Caffeine quantification from tea leaves using solvent extraction

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Research work done by my IB student Eugene Leow. Please cite and give proper referencing to him on his work if you use this material.

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  • I am an IB student. My work is also loosely related to this, as I am also researching on caffeine. Thank you very much.
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Caffeine quantification from tea leaves using solvent extraction

  1. 1. International Baccalaureate Diploma Program Sri KDU Smart School Extended Essay -Chemistry- How will the period of fermentation and brewing temperature affect the amount of caffeine extracted from brewed Camellia Sinensis by solvent extraction technique using dichloromethane? Word Count: 3992 By: LEOW YUN JIN 002206 - 009 
  2. 2. Extended Essay LEOW YUN JIN 002206-009AbstractCamellia Sinensis contains polyphenolic flavonoids which have strong antioxidant propertiesthat bring health benefits but at the same time contains caffeine, a stimulant. High caffeineconsumption causes insomnia and constant consumption can be highly addictive. Caffeineshould be taken in moderate dosage and thus factors affecting the amount of caffeine inbrewed tea were investigated.Investigation on the period of fermentation was done by fermenting fresh tea leaves for 0, 40,80 and 120min. Further investigation on the effect of brewing temperature was carried out at25.0, 60.0, 80.0 and 100.0°C. Crude caffeine was extracted with dichloromethane, CH2Cl2using liquid-liquid extraction technique. Selected samples were further purified throughsublimation. The mass of crude and purified caffeine were calculated as the differencebetween the initial and final readings of boiling tubes and beakers respectively.The results showed significant differences (P < 0.05) between the period of fermentation andbrewing temperature on the amount of caffeine. No significant increase in purified caffeinewas found from 0-80min but there is a significant increase at 120min. Amount of crudecaffeine increases as the temperature increases from 25.0–100.0°C and a line of linearregression graph was plotted (R2 = 0.9641) showing a positive correlation.The effect of period of fermentation and brewing temperature on the amount of caffeine aresignificant. As the period of fermentation and temperature increase, the amount of caffeineincreases. In order to reap the health benefits of Camellia Sinensis but at the same timeminimising caffeine intake, tea leaves should be processed at shorter period of fermentationand being brewed at a lower temperature. Tea leaves are suggested to undergo minimumfermentation for flavouring and brewed at an optimum temperature between 60.0–80.0°C. (283 words) Page 1 of 42
  3. 3. Extended Essay LEOW YUN JIN 002206-009Table of ContentsAbstract........................................................................................................................................1Table of Contents.................................................................................................................................2Acknowledgement.........................................................................................................................31.0 Introduction.....................................................................................................................42.0 Hypothesis........................................................................................................................7 2.1 How Period of Fermentation Affects the Amount of Caffeine.............................7 2.2 How Temperature Affects the Amount of Caffeine.............................................83.0 Methodology Used to Investigate How Period of Fermentation Affects the Amount ofCaffeine........................................................................................................................................93.1 Devised Methods..............................................................................................................9 3.1.1 Collection of Samples..........................................................................................9 3.1.2 Tea Processing for Different Period of Fermentation...........................................9 3.1.3 Brewing Tea........................................................................................................11 3.1.4 Concentrating the Brewed Tea...........................................................................12 3.1.5 Extraction of Crude Caffeine.............................................................................124.0 Methodology Used to Investigate the Effects of Temperature on Amount ofCaffeine......................................................................................................................................144.1 Devised Methods............................................................................................................14 4.1.1 Selection of Tea Leaves.....................................................................................14 4.1.2 Brewing Tea.......................................................................................................14 4.1.3 Concentrating the Brewed Tea and Extraction of Crude Caffeine..........................................................................................................................155.0 Purification of Crude Caffeine.....................................................................................166.0 Thin Layer Chromatography (TLC) Test....................................................................177.0 Data Collection.............................................................................................................187.1 Method of Data Collection.............................................................................................18 7.1.1 Calculating the mass of crude caffeine..............................................................18 7.1.2 Calculating the mass of purified caffeine...........................................................18 7.1.3 Identifying the purity of purified caffeine..........................................................187.2 Raw Data........................................................................................................................19 7.2.1 Amount of Crude and Purified Caffeine Extracted at Various Period of Fermentation...................................................................................................19 7.2.2 Amount of Crude Caffeine Extracted at Different Temperature.....................................................................................................................20 7.2.3 Thin Layer Chromatography (TLC) Test.............................................................217.3 Data Processing..............................................................................................................227.4 Statistical Analysis: ANOVA and Tukey’s HSD test.......................................................257.5 Percentage of Caffeine Extracted by Liquid-liquid Extraction........................................288.0 Evaluation......................................................................................................................29 8.1 Significant of Studies.........................................................................................31 8.2 Limitations..........................................................................................................32 8.3 Ways of Improvements......................................................................................33 8.4 Unresolved Questions for Further Investigations................................................349.0 Conclusion......................................................................................................................3510.0 Appendix........................................................................................................................3611.0 Bibliography...................................................................................................................42 Page 2 of 42
  4. 4. Extended Essay LEOW YUN JIN 002206-009 Acknowledgment Special thanks to: Lawrence Kok (Supervisor) Joel Micheal Lim Ju Anne Kalai Leow Wooi Hian Page 3 of 42
  5. 5. Extended Essay LEOW YUN JIN 002206-0091.0 Introduction Figure 1: Molecular structure of caffeine1,3,7–trimethylxanthine (C8H10N4O2) or best known as caffeine is a naturally occurringalkaloid1 that belongs to the xanthine2 chemical group. Caffeine is known as a drug. Itstimulates the cardiovascular and central nervous systems of the body. Caffeine consumedthrough foods and beverages is readily absorbed into the blood stream. It takes effectsrapidly, about 15 minutes after consumption and its level peaks after an hour. It will stay inthe body for several hours and to eliminate half the amount of caffeine will takes up to sixhours3. In the human brain, a natural occurring xanthine called adenosine4 serves as aneurotransmitter. Adenosine is responsible for sleepiness as it will binds with its receptors inthe brain cells causing the nerve cells activity to slow down. Caffeine is able to interfere withadenosine at various sites in the brain including the reticular formation by binding withadenosine receptors thus speeding up nerve cells. This results in decreased fatigue and1 A class of naturally occurring compounds containing nitrogen and having the properties of an organic amine base2 A purine base found in most body tissues, fluids and in other organisms.3 Caffeine has a half-life of 6 hours(http://www.psych.umn.edu/courses/spring05/dionisiod/psy3061/caffeine.htm)4 An inhibitory neurotransmitter that plays a role in promoting sleep Page 4 of 42
  6. 6. Extended Essay LEOW YUN JIN 002206-009increased alertness thus allowing a person to stay awake. Besides that, caffeine has the abilityto increase the heart rate, blood pressure and basal metabolic rate (BMR) for several hours. Italso acts as a diuretic which increases urination. Caffeine, a plant–based alkaloid can be found in tea, coffee and Cocoa. Tea, a populardrink among all ages is rich in polyphenolic flavonoids5 which have strong antioxidantsproperties despites containing caffeine. Flavonoids play a role in preventing cancer byprotecting cells from free radical damage. It also helps in keeping the heart healthy andresearch in Europe shows that drinking three or more cups of tea per day reduces the risk ofheart diseases. [1] Despites all the goodness of drinking tea, the negative effects of caffeine cannot beignored. Caffeine can cause insomnia, headache, nervousness and dizziness when consume inhigh doses. It also causes addiction and without it the addict will be unable to concentratewell and suffers depression. Thus, caffeine should be taken in moderate dosage.6 Due to thesereasons, I choose to focus my research on tea. Tea is produced from the leaves and buds ofthe Camellia Sinensis plant through a series of processes. Tea leaves will start to oxidise afterplucking and will take about one and the half hour to two hours to be fully fermented7. Tealeaves can be left unfermented or fermented as it will produce a range of teas from white teato black tea.5 Chemical substances that have antioxidant properties6 See appendix 17 Result from oxidation reaction but known as fermentation in the tea industry Page 5 of 42
  7. 7. Extended Essay LEOW YUN JIN 002206-009Figure 2: Camellia Sinensis plant which is use to produce tea. Tea promotes health but at the same time it causes health problems due to its caffeinecontent. Thus, to be able to reap the benefits of tea but at the same time minimising theadverse effects of caffeine is by consuming the lowest possible amount of caffeine. There areseveral factors that affect the amount of caffeine in brewed tea. I will explore the possibilityeffects of period of fermentation and brewing temperature on the amount of caffeine inbrewed tea. Processed tea leaves will be brewed using water and the caffeine will beextracted by liquid-liquid extraction using dichloromethane, CH2Cl28. Therefore, my research question is:How will the period of fermentation and brewing temperature affects the amount ofcaffeine extracted from brewed Camellia Sinensis by solvent extraction technique usingdichloromethane?8 An organic solvent used to extract caffeine Page 6 of 42
  8. 8. Extended Essay LEOW YUN JIN 002206-0092.0 Hypotheses2.1 How Period of Fermentation Affects the Amount of CaffeineCaffeine is an alkaloid known to bind with other compounds that can be found in tea such astheine9 and other polyphenols, to form insoluble complexes. The breaking of cell walls whichis done by bruising the tea leaves causes the enzymes present in the tea leaves to mix withother chemical substances within the cells allowing fermentation. A series of complexchemical reactions begins, one of the most important reactions being the oxidation ofpolyphenols by the enzyme, polyphenoloxidase. In this reaction, an oxygen atom which isderived directly from the air is added into the polyphenol in the presence ofpolyphenoloxidase. The oxidised polyphenols will no longer be able to combine with caffeineto form insoluble complexes thus affecting the amount of extractable caffeine in tea leaves.The fermentation process can be stopped by subjecting the tea leaves to 100°C throughheating. Polyphenoloxidase will be denatured causing the oxidation of polyphenols to stop aswell. Thus, my hypothesis is that as the period of fermentation increases, more polyphenolsare being oxidised by polyphenoloxidase causing less insoluble complexes to be formed withcaffeine. This means that the amount of extractable caffeine through liquid-liquid extractionwill increase as the period of fermentation increases. [2]9 A type of polyphenol that exists in Camellia Sinensis Page 7 of 42
  9. 9. Extended Essay LEOW YUN JIN 002206-009 ↑ Period of fermentation ↑ Polyphenols is oxidised by polyphenoloxidase ↓ Polyphenols combine with caffeine to form insoluble complexes ↑ Extractable caffeine Scheme 1: A summary on the effects of period of fermentation.2.2 How Temperature Affects the Amount of CaffeineTemperature of the water used during tea brewing will also affects the amount of caffeineextracted from the tea. My hypothesis is that as the temperature increases, the rate ofdiffusion of caffeine into the water increases as the caffeine particles gain more kinetic 1energy. The kinetic theory states that average Kinetic Energy, = mv2 is directly 2 3proportional to absolute temperature, T where = . Thus, the average velocity, is 2directly proportional to the T as the mass, m remains constant. The relates to the averagevelocity of caffeine molecules diffusing out from the tea leaves into the water. Therefore, asT increases, caffeine molecules gain more causing an increased in thus allowing ahigher rate of diffusion. I strongly believed that as the brewing temperature increases, theamount of caffeine in brewed tea increases. Page 8 of 42
  10. 10. Extended Essay LEOW YUN JIN 002206-0093.0 Methodology Used to Investigate How Period of Fermentation Affects theAmount of Caffeine.3.1 Devised Methods3.1.1 Collection of Samples 1. Fresh tea leaves were collected from BOH Plantation10 and were kept in air tight containers. BulbSecond Leaf First Leaf Third LeafForth Leaf Figure 3: The shoot of Camellia Sinesis plant which is use to produce tea.3.1.2 Tea Processing for Different Period of Fermentation 1. Some sample of fresh tea leaves were heated in the oven at 100°C; 2. Tea leaves were mix at every 10minutes and once dried they were kept; 3. The remaining samples were being separated evenly into three portions;10 http://www.boh.com.my/ Page 9 of 42
  11. 11. Extended Essay LEOW YUN JIN 002206-009 4. All three portions of the samples were bruised11 mechanically using hands and being spread out on a flat surface exposing them to air. 5. The three portions were heated under the same condition after being left at different time intervals of 40, 80 and 120minutes and were kept.Figure 4: Tea leaves being heated in the oven at 100°CFigure 5: Tea leaves being exposed to the air for fermentation.11 The breaking of the cell walls to release the cell contents including enzyme for fermentation. Page 10 of 42
  12. 12. Extended Essay LEOW YUN JIN 002206-0093.1.3 Brewing Tea 1. Triplicate samples for different period of fermentation containing 3.000g of tea leaves each were weighed using electronic balance (±0.001g); 2. 100cm3 of distilled water at room temperature, 27.0°C was added; 3. All samples were heated using Bunsen burner for 20minutes; 4. 5cm3 of sodium carbonate solution12 was being added into each beaker and the mixtures were stir for 15seconds; 5. Tea solution was filtered using sieve into conical flaks; 6. All samples were kept and being labelled.Figure 6: Triplicate samples being brewed.12 Used to neutralise the tannin acid from tea leaves Page 11 of 42
  13. 13. Extended Essay LEOW YUN JIN 002206-0093.1.4 Concentrating the Brewed Tea 1. Triplicate samples at four different period of fermentation were being heated using Bunsen burners; 2. Tea solutions were left to boil until 25cm3 was left.3.1.5 Extraction of Crude Caffeine Using Dichloromethane 1. All boiling tubes were pre-weigh using the electronic balance (±0.001g) and labelled; 2. The first set of triplicate samples were transferred into 50cm3 conical flasks and wash bottle was used to wash down all the tea solution; 3. 2cm3 of dichloromethane (DCM) was added into each conical flask using a calibrated glass pipette and conical flasks were being swirled slowly for a constant time period; 4. Mixtures were left tilted to settle the emulsion formed; 5. Glass pipette was used to draw the lower organic layer into its respective boiling tube. Then, another 2cm3 of DCM is added; 6. Caffeine was extracted three times from each sample; 7. Steps 3-6 were repeated for the other two samples and the organic layer was transferred into their respective boiling tubes; 8. Boiling tubes were placed in a water bath of 100°C to evaporate the DCM leaving behind the crude caffeine; 9. A small amount of DCM was then used to wash down all the crude caffeine13 to the bottom of the boiling tube and DCM was evaporated again; 10. Steps 2-9 were repeated for the other three sets of triplicate samples; 11. All boiling tubes were weighed.13 Caffeine with the presence of other impurities such as chlorophyll Page 12 of 42
  14. 14. Extended Essay LEOW YUN JIN 002206-009 Aqueous Layer Organic LayerFigure 7: Liquid-liquid extraction using Dichloromethane (DCM). Page 13 of 42
  15. 15. Extended Essay LEOW YUN JIN 002206-0094.0 Methodology Used to Investigate the Effects of Temperature on Amount ofCaffeine4.1 Devised Methods4.1.1 Selection of Tea Leaves 1. BOH tea that can be purchase in the store will be used. This allows the research to be relevant to a bigger population.4.1.2 Brewing Tea 1. Triplicate samples for four different temperatures containing 5.000g of tea leaves each were weighed using electronic balance (±0.001g); 2. 100cm3 of distilled water at room temperature, 27.0°C was added into the first set of triplicate and were stirred; 3. After 20minutes, 5cm3 of sodium carbonate solution was added into each beaker and were stir for 15seconds; 4. Tea solution was filtered using sieve into conical flaks; 5. Steps 2-4 were repeated for another three sets of triplicate samples using distilled water at 60.0°C, 80.0°C and 100.0°C which were left in water baths set at their respective temperatures. 7. All samples were kept in conical flasks. Page 14 of 42
  16. 16. Extended Essay LEOW YUN JIN 002206-009Figure 8: Water baths set at temperature of 60°C, 80°C and 100°C.4.1.3 Concentrating the Brewed Tea and Extraction of Crude CaffeineMethods used to concentrate the brewed tea and crude caffeine extraction were the same as in3.1.4 and 3.1.5 on page 12. Page 15 of 42
  17. 17. Extended Essay LEOW YUN JIN 002206-0095.0 Purification of Crude Caffeine14 1. 1cm3 of DCM was added into the boiling tube to dissolve the crude caffeine; 2. It is then transferred into a larger 25cm3 beaker and DCM was evaporated using water bath; 3. A pre-weighed smaller size 25cm3 beaker is place into the larger beaker. They are then heated using Bunsen burner; 4. The Bunsen burner is taken away from the beakers occasionally to keep the temperature below 200°C; 5. Heating is stopped after 20minutes. The smaller beaker is slowly taken out and left to cool, (Air was used to condense the caffeine instead of ice as water vapour will be formed on the outer wall of the smaller beaker thus affecting the amount of caffeine that sublimed on the surface); 6. The smaller beaker is then weighed by inverting it on the balance; 7. Steps 1-6 are repeated for the other samples.14 Conducted for samples from different period of fermentation only Page 16 of 42
  18. 18. Extended Essay LEOW YUN JIN 002206-009 Smaller beaker Larger beaker Sublimed caffeineFigure 9: Purifying the crude caffeine by sublimation.6.0 Thin Layer Chromatography (TLC) Test 1. Samples of pure caffeine15, crude caffeine and purified caffeine were dissolved in DCM; 2. TLC plates were prepared and pure caffeine was spotted on the top left, crude caffeine at the centre while purified caffeine on the right of a TLC plate using a new capillary spotter each time; 3. TLC plate was place into a chromatogram prepared from ethyl acetate16 with a few drops of concentrated sulphuric acid, placing the spotted end into the mixture; 4. TLC plate was left to dry and then viewed under UV light; 5. Observations were noted.15 Purchased from Sarget Welch, US16 A mobile phase used in this TLC test Page 17 of 42
  19. 19. Extended Essay LEOW YUN JIN 002206-0097.0 Data Collection7.1 Method of data collection7.1.1 Calculating the mass of crude caffeineAll the boiling tubes were weighed on the electronic balance (±0.001g) before theexperiment. The boiling tubes containing crude caffeine were being weighed again. The massof crude caffeine is the difference between the initial and final readings.7.1.2 Calculating the mass of purified caffeineThe smaller beakers used for the sublimation process were weighed on the electronic balance(±0.001g) before the experiment. After the purification, the beaker with caffeine sublimed onits outer wall was being weighed again. The mass of purified caffeine is the differencebetween the initial and final readings.7.1.3 Identifying the purity of purified caffeineSpots viewed under the UV light were noted. The distance of the final spot from the startingpoint of the purified caffeine was compared to the pure caffeine’s. Page 18 of 42
  20. 20. Extended Essay LEOW YUN JIN 002206-0097.2 Raw Data7.2.1 Amount of Crude and Purified Caffeine extracted at Various Period of Fermentation Before Purification Period of Mass / g (±0.001g) (a)Fermentation Mean ± S.D(e) (b) Mean ± S.D 1 2 3 Mean ± S.D / g / min / mg for 3.000g / mg g-1 A B A-B A B A-B A B A-B for 3.000g 0 40.097 40.079 0.018 39.684 39.669 0.015 38.195 38.179 0.016 0.0163 ± 0.0015 16.3 ± 1.5 5.4 ± 0.5 40 39.860 39.848 0.012 39.966 39.950 0.016 39.004 38.991 0.013 0.0137 ± 0.0021 13.7 ± 2.1 4.6 ± 0.7 80 42.666 42.650 0.016 41.770 41.752 0.018 43.035 43.020 0.015 0.0163 ± 0.0015 16.3 ± 1.5 5.4 ± 0.5 120 37.687 37.669 0.018 43.030 43.010 0.020 43.179 43.158 0.021 0.0197 ± 0.0015 19.7 ± 1.5 6.6 ± 0.5 After Purification Mass / g (±0.001g) (c) (d) Mean ± S.D / Mean ± S.D 1 2 3 Mean ± S.D / g mg for 3.000g / mg g-1 C D C-D C D C-D C D C-D for 3.000g 0 22.839 22.834 0.005 22.843 22.834 0.009 22.842 22.834 0.008 0.0073 ± 0.0021 7.3 ± 2.1 2.4 ± 0.7 40 36.874 36.864 0.010 36.870 36.864 0.006 36.871 36.864 0.007 0.0077 ± 0.0021 7.7 ± 2.1 2.6 ± 0.7 80 48.442 48.432 0.010 48.440 48.432 0.008 48.439 48.432 0.007 0.0083 ± 0.0015 8.3 ± 1.5 2.8 ± 0.5 120 34.139 34.126 0.013 34.142 34.126 0.016 34.140 34.126 0.014 0.0143 ± 0.0007 14.3 ± 0.7 4.8 ± 0.5Table 1: Amount of Crude and Purified Caffeine for Various Period of Fermentationa Mean amount of crude caffeine extracted from 3.000g of tea leaves ±S.D. for triplicate samples A: Mass of Boling Tube (Final)b Mean amount of crude caffeine per gram of tea leaves ±S.D. for triplicate samples B: Mass of Boling Tube (Initial)c Mean amount of purified caffeine from 3.000g of tea leaves ±S.D. for triplicate samples B-A: Mass of Crude Caffeined Mean amount of purified caffeine per gram of tea leaves ±S.D. for triplicate samples C: Mass of Beaker (Final)e S.D : Standard deviation D: Mass of Beaker (Initial) C-D: Mass of Purified Caffeine Page 19 of 42
  21. 21. Extended Essay LEOW YUN JIN 002206-0097.2.2 Amount of Crude Caffeine Extracted at Different Temperature Mass / g (±0.001g) (a) (b) Mean ± MeanTemperature / S.D(c) / mg ± S.D / °C (±0.1°C) 1 2 3 Mean ± S.D / g for 5.000g mg g-1 for 5.000g A-B A-B A-B A B A B A B 25.0 42.397 42.387 0.010 42.37 42.358 0.012 42.074 42.059 0.015 0.0123 ± 0.0025 12.3 ± 2.5 2.5 ± 0.5 60.0 40.547 40.531 0.016 39.514 39.495 0.019 43.206 43.184 0.022 0.0190 ± 0.0030 19.0 ± 3.0 3.8 ± 0.6 80.0 39.174 39.147 0.027 43.155 43.133 0.022 40.018 39.995 0.023 0.0240 ± 0.0026 24.0 ± 2.6 4.8 ± 0.5 100.0 38.437 38.412 0.025 39.095 39.071 0.024 42.756 42.726 0.030 0.0263 ± 0.0032 26.3 ± 3.2 5.3 ± 0.6Table 2: Amount of Crude Caffeine Extracted at Various Temperature.a Mean amount of crude caffeine extracted from 5.000g of tea leaves ±S.D. for triplicate samples A: Mass of Boling Tube (Final)b Mean amount of crude caffeine per gram of tea leaves ±S.D. for triplicate samples B: Mass of Boling Tube (Initial)c S.D : Standard deviation B-A: Mass of Crude Caffeine Page 20 of 42
  22. 22. Extended Essay LEOW YUN JIN 002206-0097.2.3 Thin Layer Chromatography (TLC) Test Solvent FrontPure Caffeine ImpuritiesStarting Point Pure Caffeine Crude Caffeine Purified CaffeineFigure 10: TLC plate spotted with pure caffeine, crude caffeine and purified caffeine wasviewed under UV light. Page 21 of 42
  23. 23. Extended Essay LEOW YUN JIN 002206-0097.3 Data Processing Mean Amount of Crude and Purified Caffeine / mg g-1 against Period of Fermentation / min 8.0 Mean Amount of Crude/Purified Caffeine /mg g -1 7.0 6.0 (c) 5.0 4.0 (a) Crude Caffeine 3.0 (b) Purified Caffeine 2.0 1.0 0.0 0 40 80 120 Period of Fermentation / minGraph 1: Mean amount of crude caffeine and purified caffeine against period of fermentation. a Mean amount of crude caffeine ±S.D. for triplicate samples b Mean amount of purified caffeine ±S.D. for triplicate samples c Error bar denotes ±S.D. for triplicate samples Page 22 of 42
  24. 24. Extended Essay LEOW YUN JIN 002206-009 Mean Amount of Crude Caffeine / mg g-1 against Temperature / °C 7.0 6.0 Mean Amount of Caffeine / mg g -1 5.0 (b) 4.0 3.0 (a) Crude Caffeine 2.0 1.0 0.0 25.0 60.0 80.0 100.0 Temperature / °CGraph 2: Mean amount of crude caffeine against temperature. a Mean amount of crude caffeine ±S.D. for triplicate samples b Error bar denotes ±S.D. for triplicate samples Page 23 of 42
  25. 25. Extended Essay LEOW YUN JIN 002206-009 Mean Amount of Crude Caffeine / mg g-1 against Temperature / °C 7.0 6.0 Mean Amount of Caffeine / mg g -1 5.0 (b) 4.0 y = 0.0385x + 1.5525 R² = 0.9907 3.0 Linear ((a) Crude Caffeine) 2.0 1.0 0.0 0.0 20.0 40.0 60.0 80.0 100.0 120.0 Temperature / °CGraph 3: Mean amount of crude caffeine against temperature. a Mean amount of crude caffeine ±S.D. for triplicate samples b Error bar denotes ±S.D. for triplicate samples Page 24 of 42
  26. 26. Extended Essay LEOW YUN JIN 002206-0097.4 Statistical Analysis: ANOVA and Tukey’s HSD testAnalysis of variance (ANOVA) is conducted on the obtained data. ANOVA partitions thevariance of all observations into variations between group and variation within each group.ANOVA’s result will show whether the manipulated variable (temperature and fermentationperiod) causes a significant difference in the amount of caffeine.Firstly, a null hypothesis, H0 is made: H0 = μ1 = μ2 = μ3 = μ4 (There is no difference betweenmeans of the four different groups).The statistic test will be carried out to find the: F ratio = The null hypothesis will be rejected if the F-ratio is greater than the F-critical value at thesignificant level of 0.05 (α = 0.05) as there is a significant difference between one or moregroups. [3] Microsoft Office Excel 2007 will be used to conduct the ANOVA.If there is a significant difference, a post hoc analysis, Tukey’ HSD test is carried out.ANOVA does not indicate specifically which pairs have significant difference. Tukey’s HSDtest is a multiple comparison test by comparing to an overall significant level in order to testout the null hypothesis. A critical value, HSD will be calculated. There will be a significantdifference between the pairs if the mean difference between groups is greater than HSDcritical value. This test will be calculated manually. Page 25 of 42
  27. 27. Extended Essay LEOW YUN JIN 002206-009The results obtained are shown below: Variable F - value17 F - critical18 Result Crude All the three F - value are greater 6.39 4.07 Period of Caffeine than their respective F - critical Fermentation Purified value. This indicates that there is at 9.83 4.07 Caffeine least one significant difference between two groups in each of the Temperature 14.11 4.07 variable.Table 3: The results obtained from ANOVA on the temperature and period of fermentation.17 Refer to appendix 2 for further calculations18 Refer to appendix 2 for further calculations Page 26 of 42
  28. 28. Extended Essay LEOW YUN JIN 002206-009Tukey’s HSD test19: Group Combination, min Mean HSD (Mean Amount of Crude Caffeine Difference, critical Implication Extracted at Different Period of g value Fermentation) 0 40 0.80 1.46 no significant difference 0 80 0.00 1.46 no significant difference 0 120 1.20 1.46 no significant difference 40 80 0.80 1.46 no significant difference 40 120 2.00 1.46 significant difference 80 120 1.20 1.46 no significant differenceTable 4: Results obtained from Tukey’s HSD test on mean amount of crude caffeine extracted fordifferent period of fermentation. Group Combination, min Mean HSD (Mean Amount of Purified Caffeine at Difference, critical Implication Different Period of Fermentation) g value 0 40 0.20 1.59 no significant difference 0 80 0.40 1.59 no significant difference 0 120 2.40 1.59 significant difference 40 80 0.20 1.59 no significant difference 40 120 2.20 1.59 significant difference 80 120 2.00 1.59 significant differenceTable 5: Results obtained from Tukey’s HSD test on mean amount of purified caffeine for differentperiod of fermentation. Group Combination, °C Mean HSD (Mean Amount of Crude Caffeine Difference, critical Implication Extracted at Different Temperature) g value 25 60 1.30 1.50 no significant difference 25 80 2.30 1.50 significant difference 25 100 2.80 1.50 significant difference 60 80 1.00 1.50 no significant difference 60 100 1.50 1.50 no significant difference 80 100 0.50 1.50 no significant differenceTable 6: Results obtained from Tukey’s HSD test on mean amount of crude caffeine extracted fordifferent temperature.19 Refer to appendix 2 for further calculations Page 27 of 42
  29. 29. Extended Essay LEOW YUN JIN 002206-0097.6 Percentage of Caffeine Extracted by Liquid-liquid ExtractionCaffeine will distribute itself between water and DCM at a constant ratio according to thepartition law20. Multiple extractions are better than a single extraction. Thus, three successiveextractions were performed.2cm3 of DCM is used to extract caffeine from 25cm3 of brewed tea for each extraction.Therefore, total amount of crude caffeine that can be extracted from 1 gram of tea leaves =0.023g (23mg).21From the calculation it shows: 23Percentage of extractable caffeine = x 100 30 = 76.6%  Using the amount of crude caffeine extracted for 120 min period of fermentation from table 1 (page 19), 6.6mg g-1 for calculation: 6.6Percentage efficiency of extraction in this research = x 100 23 = 28.7%20 Refer to Appendix 321 Refer to Appendix 3 for further calculations Page 28 of 42
  30. 30. Extended Essay LEOW YUN JIN 002206-0098.0 EvaluationTLCResults obtained from TLC test shows that sublimation process does not fully purify thecrude caffeine as many spots can be seen. This result strongly suggests that other impuritiessuch as chlorophylls are present. Furthermore, results show that there is a large decrease inmass of crude caffeine after purification indicating most of the impurities are removed. Thevalues of purified caffeine will be more reliable for a fair comparative study. Nevertheless,for the investigation on the effects of temperature, the values of crude caffeine show anobvious trend thus they were not further purified.Percentage Efficiency of ExtractionThe percentage of extractable caffeine through liquid-liquid extraction using DCM is high,76.6% but the percentage efficiency of extraction performed in this investigation is only28.7%. This method was adopted because it is easy, inexpensive and DCM is available in thelaboratory. Although the amount of crude caffeine extracted in this investigation is muchlower than the extractable value, it does not affect the trend of the results obtained. The dataare still valid as I will be comparing the relative difference of caffeine, not the total amount ofcaffeine.Period of FermentationThe mean purified caffeine extracted per gram tea leaves for period of fermentation of 0, 40and 80min are 2.4±0.7, 2.6±0.7 and 2.8±0.5mg respectively. There should be zero crudecaffeine found from the 0min as tea leaves are not bruise allowing no fermentation thus allcaffeine forms insoluble complexes. The result obtained can be due to systematic error thatoccurs during sampling as tea leaves undergo minimum fermentation during the plucking Page 29 of 42
  31. 31. Extended Essay LEOW YUN JIN 002206-009process. The samples were kept in an air tight container and the experiment was notperformed immediately allowing some polyphenols to be oxidised. This means some caffeinedoes not form complexes enabling them to be extracted. Results showed no significantdifference in the amount of purified caffeine over the period of 0-80min.The amount then increase to 4.8mg at 120min and records a 71.4% increased from 80 to120min. According to statistical analysis, 120min shows significant difference (P 0.05)with the other groups. According to graph 1, there is no significant increase at 0-80min but at120min there is a significant increase. This can be due to time lag. The enzymepolyphenoloxidase lowers the activation energy, Ea for the oxidation of polyphenols. A seriesof complex reactions take place during fermentation, thus the release of polyphenoloxidase istime dependent whereby it is release through a cascade reaction. When the period offermentation is short, only a small amount of polyphenoloxidase is available to oxidise thepolyphenols thus allowing more polyphenols to form insoluble complexes with caffeine. Theamount of polyphenoloxidase will increase exponentially overtime. Therefore, during shorterperiod of fermentation there will only be a slight increase in the caffeine but increasessignificantly at much longer period.TemperatureAs the temperature increases from 25.0-100.0°C, the mean amount of crude caffeineincreases from 2.5, 3.8, 4.8 and 5.3mg respectively. It increased at a decreasing rate as thepercentage increase drops from 52.0% to 26.3% and finally 5.3% as the temperatureincreases. Statistical analysis shows that there are significant differences (P 0.05) in theamount of crude caffeine between 25.0°C with 80.0°C and 100.0°C while there is nosignificant difference for the other groups of temperatures. Graph 2 clearly shows that theamount of crude caffeine increases at a decreasing rate. The possible reason is that most of Page 30 of 42
  32. 32. Extended Essay LEOW YUN JIN 002206-009the extractable caffeine has already diffused out from the tea leaves at 60.0°C. Thus, at 80.0and 100.0°C only a small amount of caffeine diffuses out into the brewed tea.In graph 3, a linear regression line (R2 = 0.9179) drawn shows that there is a positivecorrelation between the amount of crude caffeine and temperature. The positive correlationcan be explained by the kinetic theory: 1 3  Average Kinetic Energy, = mv2 = 2 2Average velocity, is directly proportional to absolute temperature, T. As T increases, which indicates the average velocity of caffeine diffusing out from the tea leaves into thewater increases. Therefore, as the brewing temperature increases, the caffeine moleculesgained higher Kinetic Energy which increases the rate of diffusion. Thus, more crude caffeineis extracted at higher temperature.8.1 Significant of StudiesFrom the results obtained, we can conclude that the period of fermentation and brewingtemperature do cause a significant difference in the amount of caffeine in brewed tea asindicated by the ANOVA in table 3. In order to reap the health benefits of Camellia Sinensisbut at the same time reducing the intake of caffeine, I would suggest the following: [2] 1. Fermentation is important as flavour compounds are formed but tea producers can minimise or optimise the period of fermentation to reduce the amount of caffeine in the tea brewed; 2. Consumers should brew tea at an optimum temperature that lies between 60.0°C and 80.0°C probably around 65.0°C in order to extract the beneficial substances such as flavonoids but minimising the amount of caffeine. Page 31 of 42
  33. 33. Extended Essay LEOW YUN JIN 002206-0098.2 Limitations 1. Ideally, pure caffeine should be used for comparative studies in this research. The amount of impurities in each samples obtained may vary thus affecting the trends of crude caffeine in relation with period of fermentation and temperature. 2. The extraction of crude caffeine is done using conical flask. A small amount of organic layer has to be left behind to prevent extracting the aqueous layer. The amount left behind for every extraction varies thus affecting the results. 3. The centrifuge machine found in the school can only centrifuge a maximum volume of 10cm3 while the volume of the mixtures of organic and aqueous layers are 27cm3. Emulsion forms when the mixture is swirl. Emulsion that contain caffeine is being left behind as the organic layer will be easily extracted together with it. 4. The mass of tea leaves used for brewing contains stem and it varies for every sample. The presence of stem will decrease the amount of crude caffeine extracted. 5. The sizes of tea leaves samples also vary and this affects the total surface area exposed during brewing. Larger total surface area will increase the rate of diffusion of caffeine into the water and vice versa. 6. The high variance (square of S.D.) of the results is due to the total sampling and 2 2 2 measurement steps errors, 0 = + . [4] The experiments are very tedious and time consuming thus with the limited time, only triplicate samples are used thus increasing the S.D.8.3 Ways of Improvement 1. Amount of caffeine in brewed tea can accurately measured using Reverse-Phase High Performance Liquid Chromatography (HLPC) Analysis or ultra-violet spectrophotometry. Spectrophotometer is rather affordable while an HLPC may be Page 32 of 42
  34. 34. Extended Essay LEOW YUN JIN 002206-009 too expensive. Instead of that, the extraction technique may be further improved by using a separatory funnel instead of a conical flask which allows the mixtures to be well mixed. Only a negligible amount of organic layer will be left behind thus the amount of caffeine obtained will be more accurate; 2. Different organic solvents that can improve the percentage efficiency of liquid-liquid extraction should be explored. Organic solvent used should be non-toxic and can be easily available in the laboratory; 3. Fresh tea leaves are plucked with care in order to minimise the fermentation process and being stored in a vacuum container. Without oxygen, polyphenoloxidase will not be able to oxidise the polyphenols; 4. Tea leaves that will be used for brewing have to be sorted out to ensure that other parts such as stems are removed. Tea leaves that are still intact in large pieces have to be crushed to small pieces in order to ensure the total surface area exposed during brewing will be relatively constant for all samples; 5. The number of sample size used for each condition has to be increased in order to minimise the S.D.8.4 Unresolved Questions for Further InvestigationsTea leaves can be divided into young (bulb, first and second leaf) and matured (third until thefifth leaf) ones. Their caffeine contents may differ thus the amount of caffeine in young andmatured leaves under different period of fermentation and brewing temperatures can beinvestigated.The brewing temperatures investigated are only limited to 25.0, 60.0, 80.0 and 100.0°C. Theoptimum brewing temperature that should be used to extract out most of the beneficialsubstances but minimum amount of caffeine is believed to be between 60.0–80.0°C. Thus, Page 33 of 42
  35. 35. Extended Essay LEOW YUN JIN 002206-009detailed investigation on different temperatures between this range can be carried out todetermine the optimum brewing temperature in order to reap the maximum benefits of tea.Tea leaves used for this investigation were all grown in highlands and there is a possibilitythat the lowlands tea may have different caffeine content. Samples tea leaves from lowlandscan be investigated for comparative studies with the highland tea.Camellia Sinensis can be divided into two varieties: [5] 1. Camellia sinensis var. Sinensis (small-leaved); 2. Camellia sinensis var. Assamica (large-leaved).The first variety was used in this investigation and further investigation should be conductedon the second variety to investigate whether different tea variety will have different amountof caffeine and how the period of fermentation and temperature will affects the amount ofcaffeine in its brewed tea.Investigation on how brewing temperature affects the rate of diffusion of beneficialsubstances that bring health benefits should be carried out in order to maximise the benefitsof tea.9.0 ConclusionResults show the effects of period of fermentation and brewing temperature on the amount ofcaffeine are significant. As the period of fermentation and brewing temperature increase, theamount of caffeine increases. This research shows that in order to minimise the amount ofcaffeine in brewed tea, tea leaves should undergo minimum fermentation and be brewed at alower temperature. Unfermented tea leaves may not provide any aroma and flavour thus tealeaves should be fermented at a short period of time. Brewing tea at 25.0°C shows that Page 34 of 42
  36. 36. Extended Essay LEOW YUN JIN 002206-009caffeine diffuses very slowly into the water and the same goes to other substances includingbeneficial ones. Thus it is best to brew tea within 60.0–80.0°C. Therefore, it is concluded thatunder these suggested conditions, consumers will be able to enjoy the flavour and benefits ofCamellia Sinensis but at the same time minimising caffeine intake. Page 35 of 42
  37. 37. Extended Essay LEOW YUN JIN 002206-00910.0 AppendixAppendix 1(http://kidshealth.org/teen/nutrition/general/caffeine.html#)Moderation Is the KeyCaffeine is usually thought to be safe in moderate amounts. Experts consider 200–300 mg ofcaffeine a day to be a moderate amount for adults. Teens should try to limit caffeineconsumption to no more than 100 mg of caffeine daily, and kids should get even less. Page 36 of 42
  38. 38. Extended Essay LEOW YUN JIN 002206-009Appendix 2ANOVA – Analysis of VarianceTo perform ANOVA, three assumptions are made: 1. Observations are independent (the value of one observation is not correlated to the value of another); 2. Observations in each group are normally distributed; 3. Homogeneity of variances (variance of each group is equal to that of any other group).ANOVA table is summarised below:Source of Variation Sum of Squares df Mean Squares, S2 F - ratio P-value F - critical Computer 2 Between Groups SSb k-1 = generated Fk-1, N-k −1 2 Within Groups SSw N-k = −Total SSt N-1Table 4: Summary of ANOVA table.All significances (α) for ANOVA will be at 5% or 0.05. Standard Notation Meaning SSb Sum of squares between groups SSw Sum of squares within group SSt Total sum of squares df Degree of Freedom k Total number of groups N Total number of results 2 Mean squares between groups 2 Mean squares within groupTable 5: Legend for ANOVA table. Page 37 of 42
  39. 39. Extended Essay LEOW YUN JIN 002206-009HSD – Tukey’s Honestly Significant Difference 2 HSD = q (α, k, N – k) α = represents the significant level (0.05)k = represents total number of groupsN – k = represents total number of results – total number of groupsq = the value based on (α, k, N – k).ANOVA calculations using Microsoft Office Excel 2007 and HSD values:Anova: Single factorSUMMARY Groups (Period of Fermentation, min) Count Sum Average VarianceColumn 1 (0) 3 16.3 5.44 0.26Column 2 (40) 3 13.7 4.56 0.48Column 3 (80) 3 16.3 5.44 0.26Column 4 (120) 3 19.7 6.56 0.26ANOVA Source of Variation SS df MS F P-value F critBetween Groups 6.04 3 2.01 6.39 1.61E-02 4.07Within Groups 2.52 8 0.31Total 8.56 11Table 6: ANOVA for period of fermentation (mean amount of crude caffeine extracted). 0.31 HSD = 4.53 3 = 1.46 Page 38 of 42
  40. 40. Extended Essay LEOW YUN JIN 002206-009Anova: Single factorSUMMARY Group (Period of Fermentation, min) Count Sum Average VarianceColumn 1 (0) 3 7.3 2.44 0.48Column 2 (40) 3 7.7 2.56 0.48Column 3 (80) 3 8.3 2.78 0.26Column 4 (120) 3 14.3 4.78 0.26ANOVA Source of Variation SS df MS F P-value F critBetween Groups 10.92 3 3.64 9.83 4.65E-03 4.07Within Groups 2.96 8 0.37Total 13.88 11Table 7: ANOVA for period of fermentation (mean amount of purified caffeine). 0.37 HSD = 4.53 3 = 1.59 Page 39 of 42
  41. 41. Extended Essay LEOW YUN JIN 002206-009Anova: Single factorSUMMARY Groups Count Sum Average VarianceColumn 1 (25°C) 3 7.4 2.47 0.25Column 2 (60°C) 3 11.4 3.80 0.36Column 3 (80°C) 3 14.4 4.80 0.28Column 4 (100°C) 3 15.8 5.27 0.41ANOVA Source of Variation SS df MS F P-value F critBetween Groups 13.82 3 4.61 14.11 1.47E-03 4.07Within Groups 2.61 8 0.33Total 16.44 11Table 8: ANOVA table for temperature. 0.33 HSD = 4.53 3 = 1.50 Page 40 of 42
  42. 42. Extended Essay LEOW YUN JIN 002206-009Appendix 3Partition law states that at a constant temperature, a solute distributes itself between twoimmiscible liquids so that the ratio of its concentration in each solvent is constant, regardlessof the amount of solute added. [6] = K (a constant) Values used for calculation:  Percentage of caffeine by dry weight of tea leaves range from 1.4% to 4.5%. Thus, an average amount of 3.0% is used;  Solubility of caffeine in water: 2.18g / 100cm3;  Solubility of caffeine in DCM: 18.2g / 100cm3; 18.2  Partition coefficient, k from caffeine in both solvent mixture is 100 2.18 = 8.35; 100  Using the amount of crude caffeine extracted for 120 min period of fermentation from table 1 (page 19), 6.6mg g-1. First extraction: 8.35 = 2 0.03− 25 x = 0.012g Second extraction: 8.35 = 2 0.018− 25 x = 0.007g Third extraction: 8.35 = 2 0.011− 25 x = 0.004g Page 41 of 42
  43. 43. Extended Essay LEOW YUN JIN 002206-00911.0 Bibliography 1. Maria Fitzpatrick; Healthy drinking: Tea total (http://www.telegraph.co.uk/health/3356550/Healthy-drinking-Tea-total.html). 2. P. Sivapalan, S. Kulasegaram, A. Kathiravetpillai; Handbook On Tea; Tea Research Institute of Sri Lanka, Talawakele, Sri Lanka, 1986; Printed by Aitken Spence Co. Ltd. 3. Jan W. Kuzma, Stephen E. Bohnenblust; Basic Statistics for the Health Sciences; Mayfield Publishing Company, 2001. 4. Gary D. Christian; Analytical Chemistry; Printed by Courier Westford, 2004. 5. Integrated Taxonomic Information System Report; Camellia Sinensis; Taxonomic Serial No. :506801 (http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSNsearch_value= 506801). 6. Yin Toon, Tan; Physical Chemistry for STPM; Published by Penerbit Fajar Bakti Sdn. Bhd., 2003.Conrad Astill, Mark R. Birch, Clive Dacombe, Philip G. Humphrey, and Philip T. Martin;Factors Affecting the Caffeine and Polyphenol Contents of Black and Green Tea Infusions; J.Agric. Food Chem., 2001.Yung-Sheng Lin, Yao-Jen Tsai, Jyh-Shyan Tsay, and Jen-Kun Lin; Factors Affecting theLevels of Tea Polyphenols and Caffeine in Tea Leaves; J. Agric. Food Chem., 2003. Page 42 of 42

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