observation was recorded.

3.1.6Preparation of Caffeine Solution from Crude CaffeineCrude 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.

3.1.7Testing 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.

3.2MATERIALS3.2.1ApparatusBeakersThermometer250ml Extraction funnelFilter funnelSmall air tight bottleRound bottom flaskRotary evaporatorSpatulaAnalytical balancePetri dishWeighing boatLight microscopePipetteCounter

3.2.2ChemicalsTea bagsCoffeeDistilled waterMethylene chlorideAluminum foilAnhydrous sodium sulphatePallet sodium hydroxideFilter paperLens paper