Caffeine quantification from tea leaves using solvent extraction

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

Extended Essay LEOW YUN JIN 002206-009

Page 1 of 42

Abstract

Camellia Sinensis contains polyphenolic flavonoids which have strong antioxidant properties

that bring health benefits but at the same time contains caffeine, a stimulant. High caffeine

consumption causes insomnia and constant consumption can be highly addictive. Caffeine

should be taken in moderate dosage and thus factors affecting the amount of caffeine in

brewed 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 at

25.0, 60.0, 80.0 and 100.0°C. Crude caffeine was extracted with dichloromethane, CH2Cl2

using liquid-liquid extraction technique. Selected samples were further purified through

sublimation. The mass of crude and purified caffeine were calculated as the difference

between the initial and final readings of boiling tubes and beakers respectively.

The results showed significant differences (P < 0.05) between the period of fermentation and

brewing temperature on the amount of caffeine. No significant increase in purified caffeine

was found from 0-80min but there is a significant increase at 120min. Amount of crude

caffeine increases as the temperature increases from 25.0–100.0°C and a line of linear

regression graph was plotted (R2 = 0.9641) showing a positive correlation.

The effect of period of fermentation and brewing temperature on the amount of caffeine are

significant. As the period of fermentation and temperature increase, the amount of caffeine

increases. In order to reap the health benefits of Camellia Sinensis but at the same time

minimising caffeine intake, tea leaves should be processed at shorter period of fermentation

and being brewed at a lower temperature. Tea leaves are suggested to undergo minimum

fermentation for flavouring and brewed at an optimum temperature between 60.0–80.0°C.

(283 words)


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Table of Contents

Abstract........................................................................................................................................1

Table of Contents.................................................................................................................................2

Acknowledgement.........................................................................................................................3

1.0 Introduction.....................................................................................................................4

2.0 Hypothesis........................................................................................................................7

2.1 How Period of Fermentation Affects the Amount of Caffeine.............................7

2.2 How Temperature Affects the Amount of Caffeine.............................................8

3.0 Methodology Used to Investigate How Period of Fermentation Affects the Amount of

Caffeine........................................................................................................................................9

3.1 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.............................................................................12

4.0 Methodology Used to Investigate the Effects of Temperature on Amount of

Caffeine......................................................................................................................................14

4.1 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..........................................................................................................................15

5.0 Purification of Crude Caffeine.....................................................................................16

6.0 Thin Layer Chromatography (TLC) Test....................................................................17

7.0 Data Collection.............................................................................................................18

7.1 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..........................................................18

7.2 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.............................................................21

7.3 Data Processing..............................................................................................................22

7.4 Statistical Analysis: ANOVA and Tukey’s HSD test.......................................................25

7.5 Percentage of Caffeine Extracted by Liquid-liquid Extraction........................................28

8.0 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................................................34

9.0 Conclusion......................................................................................................................35

10.0 Appendix........................................................................................................................36

11.0 Bibliography...................................................................................................................42


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Acknowledgment

Special thanks to:

Lawrence Kok (Supervisor)

Joel Micheal

Lim Ju Anne

Kalai

Leow Wooi Hian


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1.0 Introduction

Figure 1: Molecular structure of caffeine

1,3,7–trimethylxanthine (C8H10N4O2) or best known as caffeine is a naturally occurring

alkaloid1 that belongs to the xanthine

2 chemical group. Caffeine is known as a drug. It

stimulates the cardiovascular and central nervous systems of the body. Caffeine consumed

through foods and beverages is readily absorbed into the blood stream. It takes effects

rapidly, about 15 minutes after consumption and its level peaks after an hour. It will stay in

the body for several hours and to eliminate half the amount of caffeine will takes up to six

hours3.

In the human brain, a natural occurring xanthine called adenosine4 serves as a

neurotransmitter. Adenosine is responsible for sleepiness as it will binds with its receptors in

the brain cells causing the nerve cells activity to slow down. Caffeine is able to interfere with

adenosine at various sites in the brain including the reticular formation by binding with

adenosine receptors thus speeding up nerve cells. This results in decreased fatigue and

1 A class of naturally occurring compounds containing nitrogen and having the properties of an organic amine base 2 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

http://en.wikipedia.org/wiki/Purine

http://en.wikipedia.org/wiki/Base_(chemistry)


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increased alertness thus allowing a person to stay awake. Besides that, caffeine has the ability

to increase the heart rate, blood pressure and basal metabolic rate (BMR) for several hours. It

also acts as a diuretic which increases urination.

Caffeine, a plant–based alkaloid can be found in tea, coffee and Cocoa. Tea, a popular

drink among all ages is rich in polyphenolic flavonoids5 which have strong antioxidants

properties despites containing caffeine. Flavonoids play a role in preventing cancer by

protecting cells from free radical damage. It also helps in keeping the heart healthy and

research in Europe shows that drinking three or more cups of tea per day reduces the risk of

heart diseases. [1]

Despites all the goodness of drinking tea, the negative effects of caffeine cannot be

ignored. Caffeine can cause insomnia, headache, nervousness and dizziness when consume in

high doses. It also causes addiction and without it the addict will be unable to concentrate

well and suffers depression. Thus, caffeine should be taken in moderate dosage.6 Due to these

reasons, I choose to focus my research on tea. Tea is produced from the leaves and buds of

the Camellia Sinensis plant through a series of processes. Tea leaves will start to oxidise after

plucking and will take about one and the half hour to two hours to be fully fermented7. Tea

leaves can be left unfermented or fermented as it will produce a range of teas from white tea

to black tea.

5 Chemical substances that have antioxidant properties 6 See appendix 1 7 Result from oxidation reaction but known as fermentation in the tea industry


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Figure 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 caffeine

content. Thus, to be able to reap the benefits of tea but at the same time minimising the

adverse effects of caffeine is by consuming the lowest possible amount of caffeine. There are

several factors that affect the amount of caffeine in brewed tea. I will explore the possibility

effects of period of fermentation and brewing temperature on the amount of caffeine in

brewed tea. Processed tea leaves will be brewed using water and the caffeine will be

extracted by liquid-liquid extraction using dichloromethane, CH2Cl28.

Therefore, my research question is:

How will the period of fermentation and brewing temperature affects the amount of

caffeine extracted from brewed Camellia Sinensis by solvent extraction technique using

dichloromethane?

8 An organic solvent used to extract caffeine


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2.0 Hypotheses

2.1 How Period of Fermentation Affects the Amount of Caffeine

Caffeine is an alkaloid known to bind with other compounds that can be found in tea such as

theine9 and other polyphenols, to form insoluble complexes. The breaking of cell walls which

is done by bruising the tea leaves causes the enzymes present in the tea leaves to mix with

other chemical substances within the cells allowing fermentation. A series of complex

chemical reactions begins, one of the most important reactions being the oxidation of

polyphenols by the enzyme, polyphenoloxidase. In this reaction, an oxygen atom which is

derived directly from the air is added into the polyphenol in the presence of

polyphenoloxidase. The oxidised polyphenols will no longer be able to combine with caffeine

to 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 through

heating. Polyphenoloxidase will be denatured causing the oxidation of polyphenols to stop as

well. Thus, my hypothesis is that as the period of fermentation increases, more polyphenols

are being oxidised by polyphenoloxidase causing less insoluble complexes to be formed with

caffeine. This means that the amount of extractable caffeine through liquid-liquid extraction

will increase as the period of fermentation increases. [2]

9 A type of polyphenol that exists in Camellia Sinensis


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↑ 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 Caffeine

Temperature of the water used during tea brewing will also affects the amount of caffeine

extracted from the tea. My hypothesis is that as the temperature increases, the rate of

diffusion of caffeine into the water increases as the caffeine particles gain more kinetic

energy. The kinetic theory states that average Kinetic Energy, 𝐸𝐾 = 1

2 mv

2 is directly

proportional to absolute temperature, T where 𝐸𝐾 = 3

2𝑘𝐵𝑇. Thus, the average velocity, 𝑣 is

directly proportional to the T as the mass, m remains constant. The 𝑣 relates to the average

velocity of caffeine molecules diffusing out from the tea leaves into the water. Therefore, as

T increases, caffeine molecules gain more 𝐸𝐾 causing an increased in 𝑣 thus allowing a

higher rate of diffusion. I strongly believed that as the brewing temperature increases, the

amount of caffeine in brewed tea increases.


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3.0 Methodology Used to Investigate How Period of Fermentation Affects the

Amount of Caffeine.

3.1 Devised Methods

3.1.1 Collection of Samples

1. Fresh tea leaves were collected from BOH Plantation10

and were kept in air tight

containers.

Bulb

Second Leaf

First Leaf

Third Leaf

Forth 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/


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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°C

Figure 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.


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3.1.3 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 solution

12 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


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3.1.4 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


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Aqueous Layer

Organic Layer

Figure 7: Liquid-liquid extraction using Dichloromethane (DCM).


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4.0 Methodology Used to Investigate the Effects of Temperature on Amount of

Caffeine

4.1 Devised Methods

4.1.1 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.


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Figure 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 Caffeine

Methods used to concentrate the brewed tea and crude caffeine extraction were the same as in

3.1.4 and 3.1.5 on page 12.


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5.0 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


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Smaller beaker

Larger beaker

Sublimed caffeine

Figure 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, US 16A mobile phase used in this TLC test


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7.0 Data Collection

7.1 Method of data collection

7.1.1 Calculating the mass of crude caffeine

All the boiling tubes were weighed on the electronic balance (±0.001g) before the

experiment. The boiling tubes containing crude caffeine were being weighed again. The mass

of crude caffeine is the difference between the initial and final readings.

7.1.2 Calculating the mass of purified caffeine

The 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 on

its outer wall was being weighed again. The mass of purified caffeine is the difference

between the initial and final readings.

7.1.3 Identifying the purity of purified caffeine

Spots viewed under the UV light were noted. The distance of the final spot from the starting

point of the purified caffeine was compared to the pure caffeine’s.


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7.2 Raw Data

7.2.1 Amount of Crude and Purified Caffeine extracted at Various Period of Fermentation

Period of Fermentation

/ min

Before Purification

Mass / g (±0.001g) (a)Mean ± S.D(e) / mg for 3.000g

(b)Mean ± S.D / mg g-1

1 2 3 Mean ± S.D / g for 3.000g A B A - B A B A - B A B A - B

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)Mean ± S.D / mg for 3.000g

(d)Mean ± S.D / mg g-1

1 2 3 Mean ± S.D / g for 3.000g C D C - D C D C - D C D C - D

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 ± 0.5

Table 1: Amount of Crude and Purified Caffeine for Various Period of Fermentation

a 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 Caffeine d 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


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7.2.2 Amount of Crude Caffeine Extracted at Different Temperature

Temperature / °C (±0.1°C)

Mass / g (±0.001g) (a)Mean ± S.D(c) / mg for 5.000g

(b)Mean ± S.D / mg g-1

1 2 3 Mean ± S.D / g 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 ± 0.6

Table 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


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7.2.3 Thin Layer Chromatography (TLC) Test

Solvent Front

Pure Caffeine

Impurities

Starting Point

Pure Caffeine Crude Caffeine Purified Caffeine

Figure 10: TLC plate spotted with pure caffeine, crude caffeine and purified caffeine was

viewed under UV light.


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7.3 Data Processing

Graph 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

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0 40 80 120

Me

an A

mo

un

t o

f C

rud

e/P

uri

fie

d C

affe

ine

/m

g g

-1

Period of Fermentation / min

Mean Amount of Crude and Purified Caffeine / mg g-1 against Period of Fermentation / min

(a) Crude Caffeine

(b) Purified Caffeine

(c)


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Graph 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

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

25.0 60.0 80.0 100.0

Me

an A

mo

un

t o

f C

affe

ine

/ m

g g

-1

Temperature / °C

Mean Amount of Crude Caffeine / mg g-1 against Temperature / °C

(a) Crude Caffeine

(b)


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Graph 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

y = 0.0385x + 1.5525R² = 0.9907

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0.0 20.0 40.0 60.0 80.0 100.0 120.0

Me

an A

mo

un

t o

f C

affe

ine

/ m

g g

-1

Temperature / °C

Mean Amount of Crude Caffeine / mg g-1 against Temperature / °C

Linear ((a) Crude Caffeine)

(b)


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7.4 Statistical Analysis: ANOVA and Tukey’s HSD test

Analysis of variance (ANOVA) is conducted on the obtained data. ANOVA partitions the

variance of all observations into variations between group and variation within each group.

ANOVA’s result will show whether the manipulated variable (temperature and fermentation

period) 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 between

means of the four different groups).

The statistic test will be carried out to find the:

F ratio = 𝑚𝑒𝑎𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝑏𝑒𝑡𝑤𝑒𝑒𝑛 𝑔𝑟𝑜𝑢𝑝𝑠

𝑚𝑒𝑎𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝑤𝑖𝑡 𝑕𝑖𝑛 𝑔𝑟𝑜𝑢𝑝𝑠

The null hypothesis will be rejected if the F-ratio is greater than the F-critical value at the

significant level of 0.05 (α = 0.05) as there is a significant difference between one or more

groups. [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 HSD

test is a multiple comparison test by comparing to an overall significant level in order to test

out the null hypothesis. A critical value, HSD will be calculated. There will be a significant

difference between the pairs if the mean difference between groups is greater than HSD

critical value. This test will be calculated manually.


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The results obtained are shown below:

Variable F - value17 F - critical18 Result


Crude Caffeine

6.39 4.07 All the three F - value are greater than their respective F - critical

value. This indicates that there is at least one significant difference

between two groups in each of the variable.

Purified Caffeine

9.83 4.07

Temperature 14.11 4.07

Table 3: The results obtained from ANOVA on the temperature and period of fermentation.

17 Refer to appendix 2 for further calculations 18 Refer to appendix 2 for further calculations


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Tukey’s HSD test19

:

Group Combination, min (Mean Amount of Crude Caffeine Extracted at Different Period of

Fermentation)

Mean Difference,

g

HSD critical value

Implication

0 40 0.80 1.46 no significant difference




40 120 2.00 1.46 significant difference

80 120 1.20 1.46 no significant difference Table 4: Results obtained from Tukey’s HSD test on mean amount of crude caffeine extracted for

different period of fermentation.

Group Combination, min (Mean Amount of Purified Caffeine at

Different Period of Fermentation)

Mean Difference,

g

HSD critical value

Implication






80 120 2.00 1.59 significant difference Table 5: Results obtained from Tukey’s HSD test on mean amount of purified caffeine for different

period of fermentation.

Group Combination, °C (Mean Amount of Crude Caffeine

Extracted at Different Temperature)

Mean Difference,

g

HSD critical value

Implication






80 100 0.50 1.50 no significant difference Table 6: Results obtained from Tukey’s HSD test on mean amount of crude caffeine extracted for

different temperature.

19 Refer to appendix 2 for further calculations


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7.6 Percentage of Caffeine Extracted by Liquid-liquid Extraction

Caffeine will distribute itself between water and DCM at a constant ratio according to the

partition law20

. Multiple extractions are better than a single extraction. Thus, three successive

extractions were performed.

2cm3 of DCM is used to extract caffeine from 25cm

3 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).21

From the calculation it shows:

Percentage of extractable caffeine = 23

30 x 100

= 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:

Percentage efficiency of extraction in this research = 6.6

23 x 100

= 28.7%

20 Refer to Appendix 3 21 Refer to Appendix 3 for further calculations


Page 29 of 42

8.0 Evaluation

TLC

Results obtained from TLC test shows that sublimation process does not fully purify the

crude caffeine as many spots can be seen. This result strongly suggests that other impurities

such as chlorophylls are present. Furthermore, results show that there is a large decrease in

mass of crude caffeine after purification indicating most of the impurities are removed. The

values 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 an

obvious trend thus they were not further purified.

Percentage Efficiency of Extraction

The 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 only

28.7%. This method was adopted because it is easy, inexpensive and DCM is available in the

laboratory. Although the amount of crude caffeine extracted in this investigation is much

lower than the extractable value, it does not affect the trend of the results obtained. The data

are still valid as I will be comparing the relative difference of caffeine, not the total amount of

caffeine.


The mean purified caffeine extracted per gram tea leaves for period of fermentation of 0, 40

and 80min are 2.4±0.7, 2.6±0.7 and 2.8±0.5mg respectively. There should be zero crude

caffeine found from the 0min as tea leaves are not bruise allowing no fermentation thus all

caffeine forms insoluble complexes. The result obtained can be due to systematic error that

occurs during sampling as tea leaves undergo minimum fermentation during the plucking


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process. The samples were kept in an air tight container and the experiment was not

performed immediately allowing some polyphenols to be oxidised. This means some caffeine

does not form complexes enabling them to be extracted. Results showed no significant

difference 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 to

120min. 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 at

120min there is a significant increase. This can be due to time lag. The enzyme

polyphenoloxidase lowers the activation energy, Ea for the oxidation of polyphenols. A series

of complex reactions take place during fermentation, thus the release of polyphenoloxidase is

time dependent whereby it is release through a cascade reaction. When the period of

fermentation is short, only a small amount of polyphenoloxidase is available to oxidise the

polyphenols thus allowing more polyphenols to form insoluble complexes with caffeine. The

amount of polyphenoloxidase will increase exponentially overtime. Therefore, during shorter

period of fermentation there will only be a slight increase in the caffeine but increases

significantly at much longer period.

Temperature

As the temperature increases from 25.0-100.0°C, the mean amount of crude caffeine

increases from 2.5, 3.8, 4.8 and 5.3mg respectively. It increased at a decreasing rate as the

percentage increase drops from 52.0% to 26.3% and finally 5.3% as the temperature

increases. Statistical analysis shows that there are significant differences (P < 0.05) in the

amount of crude caffeine between 25.0°C with 80.0°C and 100.0°C while there is no

significant difference for the other groups of temperatures. Graph 2 clearly shows that the

amount of crude caffeine increases at a decreasing rate. The possible reason is that most of


Page 31 of 42

the extractable caffeine has already diffused out from the tea leaves at 60.0°C. Thus, at 80.0

and 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 positive

correlation between the amount of crude caffeine and temperature. The positive correlation

can be explained by the kinetic theory:

Average Kinetic Energy, 𝐸𝐾 = 1

2 mv

2 =

3

2𝑘𝐵𝑇

Average velocity, 𝑣 is directly proportional to absolute temperature, T. As T increases, 𝑣

which indicates the average velocity of caffeine diffusing out from the tea leaves into the

water increases. Therefore, as the brewing temperature increases, the caffeine molecules

gained higher Kinetic Energy which increases the rate of diffusion. Thus, more crude caffeine

is extracted at higher temperature.

8.1 Significant of Studies

From the results obtained, we can conclude that the period of fermentation and brewing

temperature do cause a significant difference in the amount of caffeine in brewed tea as

indicated by the ANOVA in table 3. In order to reap the health benefits of Camellia Sinensis

but at the same time reducing the intake of caffeine, I would suggest the following:

1. Fermentation is important as flavour compounds are formed [2]

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.


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8.2 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 27cm

3.

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

measurement steps errors, 𝑠02 = 𝑠𝑠

2 + 𝑠𝑎2.

[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 33 of 42

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 Investigations

Tea leaves can be divided into young (bulb, first and second leaf) and matured (third until the

fifth leaf) ones. Their caffeine contents may differ thus the amount of caffeine in young and

matured leaves under different period of fermentation and brewing temperatures can be

investigated.

The brewing temperatures investigated are only limited to 25.0, 60.0, 80.0 and 100.0°C. The

optimum brewing temperature that should be used to extract out most of the beneficial

substances but minimum amount of caffeine is believed to be between 60.0–80.0°C. Thus,


Page 34 of 42

detailed investigation on different temperatures between this range can be carried out to

determine 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 possibility

that the lowlands tea may have different caffeine content. Samples tea leaves from lowlands

can 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 conducted

on the second variety to investigate whether different tea variety will have different amount

of caffeine and how the period of fermentation and temperature will affects the amount of

caffeine in its brewed tea.

Investigation on how brewing temperature affects the rate of diffusion of beneficial

substances that bring health benefits should be carried out in order to maximise the benefits

of tea.

9.0 Conclusion

Results show the effects of period of fermentation and brewing temperature on the amount of

caffeine are significant. As the period of fermentation and brewing temperature increase, the

amount of caffeine increases. This research shows that in order to minimise the amount of

caffeine in brewed tea, tea leaves should undergo minimum fermentation and be brewed at a

lower temperature. Unfermented tea leaves may not provide any aroma and flavour thus tea

leaves should be fermented at a short period of time. Brewing tea at 25.0°C shows that


Page 35 of 42

caffeine diffuses very slowly into the water and the same goes to other substances including

beneficial ones. Thus it is best to brew tea within 60.0–80.0°C. Therefore, it is concluded that

under these suggested conditions, consumers will be able to enjoy the flavour and benefits of

Camellia Sinensis but at the same time minimising caffeine intake.


Page 36 of 42

10.0 Appendix

Appendix 1

(http://kidshealth.org/teen/nutrition/general/caffeine.html#)

Moderation Is the Key

Caffeine is usually thought to be safe in moderate amounts. Experts consider 200–300 mg of

caffeine a day to be a moderate amount for adults. Teens should try to limit caffeine

consumption to no more than 100 mg of caffeine daily, and kids should get even less.


Page 37 of 42

Appendix 2

ANOVA – Analysis of Variance

To 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

Between Groups SSb k - 1 𝑀𝑆𝑏2 =

𝑆𝑆𝑏

𝑘−1

𝑀𝑆𝑤𝑀𝑆𝑏

Computer generated Fk-1, N-k

Within Groups SSw N - k 𝑀𝑆𝑤2 =

𝑆𝑆𝑤

𝑁−𝑘

Total SSt N - 1 Table 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

𝑀𝑆𝑏2

Mean squares between groups

𝑀𝑆𝑤2

Mean squares within group

Table 5: Legend for ANOVA table.


Page 38 of 42

HSD – Tukey’s Honestly Significant Difference

HSD = q (α, k, N – k) 𝑀𝑆𝑤

2

𝑛

α = represents the significant level (0.05)

k = represents total number of groups

N – k = represents total number of results – total number of groups

q = the value based on (α, k, N – k).

ANOVA calculations using Microsoft Office Excel 2007 and HSD values:

Anova: Single factor

SUMMARY Groups

(Period of Fermentation, min) Count Sum Average Variance Column 1 (0) 3 16.3 5.44 0.26 Column 2 (40) 3 13.7 4.56 0.48 Column 3 (80) 3 16.3 5.44 0.26 Column 4 (120) 3 19.7 6.56 0.26

ANOVA Source of Variation SS df MS F P-value F crit

Between Groups 6.04 3 2.01 6.39 1.61E-02 4.07

Within Groups 2.52 8 0.31

Total 8.56 11 Table 6: ANOVA for period of fermentation (mean amount of crude caffeine extracted).

HSD = 4.53 0.31

3

= 1.46


Page 39 of 42


SUMMARY Group

(Period of Fermentation, min) Count Sum Average Variance Column 1 (0) 3 7.3 2.44 0.48 Column 2 (40) 3 7.7 2.56 0.48 Column 3 (80) 3 8.3 2.78 0.26 Column 4 (120) 3 14.3 4.78 0.26


Between Groups 10.92 3 3.64 9.83 4.65E-03 4.07


Total 13.88 11 Table 7: ANOVA for period of fermentation (mean amount of purified caffeine).

HSD = 4.53 0.37

3

= 1.59


Page 40 of 42


SUMMARY Groups Count Sum Average Variance

Column 1 (25°C) 3 7.4 2.47 0.25 Column 2 (60°C) 3 11.4 3.80 0.36 Column 3 (80°C) 3 14.4 4.80 0.28 Column 4 (100°C) 3 15.8 5.27 0.41


Between Groups 13.82 3 4.61 14.11 1.47E-03 4.07


Total 16.44 11 Table 8: ANOVA table for temperature.

HSD = 4.53 0.33

3

= 1.50


Page 41 of 42

Appendix 3

Partition law states that at a constant temperature, a solute distributes itself between two

immiscible liquids so that the ratio of its concentration in each solvent is constant, regardless

of the amount of solute added. [6]

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑖𝑛 𝐷𝐶𝑀

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟 = 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;

Partition coefficient, k from caffeine in both solvent mixture is

18.2

1002.18

100

= 8.35;

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 =

𝑥

20.03− 𝑥

25

x = 0.012g

Second extraction: 8.35 =

𝑥

20.018− 𝑥

25

x = 0.007g

Third extraction: 8.35 =

𝑥

20.011− 𝑥

25

x = 0.004g


Page 42 of 42

11.0 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=TSN&search_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 the

Levels of Tea Polyphenols and Caffeine in Tea Leaves; J. Agric. Food Chem., 2003.

http://www.telegraph.co.uk/health/3356550/Healthy-drinking-Tea-total.html

http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=506801

http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=506801

Education

Caffeine quantification from tea leaves using solvent extraction