[Luan van] trích ly và cô lập caffeine từ trà xanh

VIETNAM NATIONAL UNIVERSITY-HO CHI MINH CITY

UNIVERSITY OF TECHNOLOGY

-------------------

VO THI KIM NGAN

STUDY ON THE EXTRACTION AND ISOLATION OF

CAFFEINE FROM GREEN TEA Camellia sinensis (L.)

FIELD : ORGANIC CHEMISTRY

MASTERS THESIS

HO CHI MINH CITY, July 2010

CÔNG TRÌNH ĐƯỢC HOÀN THÀNH TẠI

TRƯỜNG ĐẠI HỌC BÁCH KHOA

ĐẠI HỌC QUỐC GIA TP HỒ CHÍ MINH

Cán bộ hướng dẫn khoa học: TS. PHẠM THÀNH QUÂN

Cán bộ chấm nhận xét 1: TS. PHẠM S

Cán bộ chấm nhận xét 2: TS. NGUYỄN THỊ LAN PHI

Luận văn thạc sĩ được bảo vệ tại Trường Đại học Bách Khoa, ĐHQG Tp. HCM ngày

07 tháng 08 năm 2010

Thành phần Hội đồng đánh giá luận văn thạc sĩ gồm:

1. PGS.TS Trần Thi Việt Hoa

2. TS. Phạm Thành Quân

3. TS. Trần Thị Kiều Anh

4. TS. Phạm S

5. TS. Trần Lê Quan

Xác nhận của Chủ tịch Hội đồng đánh giá LV và Bộ môn quản lý chuyên ngành sau

khi luận văn đã được sửa chữa (nếu có).

Chủ tịch Hội đồng đánh giá LV Bộ môn quản lý chuyên ngành

TRƯỜNG ĐẠI HỌC BÁCH KHOA TP. HCM CỘNG HÒA XÃ HỘI CHỦ NGHĨA VIỆT NAM

PHÒNG ĐÀO TẠO SAU ĐẠI HỌC Độc Lập - Tự Do - Hạnh Phúc

Tp.HCM, ngày 0 5 tháng 0

7 năm 2010

NHIỆM VỤ LUẬN VĂN THẠC SĨ

Họ và tên học viên : VÕ THỊ KIM NGÂN Phái: Nữ

Ngày tháng năm sinh: 06/04/1982

Nơi sinh : Tiền Giang

Chuyên ngành : CÔNG NGHỆ HỮU CƠ MSHV : 00507378

I.TÊN ĐỀ TÀI

Nghiên cứu trích ly và tách caffeine từ trà xanh

II. NHIỆM VỤ VÀ NỘI DUNG

Khảo sát ảnh hưởng của các yếu tố nhiệt độ, thời gian, tỷ lệ rắn-

lỏng và số lần trích đến lượng caffeine trong dịch trích từ trà bằng

nước.

Khảo sát sự hấp phụ caffeine khi cho dịch trích chảy qua cột hấp phụ với bốn

loại chất hấp phụ khác nhau: XAD-4, XAD-7, IR 120H và than hoạt tính.

Khảo sát sự giải hấp caffeine từ các cột hấp phụ nói trên với các dung môi giải

hấp khác nhau: ethanol, acetone, ethyl acetate, chloroform và hexane.

III. NGÀY GIAO NHIỆM VỤ: 01/2010

IV. NGÀY HOÀN THÀNH NHIỆM VỤ: 06/2010

V. CÁN BỘ HƯỚNG DẪN: TS. PHẠM THÀNH QUÂN

CÁN BỘ HƯỚNG DẪN CN BỘ MÔN QL CHUYÊN NGÀNH

ACKNOWLEDGEMENTS

I would like to acknowledge the following people for their contributions to the project:

My supervisor, Dr. PHAM THANH QUAN for his time, guidance and enthusiasm

throughout the project.

Professors and staffs of the Department of Organic Chemistry and Faculty of Chemical

Engineering for their help and useful advice.

My friends in the Laboratory of Organic Chemistry for their help.

My family for their support and encouragement.

i

ABSTRACT

Caffeine is the world’s most popular drug and consumed everyday by millions of

people in the world. It is also used in many beverages and food. Due to its ability

to relieve headache and stimulate breathing, caffeine has been used in headache

relieving medicine, treatment of cessation of breathing for newborn babies and as

an antidote against the depression of breathing by overdoses of heroin. Caffeine

was found in tea with a content of 3-4 %. Tea has been widely grown in Vietnam

and is a large potential source of caffeine production.

In this project, the extraction and isolation of caffeine from Vietnamese green tea

were intensively studied and several results were obtained as below.

Green tea was extracted by hot distilled water and the optimal caffeine

extraction was established for 5g of tea: 10 min, 75oC, solid-liquid ratio of

1/20, one-time extraction. The caffeine amount in the tea extract is 3.2

times that of EGCG.

Caffeine in the tea extracts were adsorbed onto four adsorbent columns

(XAD-4, XAD-7, IR-120H, activated carbon) by passing the extracts

through the columns. XAD-4 was found to have the highest adsorption

affinity for caffeine while IR-120H has the highest adsorption ability for

EGCG.

Caffeine was desorbed from the columns by different solvents: ethanol,

acetone, ethyl acetate, chloroform and hexane. Acetone showed the best

desorption capability for caffeine compared to other solvents. EGCG was

not found in the desorption solutions from XAD-4, XAD-7, activated

carbon but was detected in the desorption solutions by ethanol, acetone

and ethyl acetate from IR-120H column.

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS...............................................................................................................i

ABSTRACT.....................................................................................................................................ii

TABLE OF CONTENTS................................................................................................................iii

LIST OF TABLES............................................................................................................................v

LIST OF FIGURES.......................................................................................................................viii

1. CHAPTER 1: INTRODUCTION.............................................................................................1

2. CHAPTER 2: LITERATURE REVIEW.................................................................................2

2.1. Green tea and caffeine......................................................................................................2

2.1.1. Overview...................................................................................................................2

2.1.2. Green tea’s composition...........................................................................................3

2.1.3. Main components in green tea..................................................................................5

2.1.4. Tea production in the world......................................................................................9

2.1.5. Tea in Vietnam........................................................................................................11

2.2. Extraction and extraction of caffeine from green tea.....................................................11

2.2.1. Extraction................................................................................................................11

2.2.2. Extraction of caffeine from green tea.....................................................................14

2.2.2.1. Extraction by organic solvents................................................................................14

2.3 Adsorption and adsorption in caffeine isolation.............................................................16

2.3.1 Adsorption..............................................................................................................16

2.3.2 Adsorbents..............................................................................................................18

3. CHAPTER 3: EXPERIMENTAL PROCEDURES..............................................................23

3.1. Chemicals and reagents..................................................................................................23

3.2. Preparation of standard solutions....................................................................................23

3.3 Sample preparation.........................................................................................................24

3.4 Apparatus........................................................................................................................25

3.5 Description of extraction and purification procedure.....................................................25

4. CHAPTER 4: RESULTS & DISCUSSION...........................................................................27

iii

4.1 Standard curves...............................................................................................................27

4.2. Caffeine and EGCG extraction from green tea leaves by pure water:............................32

4.2.1. Comparison between HPLC and UV-VIS method for determination of caffeine amount …………………………………………………………………………………….32

4.2.2. Comparison between HPLC and UV-VIS method for determination of EGCG amount …………………………………………………………………………………….34

4.2.3. Effect of extraction time to extracted caffeine and EGCG amount (solid/liquid 1/20; 50oC)..............................................................................................................................36

4.2.4. Effect of temperature to extracted caffeine and EGCG amount ( solid/liquid 1/20; 10 min) …………………………………………………………………………………….39

4.2.5. Effect of solid-liquid (tea-water) ratio to extracted caffeine and EGCG amount (75oC, 10min)..........................................................................................................................41

4.2.6. Effect of number of extraction times to extracted caffeine and EGCG amount (75oC, 10min, 1/20) :..............................................................................................................43

4.3. Caffeine isolation by column adsorption and desorption...............................................45

4.3.1. XAD-4 column.......................................................................................................45

4.3.2. Adsorption affinity of different adsorbents for caffeine.........................................51

4.3.3. XAD-7 column:......................................................................................................53

4.3.4. Activated carbon column........................................................................................54

4.3.5. IR-120H column.....................................................................................................56

5. CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS..........................................58

REFERENCES...............................................................................................................................60

APPENDICES................................................................................................................................63

1. Tables of data......................................................................................................................63

2. Calculation formulas...........................................................................................................81

3. Typical HPLC and UV-VIS spectra...................................................................................83

iv

LIST OF TABLES

Table 2. 1 Green tea’s chemical composition......................................................................4

Table 2. 2 Caffeine in some commercial products..............................................................7

Table 2. 3 Tea production in the world (tons)...................................................................10

Table 2. 4 Summary of selected extraction techniques by phases involved and the basic

for separation.....................................................................................................................12

Table 2. 5 Parameters of physisorption and chemisorptions.............................................17

Table 2. 6 Typical properties of Amberlites XAD-4 and XAD-7.....................................19

Table 4. 1 Equations of standard curves............................................................................29

Table 4. 2 Data of caffeine determination by UV-VIS....................................................32

Table 4. 3 Data of caffeine determination by HPLC method............................................33

Table 4. 4 Comparison of UV-VIS and HPLC results......................................................33

Table 4. 5 Data of EGCG determination by UV-VIS........................................................34

Table 4. 6 Data of EGCG determination by HPLC...........................................................34

Table 4. 7 Comparison of UV-VIS and HPLC results......................................................35

Table 4. 8 Effect of extraction time to extracted caffeine amount (solid/liquid 1/20; 50oC)

...........................................................................................................................................36

Table 4. 9 Effect of extraction time to extracted EGCG amount (solid/liquid 1/20; 50oC)

...........................................................................................................................................37

Table 4. 10 Effect of temperature to extracted caffeine amount (solid/liquid 1/20; 10 min)

...........................................................................................................................................39

v

Table 4. 11 Effect of temperature to extracted EGCG amount (solid/liquid 1/20; 10 min)

...........................................................................................................................................39

Table 4. 12 Effect of solid-liquid (tea-water) ratio to extracted caffeine amount (75oC,

10min)................................................................................................................................41

Table 4. 13 Effect of solid-liquid (tea-water) ratio to extracted EGCG amount (75oC,

10min)................................................................................................................................41

Table 4. 14 Effect of number of extraction times to extracted caffeine amount...............43

Table 4. 15 Effect of number of extraction times to extracted EGCG amount.................43

Table 4. 16 Effect of XAD-4 polymer mass to caffeine amount left in the mother solution

...........................................................................................................................................46

Table 4. 17 Effect of desorption solvent volume to desorbed caffeine amount with ethanol

as desorption solvent, 7g XAD-4.......................................................................................47

Table 4. 18 Effect of different desorption solvents to desorbed caffeine amount using

XAD-4 (7g), 125 ml solvent..............................................................................................49

Table 4. 19 Caffeine contents left in the mother solutions after passing adsorbent columns

...........................................................................................................................................51

Table 4. 20 EGCG contents left in the mother solutions after passing adsorbent columns

...........................................................................................................................................51


XAD-7 (7g), 125 ml solvent..............................................................................................53


activated carbon (7g), 125 ml solvent................................................................................54

Table 4. 23 Effect of different desorption solvents to desorbed caffeine amount using IR-

120H (7g), 125 ml solvent.................................................................................................56

vi

Table 4. 24 Effect of different desorption solvents to desorbed EGCG amount using IR-

120H (7g), 125 ml solvent.................................................................................................56

vii

LIST OF FIGURES

Figure 2. 1 Pictures of a tea bush and tea leaves.................................................................2

Figure 2. 2 Chemical structure of caffeine, theobromin and theophyllin............................5

Figure 2. 3 Tea distribution in the world.............................................................................9

Figure 2. 4 Amberlite XAD-4 and XAD-7........................................................................19

Figure 2. 5 Amberlite IR120H..........................................................................................20

Figure 4. 1 UV-VIS standard curve of caffeine.................................................................27

Figure 4. 2 UV-VIS standard curve of EGCG...................................................................27

Figure 4. 3 HPLC standard curve of Caffeine..................................................................28

Figure 4. 4 HPLC standard curve of EGCG.....................................................................28

Figure 4. 5 (a) UV-VIS spectrum and (b) HPLC chromatogram of standard Caffeine.....29

Figure 4. 6 (a) UV spectrum and (b) HPLC chromatogram of standard EGCG...............30

Figure 4. 7 Effect of extraction time on the extracted amount of caffeine and EGCG.....37

Figure 4. 8 Effect of temperature to extracted caffeine and EGCG amount.....................40

Figure 4. 9 Effect of solid-liquid (tea-water) ratio to extracted caffeine and EGCG

amount (75oC, 10min)........................................................................................................42

Figure 4. 10 Effect of number of extraction times to extracted caffeine and EGCG

amount (75oC, 10min, 1/20)...............................................................................................44

Figure 4. 11 HPLC chromatogram of the tea extract (10 min, solid/liquid ratio of 1/20;

75oC, 5g tea) before passing absorbent column.................................................................45

Figure 4. 12 Effect of XAD-4 polymer mass to caffeine amount left in the mother

solution..............................................................................................................................46

viii

Figure 4. 13 Effect of desorption solvent volume to desorbed caffeine amount with

ethanol as desorption solvent, 7g XAD-4..........................................................................48

Figure 4. 14 Effect of different desorption solvents to desorbed caffeine amount using

XAD-4 (7g), 125 ml solvent..............................................................................................49

Figure 4. 15 Adsorption yield of different adsorbent columns for caffeine and EGCG. . .52

Figure 4. 16 Effect of different desorption solvents to desorption yield of caffeine, using

XAD-7 (7g), 125 ml solvent..............................................................................................53


activated carbon (7g), 125 ml solvent................................................................................55

Figure 4. 18 Effect of different desorption solvents to caffeine and EGCG desorption

yield using IR-120H..........................................................................................................57

ix

1. CHAPTER 1: INTRODUCTION

Caffeine is one of the most popular compounds which are taken everyday by millions of

people all around the world. Due to its pleasant flavor and stimulating effect, caffeine is

more common than any chemicals and has been consumed for hundreds of years. It is

also a key component of many popular drinks and food, such as tea, coffee, soft drinks,

energy drinks and chocolate. Recently, caffeine has been used as a drug. It can stimulate

the central nervous system and make people more alert, less drowsy and improve

coordination. With its unique properties, caffeine has been combined with certain pain

relievers or medicines for treating headaches because it makes those drugs work more

quickly and effectively. Therefore, caffeine is becoming more and more important to food

and pharmaceutical industries.

Green tea (Camellia sinensis) has a long tradition of being used as a drink in Asian

countries including Vietnam, and has become one of the most popular drinks in the

world. Caffeine was discovered in green tea in the 1820s. Caffeine content in green tea

leaves was found to be 3-4 %, which is higher than that in coffee bean (1.1-2.2%). Tea

plants have been intensively grown in many areas in Vietnam, such as Thai Nguyen,

Tuyen Quang, Lam Dong. This is a large potential supply of caffeine. However, up-to-

date, most of this green tea source has been only used for exportation or beverage

production. So, it is necessary to develop a method to extract and isolate caffeine from

Vietnamese green tea for large scale application.

1

2. CHAPTER 2: LITERATURE REVIEW

2.1. Green tea and caffeine

2.1.1. Overview

More than twelve centuries ago, green tea became a popular drink in China. When

sailors began to bring tea to England from Asia in 1644, tea began to replace ale

as the national drink of England. Tea shrubs were introduced in the United States

in 1799. Tea is now one of the most widely consumed beverages in the world,

second only to water [1].

Figure 2. 1 Pictures of a tea bush and tea leaves

Tea is known as Camellia sinensis (L.) O.Kuntze. It belongs to Dicotyladoneae

band, rank of Theales, family of Theaceae, class of Dicotyladoneae, branch of

agio Sperimae, variety of agio Sperimae, species of Thea Sinensis L. Camellia

sinensis is a green plant that grows mainly in tropical and sub-tropical climates.

Nevertheless, some varieties can also tolerate marine climates and are cultivated

as far north as Pembrokeshirein the British mainland. Tea plants require at least

127 cm of rainfall a year and prefer acidic soils [1-3].

2

Leaves of Camellia sinensis soon begin to wilt and oxidize, if they are not dried

quickly after picking. The leaves turn progressively darker as their chlorophyll

breaks down and tannins are released. This process, enzymatic oxidation, is called

fermentation in the tea industry, although it is not a true fermentation. It is not

caused by micro-organisms, and is not an anaerobic process. The next step in

processing is to stop oxidation at a predetermined stage by heating, which

deactivates the enzymes responsible. Without careful moisture and temperature

control during manufacture and packaging, the tea will grow fungi. The fungus

causes real fermentation that will contaminate the tea with toxic and sometimes

carcinogenic substances, as well as off-flavors. Tea is traditionally classified

based on the techniques with which it is produced and processed [1-3]:

White tea: Wilted and unoxidized

Yellow tea: Unwilted and unoxidized, but allowed to yellow

Green tea: Unwilted and unoxidized

Oolong: Wilted, bruised, and partially oxidized

Black tea: Wilted, sometimes crushed, and fully oxidized

Post-fermented tea: Green tea that has been allowed to ferment/compost

2.1.2. Green tea’s composition

As mentioned, green tea production does not involve oxidation of young tea

leaves. Therefore, green tea’s chemical composition is very similar to that of fresh

leaf and presented in table 2.1 [1-8].

Green tea contains catechins, a type of antioxidant with EGCG as the main

component, which can compose up to 30 % of the dry weight. Beside catechins,

tea contains caffeine at about 3-4 % of its dry weight. Tea also contains

theobromine, theophylline, amino acids, vitamins, minerals, etc.

3

Table 2. 1 Green tea’s chemical composition

Compound Percentage (%)

Caffeine 3-4

Catechin 25-30

Flavonol and flavonol glucoside 3-4

Polyphenolic acid and depside 3-4

Leucoanthocyanin 2-3

Chlorophyll & other color substances 0.5 – 0.6

Mineral 5-6

Theobromine 0.2

Theophylline 0.5

Amino acid 4-5

Organic acid 0.5 – 0.6

Monosaccharide 4-5

Polysaccharide 14-22

Cellulose & hemicellulose 4-7

Pectin 5-6

Lignin 5-6

Protein 14-17

Lipid 3-5

4

Volatile substances 0.01 – 0.02

5

2.1.3. Main components in green tea2.1.3.1. Caffeine

Caffeine (1,3,7-trimethylxanthine) is a plant alkaloid found in coffee, tea, cocoa,

etc. It acts as natural pesticide, protecting plants against certain insects feeding on

them [1-4, 9, 10]. Green tea also contains two caffeine-like substances:

theophylline, which is a stronger stimulant than caffeine, and theobromine, which

is slightly weaker than caffeine.

The most important sources of caffeine are coffee (Coffea spp.), tea (Camellia

sinensis), guarana (Paullinia cupana), maté (Ilex paraguariensis), cola nuts (Cola

vera), and cocoa (Theobroma cacao). The amount of caffeine found in these

products varies – the highest amounts are found in guarana (4–7%), followed by

tea leaves (3-4%), maté tea leaves (0.89–1.73%), coffee beans (1.1–2.2%), cola

nuts (1.5%), and cocoa beans (0.03%) [11].

Figure 2. 2 Chemical structure of caffeine, theobromin and theophyllin

(from left to right)

6

Some basic information about caffeine is displayed as below:

Molecular formula: C8H10N4O2

Molar mass: 194.19 g/mol

Appearance: odorless in liquid, white needles or powder.

Density: 1.23 g/cm3

Melting point: 227oC

Boiling point: 178oC

Solubility in water: 2.17 g/100 ml (25oC), 18.0 g/100ml (80oC), 67.0g/100

ml(100oC)

Caffeine is a legal drug which is taken everyday by millions of people all around

the world. It is more common than any medicine. The average daily caffeine

intake in the United States is about 200 mg per individual [12].

Caffeine is widely used in beverage industry. Soft drinks typically contain about

10 to 50 milligrams of caffeine per serving. By contrast, energy drinks such as

Red Bull can start at 80 milligrams of caffeine per serving. The caffeine in these

drinks either originates from the ingredients used or is an additive derived from

the product of decaffeination or from chemical synthesis. Guarana, a prime

ingredient of energy drinks, contains large amounts of caffeine with small

amounts of theobromine and theophylline. Chocolate derived from cocoa beans

contains a small amount of caffeine. The weak stimulant effect of chocolate may

be due to a combination of theobromine and theophylline as well as caffeine. A

typical 28-gram serving of a milk chocolate bar has about as much caffeine as a

cup of decaffeinated coffee, although some dark chocolate currently in production

contains as much as 160 mg per 100g. It is also used as a flavor enhancer in food

7

and as a flavoring agent in baked goods, frozen dairy desserts, gelatins, puddings

and soft candy [4].

Caffeine is a substance that can stimulate the central nervous system. It makes

people more alert, less drowsy and improves coordination. Combined with certain

pain relievers or medicines for treating migraine headache, caffeine makes those

drugs work more quickly and effectively. Caffeine alone can also help to relieve

headaches. Antihistamines are sometimes combined with caffeine to weaken the

drowsiness that those drugs cause. Caffeine is also used to treat breathing

problems in newborns and in young babies after surgery [1, 12]. Caffeine content

in some commercial products is shown in table 2.2. In recent years, various

manufacturers have begun putting caffeine into shower products such as shampoo

and soap, claiming that caffeine can be absorbed through the skin. However, the

effectiveness of such products has not been proven, and they are likely to have

little stimulatory effect on the central nervous system because caffeine is not

readily absorbed through the skin.

Table 2. 2 Caffeine in some commercial products

ProductServing size Caffeine per serving

(mg)

Caffeine tablet (regular-strength) 1 tablet 100

Caffeine tablet (extra-strength) 1 tablet 200

Excedrin tablet 1 tablet 65

Excedrin 1 tablet 65

Bayer Select Maximum Strength1 tablet

65.4

Midol Menstrual Maximum

Strength

1 tablet 60

8

NoDoz 100 mg 1 tablet 32.4

Pain Reliever Tablets1 tablet

65

Vivarin1 tablet

200

Panadol 500mg 1 tablet 65

2.1.3.2. Catechins

As stated above, green tea can contain up to 30 % of catechins. The four main

catechins in tea are:

Epicatechin (EC)

Epicatechin-3-gallate (ECG)

Epigallocatechin (EGC)

Epigallocatechin-3-gallate (EGCG): major component of tea catechin

EGCG has the highest content compared to other tea catechins and is a strong

antioxidant. It has been found to be over 100 times more effective in neutralizing

free radicals than vitamin C and 25 times more powerful than vitamin E [4].

2.1.3.3. Amino acid

Amino acid is another important constituent of green tea and there are about 20

different types of amino acids found in green tea. Theanine is the major form of

amino acid, which is unique to green tea because the steaming process does not

eliminate it. It gives the elegant taste and sweetness to green tea. As a natural

process, tea plant converts some amino acids into catechins. This means that the

theanine content of green tea varies greatly according to the harvesting season of

tea leaves [1, 2].

2.1.3.4. Vitamins, minerals and other components

9

Green tea contains several B vitamins and C vitamin. These vitamins are left

intact in the tea-making process. Other green tea ingredients include 6% to 8% of

minerals such as aluminium, fluoride and manganese. Green tea also contains

organic acids such as gallic and quinic acids, and 10% to 15% of carbohydrate

and small amount of volatiles [3].

10

2.1.4. Tea production in the world

Figure 2. 3 Tea distribution in the world

Tea is produced in many countries. China is the largest tea producing country that

produces green tea, oolong tea and black tea. Other than China, tea is also

produced in India, Kenya, Russia, Sri Lanka, Indonesia, Thailand, Vietnam,

Japan, Turkey, etc. The annual production of tea is about 2.9-3.9 million tons.

Table 2.3 shows the tea production data in the world in 2000-2007 [13].

11

Table 2. 3 Tea production in the world (tons)

2.1.5. Tea in Vietnam

Vietnam has a strong tea culture dating back thousands of years. Tea has been produced

commercially since the beginning of the 20th century. Tea plantations are most plentiful in

the north but are also found in central Vietnam. Vietnam has traditionally been an

exporter of black tea – most of which ends up in blends. The Vietnamese people,

however, have a long tradition of drinking green tea, and this green tea is gaining a

reputation as some of the finest green tea available.

There are many different types of Vietnam tea. Black tea is the leader in exports, but it

has a reputation as being a “cheap tea” that can only be used for blending. Vietnam also

produces oolong tea and white tea. The best Vietnam tea, however, is green tea. Vietnam

has been producing green tea for thousands of years and this long history shows in the

quality of the tea. The climate and soil are ideal for growing tea, and there are many

regional variations and methods of production. Since 1995 tea production in Vietnam has

doubled and exports have increased almost 300%. Taiwan and Japan are the biggest

Asian importers of Vietnamese green tea, and western countries like the USA, France,

and Australia are also major importers [8, 14].

2.2. Extraction and extraction of caffeine from green tea

2.2.1. Extraction

An extraction is the process of moving one on more compounds from one phase to

another. Solid-liquid extraction is the process of removing a solute from a solid by using

of liquid solvent. In general, the extraction process occurs as a series of steps. First the

extracting phase is contacted with the sample phase, usually by a diffusion process. Then

the compound of interest partitions into or is solubilized by the extracting solvent. With

liquid samples this step is generally not problematic. However, for the compound being

extracted to go into the extracting solvent the energy of interaction between the

compound of interest and the sample substrate must be overcome. That is, the material’s

affinity for the extracting solvent must be greater than its affinity for the sample. Various

extraction techniques can be classified according to the phases and applied work (or the

basis of separation), as shown in table 2.4 for several selected extraction techniques [15-

18].

Table 2. 4 Summary of selected extraction techniques by phases involved and the basic for

separation

Extraction

technique

Sample phaseExtracting phase Basis for separation

Liquid-liquid

extractionliquid liquid Partitioning

Solid-phase

extraction ( and

microextraction)

Gas, liquidLiquid or solid

stationary phasePartitioning or adsorption

Leaching solid liquid Partitioning

Soxhlet extraction solid liquid Partitioning (with applied heat)

Sonication solid liquidPartitioning (with applied

ultrasound energy)

Accelerated solvent

extractionsolid liquid Partitioning (with applied heat)

Microwave-assisted

extractionsolid liquid

Partitioning (with applied

microwave irradiation)

Supercritical fluid

extraction

Solid, liquid Supercritical fluid Partitioning (with applied heat)

Purge-and-trap solid, liquid gas Partitioning

Thermal desorption solid liquid gas Partitioning (with applied heat)

2.2.1.1. Requirements for extraction

Chemical samples requiring extraction are composed of the compound of interest and the

sample matrix, which may contain interfering species. Prior to choosing an extraction

method, knowledge must be gained about the structure (including functional group

arrangement), molecular mass, polarity, solubility, pKa, and other physical properties of

both the species of interest and potential interfering compounds [15, 16].

Some requirements of a suitable extraction solvent [15, 16]:

Selectivity, i.e. the ability to extract the material of interest in preference to other,

interfering material.

High distribution coefficient to minimize the solvent-to-feed ratio.

Solute solubility, which is usually related to polarity differences between the two

phases.

Ability to recover the extracted material. Thus the formation of emulsions and other

deleterious events must be minimized.

Capacity, the ability to load a high amount of solute per unit of solvent.

Low interfacial tension to facilitate mass transfer across the phase boundary.

Interfacial tension tends to decrease with increasing solute solubility and as solute

concentration increases. In liquid-liquid extraction low interfacial tension allows the

disruption of solvent droplets (entrained in the feed solution) with low agitation.

Low relative toxicity.

Nonreactive. In some instances, such as ion exchange extractions, known reactivity in

the extracting fluid is used. In addition to being nonreactive with the feed, the solvent

should be nonreactive with the extraction system (e.g., noncorrosive) and should be

stable.

Inexpensive. Cost considerations should emphasize the energy costs of an extraction

procedure, since, for a given extraction method, capital costs are relatively constant.

2.2.2. Extraction of caffeine from green tea

Due to the important role of caffeine in food, beverage and pharmaceutical industries,

there have been several attempts to isolate caffeine from tea by extraction.

2.2.2.1. Extraction by organic solvents

Extraction of caffeine from tea is an important industrial process and can be performed

using a number of different solvents. Benzene, chloroform, trichloroethylene and

dichloromethane have all been used over the years [6, 19-25].

In Misra et al.’s study, different organic solvents and aqueous mixtures of varying nature

were used for the screening of caffeine extraction from tea granules. Order of recovery of

caffeine with different organic solvents and aqueous mixtures was: n-hexane < ethyl

acetate < methylene dichloride< chloroform < methanol < water < 5% sulphuric acid in

water < 5% diethyl amine in water [12].

2.2.2.2. Extraction by supercritical carbon dioxide

Supercritical carbon dioxide is a good nonpolar solvent for caffeine, and is safer than

most of organic solvents. In Kim et al.’s work, caffeine and EGCG (epigallocatechin

gallate) were extracted from green tea using supercritical carbon dioxide (SCCO2) with

water as a cosolvent [26]. Various experimental conditions were explored including

temperatures ranging 40–80oC, pressure ranging 200–400 bar, and water contents ranging

4–7 wt%. At 40oC, 400 bar and the water content of 7 wt%, the caffeine extraction yield

was 54% while the EGCG extraction yield was 21%, resulting in caffeine/EGCG

extraction selectivity of 2.57.

2.2.2.3. Microwave-assisted extraction of tea polyphenols and tea caffeine

from green tea leaves

A microwave-assisted extraction (MAE) method was used for the extraction of tea

polyphenols (TP) and tea caffeine from green tea leaves. Various experimental

conditions, such as ethanol concentration (0-100%, v/v), MAE time (0.5-/8 min),

liquid/solid ratio (10:1-/25:1 ml g-1), pre-leaching time (0-/90 min) before MAE and

different solvents for the MAE procedure were investigated to optimize the extraction.

Colorimetric method was used to analyze the amounts of tea caffeine and polyphenols.

To determine caffeine amount, lead acetate was used to remove polyphenols out of the

extract. The extraction of tea polyphenols and tea caffeine with MAE for 4 min (30 and

4%) were higher than those of extraction at room temperature for 20 h, ultrasonic

extraction for 90 min and heat reflux extraction for 45 min (28 and 3.6%), respectively.

From the points of extraction time, the extraction efficiency and the percentages of tea

polyphenols or tea caffeine in extracts, MAE was more effective than the conventional

extraction methods studied [27].

2.2.2.4. Caffeine extraction from green tea leaves assisted by high pressure

processing

In Jun’s research, high pressure processing (HPP) extraction was used to extract caffeine

from green tea leaves. The effect of different parameters such as high hydrostatic

pressure (100–600 MPa), different solvents (acetone, methanol, ethanol and water),

ethanol concentration (0–100% ml/ml), pressure holding time (1–10 min) and liquid/solid

ratio (10:1 to 25:1 ml/g) were studied for the optimal caffeine extraction from green tea

leaves. The highest yields (4.0 ± 0.22%.) were obtained at 50% (ml/ml) ethanol

concentration, liquid/solid ratio of 20:1 (ml/g), and 500 MPa pressure applied for 1 min.

Experiments using conventional extraction methods (extraction at room temperature,

ultrasonic extraction and heat reflux extraction) were also conducted, which showed that

extraction using high pressure processing possessed higher yields, shorter extraction

times and lower energy consumption [28].

2.2.2.5. Decaffeination of fresh green tea leaf by hot water treatment

Hot water treatment was used to decaffeinate fresh tea leaf in Liang et al.’s study [29].

Water temperature, extraction time and ratio of tea leaf to water had a statistically

significant effect on the decaffeination. When fresh tea leaf was decaffeinated with a ratio

of tea leaf to water of 1:20 (w/v) at 100oC for 3 min, caffeine concentration was

decreased from 23.7 to 4.0 mg/g, while total tea catechins decreased from 134.5 to 127.6

mg/g; 83% of caffeine was removed and 95% of total catechins was retained in the

decaffeinated leaf. It was found that the hot water treatment is a safe, effective and

inexpensive method for decaffeinating green tea.

2.3 Adsorption and adsorption in caffeine isolation

2.3.1 Adsorption

Adsorption is a natural tendency for components of a liquid or a gas to collect - often as a

monolayer but sometimes as a multilayer - at the surface of a solid material. This is a

fundamental property of matter, having its origin in the attractive forces between

molecules. The solid material is called the adsorbent and the material adsorbed at the

surface of the adsorbent is the adsorbate.

There are two kinds of adsorption: chemisorption and physisorption, depending on the

nature of the surface forces. Physisorption is caused mainly by Van der Waals forces and

electrostatic forces between adsorbate molecules and the atoms which compose the

adsorbent surface. In chemisorption, there is significant electron transfer, equivalent to

the formation of a chemical bond between the sorbate and the solid surface. These

interactions are both stronger and more specific than the forces of physical adsorption

and are limited to monolayer coverage [30, 31].

The differences in the general features of physical and chemisorption can be seen below:

Table 2. 5 Parameters of physisorption and chemisorptions

Parameter Physical adsorption Chemisorption

Heat of adsorption (H) low, < 2 or 3 times latent heat

of evaporation

high, > 2 or 3 times latent

heat of evaporation

Specificity nonspecific highly specific

Nature of adsorbed

phase

monolayer or multilayer, no

dissociation of adsorbed

species

monolayer only, may

involve dissociation

Temperature range only significant at relatively

low temperatures

possible over a wide range

of temperature

Forces of adsorption no electron transfer, although

polarization of sorbate may

occur

electron transfer leading to

bond formation between

sorbate and surface

Reversibility rapid, nonactivated, reversible activated , may be slow and

irreversible

2.3.2 Adsorbents

There are many ways to classify adsorbents, for example, as polar and nonpolar adsorbents

(or hydrophilic and hydrophobic adsorbents). Polar adsorbents have affinity with polar

substances such as water or alcohols. So they are called “hydrophilic”. Aluminosilicates such

as zeolites, porous alumina and silica gel are examples of this type. In contrast, nonpolar

adsorbents are generally hydrophobic. Carbonaceous adsorbents, polymer adsorbents and

silicalite are typical nonpolar adsorbents. These adsorbents have more affinity with oil and

hydrocarbons than water. Adsorption is a prominent method for the treatment of effluents

containing organic substances from dilute aqueous solutions because of the high adsorbing

ability of the typical adsorbent. [30, 31].

Polymeric resins XAD-4 and XAD-7 (figure 2.4)

In comparison with classical adsorbents such as silicagels, aluminas and activated

carbons, macroporous polymeric adsorbents are more attractive alternatives because

of their wide range of pore structures and physic-chemical characteristics. Because of

its high chemically stability and excellent selectivity towards aromatic solutes,

Amberlite XAD-4 polymeric resin, a macroporous styrene-divinylbenzene

copolymer, is found to be a good polymeric adsorbent for organic compounds.

Amberlite XAD-7 is a nonionic aliphatic acrylic polymer, which derives its

adsorptive properties from its macroreticular structure (containing both a continuous

polymer phase and a continuous pore phase), high surface area and the aliphatic

nature of its surface. It is characterized as a hydrophobic adsorbent having a

somewhat more hydrophilic structure comparing to XAD-4. Its macroreticular

structure also gives to it excellent physical and thermal stability and it is also stable at

all pH range in aqueous solution. The typical properties of both resins are listed in

Table 2.6 [32-34]. Maity et al. and Saikia et al. investigated the adsorption of caffeine

onto these resins and found that XAD-4 possessed better adsorption behavior for

caffeine than XAD-7 [35, 36].

Figure 2. 4 Amberlite XAD-4 and XAD-7

Table 2. 6 Typical properties of Amberlites XAD-4 and XAD-7

Property XAD-4 XAD-7

Porosity (ml.pore/ml bead—dry basis) 0.35–0.50 ≥ 0.50

Surface area (m2/g dry basis) 750 450

Average pore diameter (Ao—dry basis) 50 90

Mean particle size (mesh) 40 40

Dipole moment 0.3 1.8

Chemical nature Polystyrene-

divinylbenzene

Methylacrylate ester

Amberlite IR-120H

Amberlite IR-120H resin is a strongly acidic cation exchange resin of the sulfonated

polystyrene type (figure 2.5) [30, 31, 37-39].

Figure 2. 5 Amberlite IR120H

The summary of its properties is described as below:

Physical form______________________________spherical beads

Matrix__________________________ Styrene divinylbenzene copolymer

Functional group ___________________________ Sulfonic acid

Ionic form ________________________________ H+

Total exchange capacity ____________________ ≥ 1.80 eq/L (H+ form)

Particle size

Uniformity coefficient __________________ ≤ 1.8

Harmonic mean size ____________________ 0.620 to 0.830 mm

< 0.300 mm ____________________________ 2 % max

Activated Carbon

Activated carbon (AC) is the most widely used sorbent. Its manufacture and use date

back to the 19th century. Its usefulness derives mainly from its large micropore and

mesopore volumes and the resulting high surface area. Compared with several other

sorbents, it is important to consider the charge of the surface because it determines

the capacity of the carbon for ion exchange. AC is dominantly used for purposes of

adsorption, a task for which it is well designed. AC is often used for adsorption of

organic solutes covers a wide spectrum of systems such as drinking water and

wastewater treatments, and applications in the food, beverage, pharmaceutical and

chemical industries.

In spite of the large market for AC, the specific mechanisms by which the adsorption

of many compounds, especially organic compounds, take place on this adsorbent are

still uncertain. Adsorption of organic compounds and of aromatics in particular, is a

complex interplay of electrostatic and dispersive interactions. This is particularly true

for phenolic compounds [40-42].

2.4. Scope of the project

As listed in previous sections, there have been some studies on extraction and isolation of

caffeine from green tea by different methods. However, most of the studies were based

on batch method, microwave energy, high pressure, and supercritical CO2, which are hard

to scale up for large application and expensive.

Therefore, in our project, we would like to study a way for extraction and isolation

caffeine from Vietnamese green tea, which is cheaper and easier to be scaled up for

industrial application.

To achieve that target, the following tasks will be done:

Extracting caffeine from green tea by distilled water and investigating the effect of

time, temperature, solid-liquid ratio and number of extraction times.

Investigating adsorption step for caffeine in the tea extract by using column

adsorption method with different adsorbents.

Investigating desorption step for caffeine from the adsorbent columns with different

desorption solvents.

Analyzing the caffeine and EGCG contents in the samples with UV-VIS and HPLC

techniques.

3. CHAPTER 3: EXPERIMENTAL PROCEDURES

3.1. Chemicals and reagents

Anhydrous caffeine and EGCG used for preparation of the standard solutions were purchased

from Sigma (St. Louis, MO, USA). Methanol for the mobile phase was HPLC grade (Fisher

Scientific, Pittsburgh, PA, USA). Deionized water was obtained from a water purification

system. XAD-4, XAD-7, IR-120H and activated carbon were purchased from Merck.

Ethanol, methanol, ethyl acetate, chloroform, hexane and acetone used in the experimental

work were all of analytical reagent grade chemicals. Green tea (Kim Tuyen) was collected

from Bao Loc-Lam Dong.

3.2. Preparation of standard solutions

The caffeine determination was accomplished by utilizing high performance liquid

chromatography (HPLC) equipped with a UV/Visible detector and by UV-VIS method with

HACH (DR-5000) UV/VIS spectrophotometer. The mobile phase for HPLC consists of

30:70 (v/v) of methanol and deionized water.

Preparation of caffeine standard solutions

Caffeine (10 mg) was weighed with an analytical balance and transferred into a 100 mL

volumetric flask. Deionized water was added to get a 100 mL bulk standard solution.

Shake was applied to completely dissolve the caffeine. From this stock solution, five

standard solutions of 1, 5, 10, 15 and 20ppm were prepared. The five standard solutions

were stored at room temperature. These solutions were analyzed to prepare the

appropriate standard curve.

Preparation of EGCG standard solutions

EGCG (10 mg) was weighed and transferred into a 100 mL volumetric flask. Deionized

water was added to get a 100 mL bulk standard solution. Shake was applied to

completely dissolve the EGCG. Dilution with deionized water was done to prepare 1, 10,

20, 30 and 40ppm solutions. 0.1 ml of each standard solution, 0.2 ml of Folin–ciocalteu

reagent, 0.5 ml of 20% Na2CO3 and water were added together to form a solution of

10ml. Keep them in dark for 1 hour. Then these five standard solutions were analyzed to

prepare the appropriate standard curve by UV/vis spectrophotometer.

3.3 Sample preparation Sample preparation for caffeine analysis by UV-VIS

5 g of ground green tea was heated with 100 ml deionized water. Then mixture was

filtered by vacuum filtration. 2 ml of each tea extract, 10 ml of 0.01M HCl and 2 ml of

lead acetate basic solution (50 g of Pb(CH3COO)2 Pb(OH)2 were mixed in 100 ml water

and then were collected to stand at least for 12 h) were mixed with water in a 100-ml

volumetric flask. The mixed solution was stand for 1 h and then was filtered. After that,

50 ml filtered solution and 0.2 ml sulfuric acid (H2SO4) solution (4.5 M) were mixed with

49.8 ml water in a 100-ml volumetric flask. The mixture was stand for 30 min and then

was filtered. The filtered solution was measured by HACH (DR-5000) UV/vis

spectrophotometer at 272nm with a 10 mm quartz cell. The measurement was performed

in triplicate.

Sample preparation for EGCG analysis by UV-VIS

0.1 ml extraction solution of each sample with 0.2 ml Folin–ciocalteu reagent, 0.5 ml

20% Na2CO3 were added with deionized water to get 10 ml. The mixtures were kept in

dark for 1 hour. Then these solutions were analyzed by HACH (DR-5000) UV/vis

spectrophotometer at 725nm.

Sample preparation for caffeine and EGCG determination by HPLC

5g of ground tea with 100 ml of pure water was heated. Then mixture was filtered by

vacuum filtration. 1ml of the filtrate was placed in a volumetric flask and diluted to 100

ml with distilled water. Then these solutions were analyzed by HPLC.

3.4 Apparatus HPLC

The caffeine and EGCG content were determined by a Shimadzu reverse-phase high

performance liquid chromatography (HPLC) system equipped with a UV/Visible

detector. The injector with a 1 μL volume introduced a known sample volume into the

system. The chromatographic separation occurred on a Prodigy 250-mm x 4.6-mm C-18

column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of 30%:70%

(v/v) methanol and deionized water. The wavelength of detection was set at 280 nm and

the flow rate was set at 1 mL/min.

Ultraviolet-visible spectroscopy (UV-VIS)

Concentrations of the caffeine and EGCG in solutions were detected using a HACH (DR-

5000) UV/vis spectrophotometer with a 10 mm pathlength quartz cuvette (Starna).

Spectra were recorded at a wave-length range of 190 to 500 nm for determination of

caffeine. Spectra were recorded at a wave-length range of 190 to 800 nm for

determination of EGCG.

3.5 Description of extraction and purification procedureGreen tea leaves were collected from Bao Loc, Lam Dong province. Then these tea leaves

were ground in 5 min at 100oC, and put into an oven at 80oC for 24 hours. These dried tea

leaves were ground by a house hold blender in order to increase extraction yield.

The first step of extraction green tea is performed by using pure water as a solvent.

Extraction time, temperature, solid/liquid ratio and number of extraction times were

investigated in this study. These tea extracts were filtered to remove residue. Then these

samples were analyzed by UV-VIS and HPLC to determine caffeine and EGCG amounts.

From this result, optimal tea extraction condition was chosen.

The second step of this study is the investigation of caffeine adsorption capability of four

adsorbent columns (XAD4, XAD7, IR-120H, activated carbon) and caffeine desorption of

organic solvents (ethanol, acetone, ethyl acetate, chloroform and hexane). In this step,

adsorbent mass and desorption solvent volume were also investigated.

The mixtures after desorption step (mostly composed of caffeine and organic solvent) were

evaporated to remove solvent. Then these samples were analyzed to determine caffeine and

EGCG content.

The extraction and purification procedure is described in the flowchart below:

Green tea, dried and ground

Extraction (with pure water)

Filtration

Filtrate

Residue

Investigate:

Extraction time

Temperature

Solid/liquid ratio

Number of extraction times.

Adsorbent column

Study four types of adsorbent:

XAD-4; XAD-7; cation exchange IR-120H; activated carbon

Desorption of caffeine using organic solvent

Preparation of samples for HPLC and UV-VIS Analysis

Evaporation of solvent

4. CHAPTER 4: RESULTS & DISCUSSION

4.1 Standard curves

Figure 4. 1 UV-VIS standard curve of caffeine

Figure 4. 2 UV-VIS standard curve of EGCG

Figure 4. 3 HPLC standard curve of Caffeine

Figure 4. 4 HPLC standard curve of EGCG

Table 4. 1 Equations of standard curves

Method Caffeine EGCGUV-VIS y=0.0619x + 0.0252

R2=0.9978y=0.004x + 0.012

R2=0.997HPLC y=24530x + 15986

R2=0.997Y=44277x + 10928

R2=0.998

Figure 4. 5 (a) UV-VIS spectrum and (b) HPLC chromatogram of standard Caffeine

(b)

(a)

Figure 4. 6 (a) UV spectrum and (b) HPLC chromatogram of standard EGCG

(b)

(a)

To determine caffeine and EGCG qualitatively and quantitatively, different standard

solutions of caffeine and EGCG with various concentrations of 1, 5, 10, 15 and 20 ppm

were prepared and measured by UV-VIS and HPLC method. The results are shown in

figures 4.1-4.4. All the standard data show good linearity with square of correlation

coefficient (R2) values more than 0.99. Equations of the standard curves are shown in

table 4.1.

Figure 4.5a shows a typical UV-VIS spectrum of pure caffeine with a peak of caffeine

appearing at ca. 272 nm. This peak position is consistent with what was found by Belay

et al. [43]. The HPLC chromatogram of standard caffeine in Figure 4.5b displays a

retention time of ca. 9.1 min. In the HPLC spectrum of standard EGCG (figure 4.6b), a

retention time value of ca. 19.2 min was found for EGCG.

4.2. Caffeine and EGCG extraction from green tea leaves by pure water:

HPLC and UV-VIS methods were used to determine caffeine and EGCG

amount in extracted solutions. The results of the two methods are compared in

section 4.2.1 and 4.2.2.

In the experiments of these two sections, different 5g tea samples were

extracted at 50oC with 100 ml of distilled water for 1, 5 and 10 min and the

final solutions were analyzed by HPLC and UV-VIS.

4.2.1. Comparison between HPLC and UV-VIS method for determination of caffeine amount

Using UV-VIS method to determine caffeine amount after

catechin removal by lead salt method

Table 4. 2 Data of caffeine determination by UV-VIS

Extraction time

(min) A C (ppm)

mcaf

(mg)

mcaf

(mg/g dry tea leaves)

1 0.512 7.86 66.82 13.36±0.09

5 0.747 11.66 99.15 19.83±0.07

10 0.880 13.81 117.38 23.48±0.16

Using HPLC method to determine caffeine amount

Table 4. 3 Data of caffeine determination by HPLC methodExtraction time

(min) A C (ppm) mcaf (mg)

mcaf (mg/g dry tea

leaves)

1 200385 7.52 63.90 12.78±0.02

5 292179 11.26 95.70 19.14±0.17

10 343110 13.34 113.35 22.67±0.24

Comparison:

Table 4. 4 Comparison of UV-VIS and HPLC results

Extraction time

UV-VIS method

HPLC

method

Difference

(%)(min)

1 66.82 63.9 4.57

5 99.15 95.7 3.61

10 117.38 113.35 3.56

Caffeine content in the extracts was determined by UV-VIS and HPLC and the results

are displayed in table 4.2-4.4. As shown in table 4.4, the variation between the two

methods is under 5%, which is small enough for UV-VIS to be used as the main

analytical method for caffeine analysis.

4.2.2. Comparison between HPLC and UV-VIS method for determination of EGCG amount

Using UV-VIS method with Folin – ciocalteu reagent to determine

EGCG amount

Table 4. 5 Data of EGCG determination by UV-VIS

Extraction time

(min) A C (ppm) mEGCG (mg)

mEGCG (mg/g dry

tea leaves)

1 0.022 2.50 21.25±0.43 4.25±0.43

5 0.025 3.25 27.63±0.74 5.53±0.74

10 0.029 4.25 36.13± 7.23±0.43

Using HPLC method to determine EGCG amount

Table 4. 6 Data of EGCG determination by HPLC

Extraction time


mEGCG (mg/g dry

tea leaves)

1 115685 2.37 20.11 4.02±0.03

5 147179 3.08 26.16 5.23±0.04

10 188010 4.00 34.00 6.80±0.03

Comparison:

Table 4. 7 Comparison of UV-VIS and HPLC results

Extraction time

UV-VIS method

HPLC

method

Difference

(%)(min)

1 21.25 20.11 5.67

5 27.63 26.16 5.62

10 36.13 34.00 6.26

As displayed in table 4.5-4.7, the EGCG amounts determined from the

extracts by UV-VIS and HPLC are not so different (under 7 %). Therefore,

UV-VIS can be employed as the main method for analyzing EGCG in the

samples.

Dry tea leaves were ground into fine powder and 5g of tea powder was used for each extraction

experiment by distilled water. Extraction time, water temperature, ratio of tea to water

(solid/liquid ratio) and the number of extraction times were investigated in sections 4.2.3-4.2.6.

4.2.3. Effect of extraction time to extracted caffeine and EGCG amount (solid/liquid 1/20; 50oC)

Table 4. 8 Effect of extraction time to extracted caffeine amount (solid/liquid 1/20; 50oC)

Table 4. 9 Effect of extraction time to extracted EGCG amount (solid/liquid 1/20; 50oC)

Extraction

time

(min) A C (ppm) mcaf (mg)

mcaf (mg/g dry

tea leaves)

1 0.512 7.86 66.82 13.36±0.09

3 0.647 10.05 85.45 17.09±0.34

5 0.747 11.66 99.15 19.83±0.07

10 0.880 13.81 117.38 23.48±0.16

15 0.866 13.58 115.40 23.08±0.07

20 0.890 13.97 118.71 23.74±0.04

30 0.861 13.50 114.71 22.94±0.12

60 0.885 13.89 118.03 23.61±0.22

90 0.883 13.86 117.78 23.56±0.20

Figure 4. 7 Effect of extraction time on the extracted amount of caffeine and EGCG

Extraction

time


mEGCG (mg/g dry

tea leaves)

1 0.022 2.50 21.25 4.25±0.43

3 0.024 3.00 25.50 5.10±1.28

5 0.025 3.25 27.63 5.53±0.74

10 0.029 4.25 36.13 7.23±0.43

15 0.028 4.00 34.00 6.80±1.12

20 0.030 4.50 38.25 7.65±0.74

30 0.029 4.25 36.13 7.23±0.43

60 0.029 4.25 36.13 7.23±0.43

90 0.028 4.00 34.00 6.80±1.12

Table 4.8, 4.9 and figure 4.7 show the effect of extraction time on the

extracted amount of caffeine and EGCG. At the first stage, the extracted

amount of caffeine increases with the extraction time. Then, the caffeine

amount in the extract becomes stable from 10 min and nearly unchanged

with increasing extraction time. At 10 min, the mass ratio of extracted

amount of caffeine and EGCG is 3.2. It indicates that extracted EGCG

amount is small compared to caffeine. This result is in high agreement with

Liang et al. and Lee et al.’s works [19, 29]. In their studies, it was found that

83% of caffeine was removed while 95% of catechin was retained in tea leaf

by hot water extraction and the majority in the extract is caffeine. Therefore,

10 min is a suitable time for caffeine extraction from green tea by distilled

water.

It is believed that the large difference in extracted amounts of caffeine and

catechin is due to their water solubility and molecular weight. The solubility

of caffeine is 21.7 g/l, which is much higher than that of EGCG (5 g/l). The

molecular weight of caffeine is 194.2, while that of EGCG is 458.4. The

higher solubility in water and smaller molecular help caffeine to diffuse and

dissolve into aqueous solvent more easily than EGCG.

4.2.4. Effect of temperature to extracted caffeine and EGCG amount ( solid/liquid 1/20; 10 min)

Table 4. 10 Effect of temperature to extracted caffeine amount (solid/liquid 1/20; 10 min)

Table 4. 11 Effect of temperature to extracted EGCG amount (solid/liquid 1/20; 10 min)

Temperature(oC) A C (ppm) mcaf (mg)

mcaf

(mg/g tea)

50 0.88 13.81 117.38 23.48±0.16

75 1.075 16.96 144.16 28.83±0.22

100 1.086 17.14 145.67 29.13±0.26

Temperature

(oC) A C (ppm) mEGCG (mg)

mEGCG

(mg/g tea)

50 0.029 4.25 36.13 7.23±0.43

75 0.032 5.00 42.50 8.50±0.85

100 0.033 5.25 44.63 8.93±0.43

Figure 4. 8 Effect of temperature to extracted caffeine and EGCG amount (solid/liquid 1/20; 10 min)

In this section, 5g of green tea was extracted at 50, 75 and 100oC for 10 min

with 100 ml of water. The results are shown in table 4.10, 4.11 and figure

4.8. The extracted caffeine amount increases when the extraction

temperature goes up from 50 to 75oC because the solubility of caffeine

increases with increasing temperature, and then the caffeine amount slightly

increases at 100oC. Despite of the temperature increase, the extracted content

of EGCG just goes up a bit due to its limited solubility. This behavior

indicates that 75oC is an appropriate temperature for caffeine extraction

process.

4.2.5. Effect of solid-liquid (tea-water) ratio to extracted caffeine and EGCG amount (75oC, 10min)

Table 4. 12 Effect of solid-liquid (tea-water) ratio to extracted caffeine amount (75oC, 10min)

Table 4. 13 Effect of solid-liquid (tea-water) ratio to extracted EGCG amount (75oC, 10min)

Solid-

liquid

ratio A C (ppm) mcaf (mg)

mcaf

(mg/g tea)

1:10 1.483 23.55 94.20 18.84±0.06

1:15 1.347 21.35 128.12 25.62±0.21

1:20 1.075 16.96 144.16 28.83±0.22

1:25 0.83 13.00 143.02 28.60±0.16

1:30 0.69 10.74 144.99 29.00±0.20

Solid-

liquid

ratio A C (ppm) m EGCG (mg)

m EGCG

(mg/g tea)

1:10 0.028 4.00 16.00 3.20±0.20

1:15 0.037 6.25 37.50 7.50±0.79

1:20 0.032 5.00 42.50 8.50±0.85

1:25 0.028 4.00 44.00 8.80±1.46

1:30 0.025 3.25 43.88 8.78±0.68

Figure 4. 9 Effect of solid-liquid (tea-water) ratio to extracted caffeine and EGCG amount

(75oC, 10min)

To investigate the effect of the solid-liquid ratio to the extracted content of

caffeine and EGCG, the extraction process was performed at various solid-

liquid ratios with constant extraction temperature and time (75oC, 10min)

(table 4.12-13, figure 4.9). The results show an increase in extracted amount

of caffeine when the solid-liquid ration increases from 1/10 to 1/20 and a

stable caffeine amount of ca. 29 mg/g was found when the ratio increases

from 1/20 to 1/30. The amount of EGCG increases with the increase of solid-

liquid ratio but is still small compared to extracted caffeine content. From

these results, it is obvious that the solid-liquid ratio of 1/20 is suitable for the

extraction of caffeine.

4.2.6. Effect of number of extraction times to extracted caffeine and EGCG amount (75oC, 10min, 1/20) :

Table 4. 14 Effect of number of extraction times to extracted caffeine amount (75oC, 10min, 1/20)

Table 4. 15 Effect of number of extraction times to extracted EGCG amount

(75oC, 10min, 1/20)

Figure 4. 10 Effect of number of extraction times to extracted caffeine and EGCG amount

(75oC, 10min, 1/20)

Extraction

times A C (ppm) m caf (mg)

mcaf

(mg/g tea)

1st 1.075 16.96 144.16 28.83±0.48

2nd 0.163 2.23 18.92 3.78±0.13

3rd 0.068 0.69 5.88 1.18±0.28

Extraction

times A C (ppm) m EGCG (mg)

m EGCG

(mg/g tea)

1st 0.032 5.00 42.50 8.50±1.28

2nd 0.018 1.50 12.75 2.55±1.12

3rd 0.015 0.75 6.38 1.28±0.74

In these experiments (table 4.14-15, figure 4.10), the extraction temperature,

time and solid-liquid ratio were kept constant at 75oC, 10 min and 1/20,

respectively. 100 ml of distilled water was used for each extraction time. The

extracted amount of caffeine in the first time is 28.83 mg/g, which is 7.6 and

24.4 times higher than that in the second and third time, respectively. This

implies that the first extraction time is more efficient and economical than

the second and the third time. As a result, one-time extraction was chosen for

further extraction experiments.

The results of experiments in sections 4.2.3-4.2.6 suggest that the optimal condition

for caffeine extraction from green tea by distilled water is as below:

5g of dry tea powder, 75oC, 10min, solid-liquid ratio of 1/20, one-time

extraction

4.3. Caffeine isolation by column adsorption and desorption

After the extraction from green tea, the extracts were passed through adsorbent

columns (adsorption step) once and then caffeine was isolated by washing the

columns with organic solvents (desorption step). Four adsorbents were studied in this

section, including XAD-4, XAD-7, cation-exchange IR-120H and activated carbon.

In adsorption step, the tea extracts in the optimal extraction condition were passed

through an adsorbent column once with a flow rate of 1ml/min. In desorption step,

several pure organic solvents including ethanol, acetone, ethyl acetate, chloroform

and hexane were used with a flow rate of 1ml/min.

UV-VIS and HPLC techniques were employed for analyzing the mother solutions

after passing through the adsorbent columns and the desorption solutions.

4.3.1. XAD-4 column

Figure 4. 11 HPLC chromatogram of the tea extract (10 min, solid/liquid ratio of 1/20; 75oC, 5g

tea) before passing absorbent column

Figure 4.11 shows the HPLC profile of a tea extract in optimal extraction condition. The

chromatogram presents a value of ca. 9.1 min for the retention time of caffeine and 19.2 min for

that of EGCG. These values are consistent with those in chromatograms of pure caffeine and

EGCG displayed in figure 4.5b and 4.6b.

4.3.1.1. Effect of XAD-4 polymer mass to caffeine amount left in the mother

solution after passing through the adsorbent column

Table 4. 16 Effect of XAD-4 polymer mass to caffeine amount left in the mother solution

Polymer mass (g) A C(ppm)

mcaf.(mg) in mother

solution

1 0.67 10.42 104.17±1.13

3 0.417 6.33 63.30±1.45

5 0.162 2.21 22.10±1.26

7 0.13 1.69 16.93±0.58

9 0.129 1.68 16.77±0.97

Figure 4. 12 Effect of XAD-4 polymer mass to caffeine amount left in the mother solution

As shown in table 4.16 and figure 4.12, the caffeine amount left in the mother solution reduces

when the XAD-4 mass increases and a stable value of ca. 17 mg/g (1ppm) of caffeine is reached

with 7g of XAD-4. With 9g of XAD-4, the caffeine amount left in the mother solution slightly

decreases compared to that with 7g of XAD-4. The possible reason is that the interaction

between the small caffeine amount left in the solution with water is stronger than that with XAD-

4 and this prevents the adsorption of this caffeine amount onto the polymeric adsorbent.

Therefore, 7g is chosen as the suitable mass of adsorbent for one-time caffeine adsorption from

tea extracts.

4.3.1.2. Effect of desorption solvent volume to desorbed caffeine amount with

ethanol as desorption solvent, 7g XAD-4

Table 4. 17 Effect of desorption solvent volume to desorbed caffeine amount with ethanol as

desorption solvent, 7g XAD-4

Ethanol volume (ml) A C (ppm) mdesorbed caf.(mg)

mdesorbed caf.

(mg/g tea)

75 0.286 4.31 43.15 8.43±0.12

100 0.338 5.34 53.39 10.11±0.25

125 0.487 7.46 74.60 14.92±0.81

150 0.486 7.44 74.44 14.89±0.32

175 0.488 7.48 74.77 14.95±0.40

Figure 4. 13 Effect of desorption solvent volume to desorbed caffeine amount with ethanol as

desorption solvent, 7g XAD-4

After caffeine was adsorbed onto the adsorbent, organic solvents were applied to separate it from

the adsorbent column with a rate of 1 ml/min. Because ethanol is often used as a non-toxic

solvent in desorption process, it was used to investigate the effect of solvent volume to the

desorbed caffeine amount. The results are presented in table 4.17 and figure 4.13. At first, the

desorbed amount of caffeine increases with the increase of ethanol volume from 75 to 125 ml.

Then, the desorbed amounts of caffeine are similar when ethanol volume increases from 125 to

175 ml. This fact shows that 125 ml is the suitable volume for one-time desorption of caffeine.

4.3.1.3. Effect of different desorption solvents to desorbed caffeine amount

using XAD-4 (7g), 125 ml solvent

Solvent A C (ppm) mdesorbed caf.(mg) mdesorbed caf.

(mg/g tea)

Desorption yield (%)

Ethanol 0.487 7.46 74.60 14.92±0.37 58.64±1.44

Ethyl acetate 0.632 9.80 98.03 19.61±0.13 77.05±0.51

Acetone 0.718 11.19 111.92 22.38±0.2 87.97±0.79

Chloroform 0.257 3.74 37.45 7.49±0.36 29.43±1.41

Hexane 0.125 1.61 16.12 3.22±0.2 12.67±0.77

Table 4. 18 Effect of different desorption solvents to desorbed caffeine amount using XAD-4

(7g), 125 ml solvent

Figure 4. 14 Effect of different desorption solvents to desorbed caffeine amount using XAD-4


The effect of desorption solvent to the desorbed caffeine amount were investigated with various

organic solvents: ethanol (EtOH), ethyl acetate, acetone, chloroform and hexane (table 4.18,

figure 4.14). Desorption efficiency of caffeine from the XAD-4 column with different solvents

was found to decrease in this order: acetone > ethyl acetate > ethanol > chloroform > hexane.

This can be explained based on the interaction of caffeine with the solvents. Because caffeine is

a polar molecule, it is more soluble in polar solvents. As described in the literature [44-46], the

polarity of the solvents is in the order: acetone > ethyl acetate > ethanol > chloroform > hexane,

which is the same as the order above. Therefore, the polarity of the solvents has the predominant

effect on the desorption efficiency of caffeine from the adsorbent. As a result, acetone is the most

powerful solvent for the desorption of caffeine from the adsorbent column. No EGCG was

detected in the desorption solutions from XAD-4 column.

4.3.2. Adsorption affinity of different adsorbents for caffeine

To compare adsorption affinity of different adsorbents for caffeine, four adsorbent columns with

7g of adsorbent in each column were prepared. The tea extracts were passed through the columns

with a rate of 1 ml/min and the caffeine content left in the mother solutions were analyzed. The

results are displayed in tables 4.19-20 and figure 4.15.

Table 4. 19 Caffeine contents left in the mother solutions after passing adsorbent columns

Adsorbent column A C (ppm) mcaf.(mg) Adsorption yield (%)

XAD-4 0.13 1.69 16.93±0.74 88.26±0.51

XAD-7 0.634 9.84 98.35±1.26 31.78±0.88

IR-120H 0.709 11.05 110.47±0.58 23.37±0.40

Activated carbon 0.652 10.13 101.26±0.70 29.76±0.49

Table 4. 20 EGCG contents left in the mother solutions after passing adsorbent columns

Adsorbent column A C (ppm) mEGCG (mg) Adsorption yield (%)

XAD-4 0.029 4.13 41.25±0.00 2.94±0.00

XAD-7 0.028 4.00 40.00±0.05 5.88±0.03

IR-120 0.017 1.25 12.50±2.50 70.59±5.88

Activated carbon 0.028 3.98 39.75±1.44 6.47±0.95

Figure 4. 15 Adsorption yield of different adsorbent columns for caffeine and EGCG

The results show that XAD-4 has the highest adsorption affinity for caffeine while IR-120H has

the strongest adsorption power for EGCG. Compared to XAD-4, XAD-7 and activated carbon

have lower adsorption strength for caffeine but higher adsorption affinity for EGCG. According

to the literature, XAD-4 is a non-polar polymer while XAD-7 is intermediate polar. Activated

carbon has several carbon-oxygen groups on its surface which make it have a high polarity. IR-

120H is known as a strong proton-exchange and very strong polar polymer [32-34, 41, 47-49].

As stated in other studies, caffeine is less polar than EGCG and this is a possible reason for the

highest adsorption strength of XAD-4 for caffeine [50]. Another possible reason is that the high

affinity between caffeine and XAD-4 is probably formed from a specific π-π interaction between

solute (π electron-deficient one) and polymer (electron-rich one) [35, 36, 51, 52]. In contrast, IR-

120H and activated carbon have high polarity and are more suitable for the adsorption of highly

polar molecules like EGCG. This trend was also observed in other studies [35, 36, 53].

4.3.3. XAD-7 column:4.3.3.1. Effect of different desorption solvents to desorbed caffeine amount

using XAD-7 (7g), 125 ml solvent


(mg/g tea)Desorption yield (%)

EtOH 0.185 2.58 25.82 5.16±0.05 56.35±0.54

Ethyl acetate 0.24 3.47 34.70 6.94±0.09 75.75±0.93

Acetone 0.265 3.87 38.74 7.75±0.18 84.57±1.96

Chloroform 0.124 1.60 15.96 3.19±0.16 34.84±1.76

Hexane 0.072 0.76 7.56 1.51±0.20 16.50±2.15

Table 4. 21 Effect of different desorption solvents to desorbed caffeine amount using XAD-7


Figure 4. 16 Effect of different desorption solvents to desorption yield of caffeine, using XAD-7


For the desorption of caffeine from XAD-7 column with different solvents (table 4.21 and figure

4.16), it was found that acetone had the strongest caffeine desorption ability while the weakest

one was observed for hexane. This tendency is similar to the case of caffeine desorption from

XAD-4 column (section 4.3.1.3). In this case, there is no EGCG found in the desorption

solutions.

4.3.4. Activated carbon column4.3.4.1. Effect of different desorption solvents to desorbed caffeine amount

using activated carbon (7g), 125 ml solvent

Table 4. 22 Effect of different desorption solvents to desorbed caffeine amount using activated carbon (7g),

125 ml solvent



EtOH 0.175 2.42 24.20 4.84±0.09 56.41±1.00

Ethyl acetate 0.189 2.65 26.46 5.29±0.13 61.68±1.51

Acetone 0.223 3.20 31.95 6.39±0.16 74.49±1.88

Chloroform 0.093 1.10 10.95 2.19±0.06 25.53±0.75

Hexane 0.064 0.63 6.27 1.25±0.23 14.61±2.64


activated carbon (7g), 125 ml solvent

Similar to the previous cases of XAD-4 and XAD-7 columns, the highest desorption strength of

caffeine was found for acetone and hexane has the lowest ability for caffeine desorption. No

EGCG was also found in the desorption solutions with different solvents.

4.3.5. IR-120H column4.3.5.1. Effect of different desorption solvents to desorbed caffeine and EGCG

amount using IR-120H (7g), 125 ml solvent:

Table 4. 23 Effect of different desorption solvents to desorbed caffeine amount using IR-120H




EtOH 0.147 1.97 19.68 3.94±0.11 58.41±1.66

Ethyl acetate 0.191 2.68 26.79 5.36±0.21 79.50±3.14

Acetone 0.208 2.95 29.53 5.91±0.19 87.66±2.88

Chloroform 0.073 0.77 7.72 1.54±0.09 22.92±1.27

Hexane 0.061 0.58 5.78 1.16±0.13 17.17±1.92

Table 4. 24 Effect of different desorption solvents to desorbed EGCG amount using IR-120H (7g), 125 ml

solvent

Solvent A C (ppm) mdesorbed EGCG(mg) mdesorbed EGCG


EtOH 0.017 1.25 12.50 2.50±0.00 41.67±0.00

Ethyl acetate 0.020 2.00 20.00 4.00±0.00 66.67±0.00

Acetone 0.021 2.25 22.50 4.50±0.5 75.00±8.33

Chloroform 0 0 0 0 0

Hexane 0 0 0 0 0

Figure 4. 18 Effect of different desorption solvents to caffeine and EGCG desorption

yield using IR-120H

The effect of different solvents to the desorption of caffeine and EGCG from IR-120H column is

displayed in table 4.23-24 and figure 4.18. The desorption of caffeine and EGCG from this

adsorbent column follow the same trend in previous cases. However, no EGCG was detected in

the desorption solutions with chloroform and hexane as the solvents. This is probably due to the

fact that these two solvents are less polar while EGCG is polar. Therefore, it is hard for these two

solvents to desorb EGCG from IR-120H column.

5. CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS

In this project, green tea leaves were extracted with hot water. The effects of extraction time,

temperature, solid-liquid ratio and number of extraction times were investigated. An optimal

extraction condition was successfully established with 5g of tea powder, 75oC, 10 min, solid-

liquid ratio of 1/20 and one-time extraction. The extracted amount of caffeine in the optimal

condition is 3.2 times that of EGCG in the extract. UV-VIS analyses of caffeine and EGCG

showed small differences in results compared to HPLC method. Therefore, UV-VIS was used as

the main technique for caffeine and EGCG determination.

After extraction step, caffeine adsorption and desorption with various adsorbent columns were

studied. The tea extracts from the optimal extraction condition were passed through different

adsorbent columns of XAD-4, XAD-7, IR-120H and activated carbon with a flow rate of 1

ml/min. XAD-4 was found to have the highest adsorption affinity for caffeine and lowest one for

EGCG while the contrary results were observed for IR-120H. It was found that the suitable

amount of XAD-4 adsorbent for one-time adsorption of caffeine is 7g. Several solvents were

used for one-time desorption of caffeine from the adsorbent columns with a flow rate of 1

ml/min and acetone was found to possess the highest strength for caffeine desorption. 125 ml of

solvent was found as the suitable volume for one-time desorption of caffeine from the adsorbent

columns. EGCG was not found in the desorption solutions from XAD-4, XAD-7 and activated

carbon. However, EGCG was found in the desorption solutions by ethanol and acetone from IR-

120H column.

To better understand the caffeine isolation from green tea by water extraction and column

adsorption, it is recommended that the following should be investigated:

As the caffeine amount of green tea is different depending on the place where it is

planted, green tea from different places in Vietnam should be studied.

The effect of pH and stirring speed for tea extraction should be investigated.

Temperature is also an important parameter for caffeine adsorption and desorption.

Therefore, it should be investigated.

More types of adsorbent columns should be tested.

Solvent selection for caffeine desorption plays an important role, especially when

caffeine is used in pharmaceutical and food industry. Therefore, it is necessary to find a

good solvent which has a high selectivity, high desorption yield for caffeine desorption

and is non-toxic.

REFERENCES

1. Hara, Y., Green Tea Health Benefits and Applications. 2001, New York: Marcel Dekker. 264.

2. Ho, C.T. and J.K. Lin, Tea and Tea Products: Chemistry and Health-promoting Properties. 2009, Boca Raton: Taylor & Francis Group. 322.

3. Graham, H.N., Green tea composition, consumption, and polyphenol chemistry. Preventive Medicine, 1992. 21(3): p. 334-350.

4. Heber, D., Nutritional Oncology. 2nd ed. 2006: Elsevier. 847.5. Finger, A., S. Kuhr, and U.H. Engelhardt, Chromatography of tea constituents. Journal of

Chromatography, 1992. 624(1-2): p. 293-315.6. Perva-Uzunalic, A., et al., Extraction of active ingredients from green tea (Camellia

sinensis): Extraction efficiency of major catechins and caffeine. Food Chemistry, 2006. 96(4): p. 597-605.

7. Nguyen, H.H., Masters thesis: Study on polyphenol extraction from tea, in Chemical Engineering. 2006, Ho Chi Minh City University of Technology: Ho Chi Minh City. p. 116.

8. Tong, V.H., Co so sinh hoa va ky thuat che bien tra. 1985: NXB Thanh pho Ho Chi Minh.9. Ashihara, H. and A. Crozier, Caffeine: A well known but little mentioned compound in

plant science. Trends in Plant Science, 2001. 6(9): p. 407-413.10. Chou, T.M. and N.L. Benowitz, Caffeine and coffee: Effects on health and cardiovascular

disease. Comparative Biochemistry and Physiology - C Pharmacology Toxicology and Endocrinology, 1994. 109(2): p. 173-189.

11. Komes, D., et al., Determination of caffeine content in tea and mate tea by using different methods. Czech Journal of Food Sciences, 2009. 27(SPEC. ISS.): p. S213-S216.

12. Misra, H., et al., Study of extraction and HPTLC - UV method for estimation of caffeine in marketed tea ( Camellia sinensis ) granules. International Journal of Green Pharmacy, 2009. 3(1): p. 47-51.

13. Alkan, I., O. Koprulu, and B. Alkan, Latest advances in world tea production and trade, Turkey's aspect. World Journal of Agricultural Sciences, 2009. 5(3): p. 345-349.

14. Pham, T.B.H., Masters thesis: Vietnam’s tea processing industry in the context of economic intergration - a case study of a regional innovative cluster. 2006, Lunds University: Sweden. p. 95.

15. Marinsky, J., Ion exchange and Solvent extraction. Vol. 9. 1985, New York: Marcel Dekker. 504.

16. Rydberg, J., Solvent Extraction Principles and Practice. 2nd ed. 2004, New York: Marcel Dekker. 480.

17. Cannell, R.J.P., Natural product Isolation. 1998, Totowa, New Jersey: Humana Press. 475.18. Colegate, S.M. and R.J. Molyneux, Bioactive Natural Products: Detection, Isolation and

Structual Determination. 2nd ed. 2008, Boca Raton, Florida: Taylor & Francis Group. 605.19. Lee, K.J. and S.H. Lee, Extraction behavior of caffeine and EGCG from green and black

tea. Biotechnology and Bioprocess Engineering, 2008. 13(5): p. 646-649.

20. Budryn, G., et al., Effect of different extraction methods on the recovery of chlorogenic acids, caffeine and Maillard reaction products in coffee beans. European Food Research and Technology, 2009. 228(6): p. 913-922.

21. Hu, X., et al., Theobromine and caffeine recovery with solvent extraction. Separation Science and Technology, 2003. 38(14): p. 3609-3624.

22. Hulbert, G.J., et al., Solid/liquid extraction of caffeine from guarana with methylene chloride. Food Science and Technology International, 1998. 4(1): p. 53-58.

23. Icen, H. and M. Guru, Extraction of caffeine from tea stalk and fiber wastes using supercritical carbon dioxide. Journal of Supercritical Fluids, 2009. 50(3): p. 225-228.

24. Nishitani, E. and Y.M. Sagesaka, Simultaneous determination of catechins, caffeine and other phenolic compounds in tea using new HPLC method. Journal of Food Composition and Analysis, 2004. 17(5): p. 675-685.

25. Yoshida, Y., M. Kiso, and T. Goto, Efficiency of the extraction of catechins from green tea. Food Chemistry, 1999. 67(4): p. 429-433.

26. Kim, W.J., et al., Selective caffeine removal from green tea using supercritical carbon dioxide extraction. Journal of Food Engineering, 2008. 89(3): p. 303-309.

27. Pan, X., G. Niu, and H. Liu, Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves. Chemical Engineering and Processing, 2003. 42(2): p. 129-133.

28. Jun, X., Caffeine extraction from green tea leaves assisted by high pressure processing. Journal of Food Engineering, 2009. 94(1): p. 105-109.

29. Liang, H., et al., Decaffeination of fresh green tea leaf (Camellia sinensis) by hot water treatment. Food Chemistry, 2007. 101(4): p. 1451-1456.

30. Rouquerol, F., Adsorption by Powders and Porous Solids. 1999: Elsevier. 467.31. Toth, J., Adsorption. Theory, Modeling, and Analysis. 2002, New York: Marcel Dekker.

878.32. Li, A., et al., Adsorption of phenolic compounds on Amberlite XAD-4 and its acetylated

derivative MX-4. Reactive and Functional Polymers, 2001. 49(3): p. 225-233.33. Kyriakopoulos, G., D. Doulia, and E. Anagnostopoulos, Adsorption of pesticides on porous

polymeric adsorbents. Chemical Engineering Science, 2005. 60(4): p. 1177-1186.34. Abdullah, M.A., L. Chiang, and M. Nadeem, Comparative evaluation of adsorption

kinetics and isotherms of a natural product removal by Amberlite polymeric adsorbents. Chemical Engineering Journal, 2009. 146(3): p. 370-376.

35. Maity, N., et al., Caffeine adsorption from aqueous solutions onto polymeric sorbents. The effect of surface chemistry on the adsorptive affinity and adsorption enthalpy. Reactive Polymers, 1992. 17(3): p. 273-287.

36. Saikia, M.D. and N.N. Dutta, Adsorption affinity of certain biomolecules onto polymeric resins: Interpretation from molecular orbital theory. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2006. 280(1-3): p. 163-168.

37. Santa Maria, L.C., et al., Preparation and characterization of manganese, nickel and cobalt ferrites submicron particles in sulfonated crosslinked networks. Polymer, 2005. 46(25): p. 11288-11293.

38. Sequi, P., G. Guidi, and G. Petruzzelli, Influence of metals on solubility of soil organic matter. Geoderma, 1975. 13(2): p. 153-161.

39. O'Dwyer, T.F. and B.K. Hodnett, Recovery of chromium from tannery effluents using a redox-adsorption approach. Journal of Chemical Technology and Biotechnology, 1995. 62(1): p. 30-37.

40. Bandosz, T.J., Activated Carbon Surfaces in Environmental Remediation. 2006, New York, USA: Elsevier. 587.

41. Bansal, R.C. and M. Goyal, Activated Carbon Adsorption. 2005, Boca Raton: Taylor & Francis Group. 487.

42. Burchell, T.D., Carbon Materials for Advanced Technologies. 1999, Oak Ridge: Pergamon.43. Belay, A., et al., Measurement of caffeine in coffee beans with UV/vis spectrometer.

Food Chemistry, 2008. 108(1): p. 310-315.44. Smallwood, I.M., Handbook of Organic Solvent Properties. 1996, New York: John Wiley &

Sons Inc. 306.45. Wypych, G., Handbook of Solvents. 2001, Toronto: ChemTec Publishing. 1704.46. Gupta, M.N., et al., Polarity index: The guiding solvent parameter for enzyme stability in

aqueous-organic cosolvent mixtures. Biotechnology Progress, 1997. 13(3): p. 284-288.47. Jianguo, C., et al., Adsorption characteristics of aniline and 4-methylaniline onto

bifunctional polymeric adsorbent modified by sulfonic groups. Journal of Hazardous Materials, 2005. 124(1-3): p. 173-180.

48. Wang, J.P., et al., Adsorption behavior of phenolic compounds onto polymericadsorbents modified with 2-carboxybenzoyl groups. Chinese Journal of Polymer Science (English Edition), 2010. 28(2): p. 241-248.

49. Fei, Z.H., et al., Adsorption of 2,4-dichlorobenzoxyacetic acid onto hypercrosslinked resin modified by phenolic hydroxyl group (AM-1). Chinese Journal of Polymer Science (English Edition), 2004. 22(6): p. 529-533.

50. Park, H.S., et al., Effects of cosolvents on the decaffeination of green tea by supercritical carbon dioxide. Food Chemistry, 2007. 105(3): p. 1011-1017.

51. Bai, X., B. Liu, and J. Yan, Adsorption behavior of water-wettable hydrophobic porous resins based on divinylbenzene and methyl acrylate. Reactive and Functional Polymers, 2005. 63(1): p. 43-53.

52. Saikia, M.D. and N.N. Dutta, Adsorption affinity of certain biomolecules onto polymeric resins: Effect of solute chemical nature. Reactive and Functional Polymers, 2008. 68(1): p. 33-38.

53. Yang, W., et al., Selective adsorption and separation of tea polyphenols and caffeine. Lizi Jiaohuan Yu Xifu/Ion Exchange and Adsorption, 2007. 23(6): p. 481-488.

APPENDICES

1. Tables of data

1.1. UV-VIS standard curve data of caffeine

1.2. UV-VIS standard curve data of EGCG

1.3. HPLC standard curve data of caffeine

1.4. HPLC Standard Curve data of EGCG

Concentration C (ppm) 1 5 10 15 20

Absorbance A 0.072 0.331 0.669 0.975 1.238


Absorbance A 0.019 0.049 0.096 0.131 0.176


Peak area 29376 146203 270986 384170 500228


Peak area 48531.9 246584 452899 659214 905529

1.5. Comparison between HPLC and UV-VIS method for determination of caffeine amount Using UV-VIS method to determine caffeine amount

Using HPLC method to determine caffeine amount

Extraction time

(min) A C (ppm) mcaf (mg) mcaf (mg/g dry tea leaves)

Overall mean+ SD

1 0.508 7.80 66.30 13.2613.36±0.091 0.513 7.88 66.98 13.40

1 0.514 7.90 67.12 13.425 0.747 11.66 99.12 19.82

19.83±0.075 0.745 11.63 98.84 19.775 0.750 11.71 99.53 19.91

10 0.880 13.81 117.38 23.4823.48±0.1610 0.886 13.91 118.20 23.64

10 0.874 13.71 116.56 23.31

Extraction time

(min) Area C (ppm) mcaf (mg) mcaf (mg/g dry tea leaves)

Overall mean+ SD

1 200265 7.51 63.86 12.77

12.78±0.021 200447 7.52 63.92 12.78

1 200685 7.53 64.00 12.805 289279 11.14 94.70 18.94

5 293785 11.32 96.26 19.25

19.14±0.175 293456 11.31 96.15 19.23

10 342209 13.30 113.04 22.61

22.67±0.2410 340150 13.22 112.33 22.47

10 346979 13.49 114.69 22.94

1.6. Comparison between HPLC and UV-VIS method for determination of EGCG amount

UV-VIS method

HPLC method

Extraction time

(min)

Extraction time

(min) A C (ppm) MEGCG (mg)

MEGCG (mg/g dry tea leaves)

10.022 2.50 21.25 4.25

4.25±0.431

0.020 2.00 17.00 3.40

10.024 3.00 25.50 5.10

5 0.025 3.25 27.63 5.53

5.53±0.745 0.023 2.75 23.38 4.68

5 0.027 3.75 31.88 6.38

10 0.027 3.75 31.88 6.38

7.23±0.4310 0.032 5.00 42.50 8.50

10 0.028 4.00 34.00 6.80

1.7. Effect of extraction time to extracted caffeine and EGCG amount (solid/liquid 1/20; 50oC)

Extraction time (min) A C (ppm) mcaf (mg) mcaf (mg/g dry tea leaves)

Overall mean+ SD

1 0.508 7.80 66.30 13.26

13.36±0.091 0.513 7.88 66.98 13.401 0.514 7.90 67.12 13.42

30.647 10.05 85.38 17.08

17.09±0.3430.635 9.85 83.74 16.75

30.660 10.26 87.17 17.43

5 0.747 11.66 99.12 19.82

19.83±0.075 0.745 11.63 98.84 19.77

5 0.750 11.71 99.53 19.91

Extraction time

(min)

Extraction time

(min) Area C (ppm) MEGCG (mg)

MEGCG (mg/g dry tea leaves)

1116385 2.38 20.24 4.05

4.02±0.031

114635 2.34 19.91 3.98

1115945 2.37 20.16 4.03

5 147434 3.08 26.21 5.24

5.23±0.045 145979 3.05 25.93 5.19

5 148264 3.10 26.36 5.27

10 188010 4.00 34.00 6.80

6.80±0.0310 187530 3.99 33.90 6.78

10 188916 4.02 34.17 6.83

10 0.880 13.81 117.38 23.48

23.48±0.1610 0.886 13.91 118.20 23.64

10 0.874 13.71 116.56 23.31

15 0.866 13.58 115.46 23.09 23.08±0.07

15 0.863 13.53 115.05 23.01

15 0.868 13.62 115.73 23.15

20 0.890 13.97 118.75 23.75 23.74±0.04

20 0.891 13.99 118.89 23.78

20 0.888 13.94 118.48 23.70

30 0.861 13.50 114.77 22.95 22.94±0.12

30 0.865 13.57 115.32 23.06

30 0.856 13.42 114.08 22.82

60 0.876 13.74 116.83 23.37

23.61±0.22

60 0.888 13.94 118.48 23.70

60 0.891 13.99 118.89 23.78

90 0.885 13.89 118.07 23.61 23.56±0.20

90 0.889 13.95 118.62 23.72

90 0.875 13.73 116.69 23.34

Extraction time (min)

A C (ppm)

MEGCG

(mg)MEGCG (mg/g dry tea

leaves) Overall mean+

SD

10.022 2.50 21.25 4.25

4.25±0.43

10.020 2.00 17.00 3.40

10.024 3.00 25.50 5.10

3 0.024 3.00 25.50 5.10

5.10±1.283 0.027 3.75 31.88 6.38

3 0.021 2.25 19.13 3.83

5 0.025 3.25 27.63 5.53

5.53±0.745 0.023 2.75 23.38 4.68

5 0.027 3.75 31.88 6.38

10 0.027 3.75 31.88 6.38

7.23±0.4310 0.032 5.00 42.50 8.50

10 0.028 4.00 34.00 6.80

15 0.031 4.75 40.38 8.08 6.80±1.12

15 0.027 3.75 31.88 6.38

15 0.026 3.50 29.75 5.95

20 0.028 4.00 34.00 6.80

7.65±0.7420 0.031 4.75 40.38 8.08

20 0.031 4.75 40.38 8.08

30 0.029 4.25 36.13 7.23

7.23±0.43

30 0.028 4.00 34.00 6.80

30 0.030 4.50 38.25 7.65

600.031 4.75 40.38 8.08 6.80±1.12

60 0.027 3.75 31.88 6.38

60 0.026 3.50 29.75 5.95

90 0.029 4.25 36.13 7.23 7.23±0.43

90 0.028 4.00 34.00 6.80

90 0.030 4.50 38.25 7.65

1.8. Effect of temperature to extracted caffeine and EGCG amount ( solid/liquid 1/20; 10 min)

Temperature(oC) A C (ppm) mcaf (mg) mcaf (mg/g dry tea leaves) Overall mean+ SD

50

0.88 13.81 117.38 23.48

23.48±0.160.87 13.65 116.01 23.20

0.89 13.97 118.75 23.75

75

1.071 16.89 143.61 28.72

28.83±0.221.081 17.06 144.98 29.00

1.073 16.93 143.88 28.78

100

1.076 16.98 144.29 28.86

29.13±0.261.095 17.28 146.90 29.38

1.087 17.15 145.80 29.16

Temperatur A C (ppm) mEGCG (mg) mEGCG

Overall

e (oC) (mg/g tea) mean+ SD

50

0.027 3.75 31.88 6.38

7.23±0.430.032 5.00 42.50 8.50

0.028 4.00 34.00 6.80

75

0.032 5.00 42.50 8.508.50±0.85

0.034 5.50 46.75 9.35

0.030 4.50 38.25 7.65

100

0.033 5.25 44.63 8.93

8.93±0.430.034 5.50 46.75 9.35

0.032 5.00 42.50 8.50

1.9. Effect of solid-liquid (tea-water) ratio to extracted caffeine amount (75oC, 10min)

Solid-liquid ratio A C (ppm) mcaf (mg)

mcaf

(mg/g tea)

Overall mean+ SD

1:101.478 23.47 93.88 18.78

18.84±0.061.486 23.60 94.40 18.88

1.485 23.58 94.33 18.87

1:151.343 21.29 127.74 25.55

25.62±0.211.338 21.21 127.25 25.45

1.359 21.55 129.29 25.86

1:201.075 16.96 144.16 28.83

28.83±0.221.083 17.09 145.26 29.05

1.067 16.83 143.06 28.61

1:250.831 13.02 143.20 28.64

28.60±0.160.825 12.92 142.13 28.43

0.834 13.07 143.73 28.75

1:300.686 10.68 144.12 28.82

29.00±0.200.689 10.72 144.77 28.95

0.695 10.82 146.08 29.22

Solid-liquid ratio A C (ppm) m EGCG (mg)

m EGCG

(mg/g tea)

Overall mean+ SD

1:100.028 4.00 16.00 3.20

3.20±0.200.027 3.75 15.00 3.00

0.029 4.25 17.00 3.40

1:15 0.038 6.50 39.00 7.80

7.50±0.790.039 6.75 40.50 8.10

0.034 5.50 33.00 6.60

1:20

0.032 5.00 42.50 8.50

8.50±0.850.034 5.50 46.75 9.35

0.030 4.50 38.25 7.65

1:250.031 4.75 52.25 10.45

8.80±1.460.027 3.75 41.25 8.25

0.026 3.50 38.50 7.70

1:300.025 3.25 43.88 8.78

8.78±0.680.026 3.50 47.25 9.45

0.024 3.00 40.50 8.10

1.10. Effect of number of extraction times to extracted caffeine amount (75oC, 10min, 1/20) :

Extraction times A C (ppm) m caf (mg)

mcaf

(mg/g tea)

Overall mean+ SD

1st

1.095 17.28 146.90 29.3828.83±0.48

1.063 16.77 142.51 28.50

1.067 16.83 143.06 28.61

2nd

0.161 2.19 18.65 3.733.78±0.13

0.168 2.31 19.61 3.92

0.159 2.16 18.37 3.67

3rd 0.078 0.85 7.25 1.45 1.18±0.28

3rd0.069 0.71 6.01 1.20

1.18±0.280.058 0.53 4.50 0.90

Extraction times A C (ppm) m EGCG (mg)

m EGCG

(mg/g tea)

Overall mean+ SD

1st

0.035 5.75 48.88 9.78

8.50±1.280.032 5.00 42.50 8.50

0.029 4.25 36.13 7.23

2nd

0.020 2.00 17.00 3.402.55±1.12

0.019 1.75 14.88 2.98

0.015 0.75 6.38 1.28

3rd

0.017 1.25 10.63 2.13

1.28±0.740.014 0.50 4.25 0.85

0.014 0.50 4.25 0.85

1.11. Effect of XAD-4 polymer mass to caffeine amount left in the mother solution after

passing through the adsorbent column

Polymer mass (g) A C(ppm)

mcaf.(mg) in mother solution

Overall mean+ SD

1

0.675 10.50 104.98104.17±1.13

0.662 10.29 102.88

0.673 10.47 104.65

3

0.426 6.47 64.7563.30±1.45

0.417 6.33 63.30

0.408 6.18 61.84

5

0.158 2.15 21.45

22.10±1.260.157 2.13 21.29

0.171 2.36 23.55

7

0.133 1.74 17.42

16.93±0.580.131 1.71 17.09

0.126 1.63 16.28

9

0.129 1.68 16.77

16.77±0.970.135 1.77 17.74

0.123 1.58 15.80

1.12. Effect of desorption solvent volume to desorbed caffeine amount with ethanol as desorption solvent, 7g XAD-4

Ethanol volume

(ml)A C (ppm) mdesorbed caf.(mg)

mdesorbed caf.

(mg/g tea)Overall mean+

SD

75

0.289 4.26 42.62 8.528.43±0.12

0.287 4.23 42.29 8.46

0.282 4.15 41.49 8.30

100

0.343 5.13 51.34 10.2710.11±0.25

0.342 5.12 51.18 10.24

0.329 4.91 49.08 9.82

125

0.515 7.91 79.13 15.83

14.92±0.810.467 7.14 71.37 14.27

0.479 7.33 73.31 14.66

150

0.475 7.27 72.67 14.5314.89±0.32

0.494 7.57 75.74 15.15

0.489 7.49 74.93 14.99

175

0.489 7.49 74.93 14.99

14.95±0.400.475 7.27 72.67 14.53

0.5 7.67 76.70 15.34

1.13. Effect of different desorption solvents to desorbed caffeine amount using XAD-4 (7g), 125 ml solvent

Solvent AC

(ppm)mdesorbed caf.

(mg)

mdesorbed

caf.

(mg/g tea)

Desorption yield %

Overall mean+ SD

Ethanol

0.474 7.25 72.50 14.50 56.99

58.64±1.440.492 7.54 75.41 15.08 59.27

0.495 7.59 75.90 15.18 59.65

Ethyl acetate

0.636 9.87 98.68 19.74 77.56

77.05±0.510.628 9.74 97.38 19.48 76.54

0.632 9.80 98.03 19.61 77.05

Acetone 0.713 11.11 111.11 22.22 87.33 87.97±0.79

0.725 11.31 113.05 22.61 88.86

0.716 11.16 111.60 22.32 87.71

Chloroform 0.259 3.78 37.77 7.55 29.69 29.43±1.41

0.267 3.91 39.06 7.81 30.70

0.245 3.55 35.51 7.10 27.91

Hexane 0.128 1.66 16.61 3.32 13.05 12.67±0.77

0.118 1.50 14.99 3.00 11.78

0.129 1.68 16.77 3.35 13.18

1.14. Caffeine content left in the mother solution after passing adsorbent column

Type of adsorbent column A C (ppm) mcaf.(mg)

Adsorption yield %

Overall mean+ SD

XAD-4

0.134 1.76 17.58 87.81

88.26±0.510.131 1.71 17.09 88.14

0.125 1.61 16.12 88.82

XAD-7

0.638 9.90 99.00 31.33

31.78±0.880.625 9.69 96.90 32.78

0.639 9.92 99.16 31.22

IR-120H 0.708 11.03 110.31 23.48 23.37±0.40

0.706 11.00 109.98 23.71

0.713 11.11 111.11 22.92

Activated carbon

0.655 10.17 101.74 29.42 29.76±0.49

0.654 10.16 101.58 29.53

0.647 10.05 100.45 30.32

Type of adsorbent column A C (ppm) mEGCG (mg)

Adsorption yield %

Overall mean+ SD

XAD-4

0.029 4.13 41.25 2.94

2.94±0.000.029 4.13 41.25 2.94

0.029 4.13 41.25 2.94

XAD-7 0.027 3.96 39.00 5.87 5.88±0.03

0.028 4.00 40.00 5.88

0.028 4.00 40.00 5.88

IR-120H 0.017 1.25 12.50 65.3 70.59±5.88

0.019 1.27 12.65 71.24

0.018 1.26 12.58 75.43

Activated carbon

0.029 3.99 39.75 7.47 6.47±0.95

0.027 3.96 38.75 6.89

0.028 3.97 38.65 6.16

1.15. Effect of different desorption solvents to desorbed caffeine amount using XAD-7 (7g),

125 ml solvent

Solvent A C (ppm)mdesorbed caf.

(mg)

mdesorbed

caf.mg (mg/g tea)


Overall mean+ SD

Ethanol0.187 2.61 26.14 5.23 57.06

56.35±0.540.184 2.57 25.65 5.13 56.00

0.185 2.58 25.82 5.16 56.35

Ethyl acetate

0.243 3.52 35.19 7.04 76.8175.75±0.93

0.239 3.45 34.54 6.91 75.40

0.238 3.44 34.38 6.88 75.04

Acetone

0.264 3.86 38.58 7.72 84.21

84.57±1.960.26 3.79 37.93 7.59 82.80

0.271 3.97 39.71 7.94 86.68

Chloroform0.124 1.60 15.96 3.19 34.84

34.84±1.760.119 1.52 15.15 3.03 33.08

0.129 1.68 16.77 3.35 36.61

Hexane 0.065 0.64 6.43 1.29 14.04 16.50±2.15

0.076 0.82 8.21 1.64 17.91

0.075 0.80 8.05 1.61 17.56

1.16. IR-120 columnEffect of different desorption solvents to desorbed caffeine and EGCG amount using IR-120 (7g), 125 ml solvent


(mg)

mdesorbed

caf.mg (mg/g tea)


Overall mean+ SD

Ethanol

0.149 2.00 20.00 4.00 59.36

58.41±1.660.143 1.90 19.03 3.81 56.49

0.149 2.00 20.00 4.00 59.36

Ethyl acetate

0.197 2.78 27.75 5.55 82.38

79.50±3.140.192 2.69 26.95 5.39 79.98

0.184 2.57 25.65 5.13 76.15

Acetone 0.208 2.95 29.53 5.91 87.66 87.66±2.88

0.214 3.05 30.50 6.10 90.53

0.202 2.86 28.56 5.71 84.78

Chloroform 0.07 0.72 7.24 1.45 21.48 22.92±1.27

0.075 0.80 8.05 1.61 23.88

0.074 0.79 7.88 1.58 23.40

Hexane 0.065 0.64 6.43 1.29 19.08 17.17±1.92

0.061 0.58 5.78 1.16 17.17

0.057 0.51 5.14 1.03 15.25

Solvent A C (ppm)mdesorbed

EGCG.(mg)

mdesorbed

EGCG (mg/g tea)


Overall mean+ SD

Ethanol

0.017 1.25 12.50 2.50 41.67

41.67±0.000.017 1.25 12.50 2.50 41.67

0.017 1.25 12.50 2.50 41.67

Ethyl acetate

0.020 2.00 20.00 4.00 66.67

66.67±0.000.020 2.00 20.00 4.00 66.67

0.020 2.00 20.00 4.00 66.67

Acetone 0.020 2.00 20.00 4.00 66.67 75.00±8.33

0.022 2.50 25.00 5.00 83.33

0.021 2.25 22.50 4.50 75.00

Chloroform 0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

Hexane 0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

1.17. Activated carbon column:

Effect of different desorption solvents to desorbed caffeine amount using activated carbon (7g), 125 ml solvent


(mg)mdesorbed caf.

mg/g teaDesorption yield (%)

Overall mean+ SD

Ethanol

0.178 2.47 24.68 4.94 57.54

56.41±1.000.173 2.39 23.88 4.78 55.66

0.174 2.40 24.04 4.81 56.03

Ethyl acetate

0.223 3.20 31.95 6.39 61.68

61.68±1.510.227 3.26 32.60 6.52 63.19

0.219 3.13 31.31 6.26 60.18

Acetone 0.223 3.20 31.95 6.39 74.49 74.49±1.88

0.228 3.28 32.76 6.55 76.37

0.218 3.11 31.15 6.23 72.60

Chloroform 0.093 1.10 10.95 2.19 25.53 25.53±0.75

0.095 1.13 11.28 2.26 26.28

0.091 1.06 10.63 2.13 24.78

Hexane 0.064 0.63 6.27 1.25 14.61 14.61±2.64

0.057 0.51 5.14 1.03 11.98

0.071 0.74 7.40 1.48 17.25

2. Calculation formulasAll samples were carried out in triplicate, the average values were chose.

Standard deviation was calculated by the following equation

Where S= standard deviation

∑ = summation

N=number of tests

Xi= each individual test

= average value.

Percentage of tea caffeine or tea polyphenols in extracts (% w/w)=

C= concentration of caffeine (polyphenol)

α = diluted coefficient

Vdd= volume of filtrate after extracting

mt= mass of green tea

Standard curves of caffeine (using UV-VIS method)

A=absorption

C=concentration of caffeine (ppm)

Standard curves of EGCG (using UV-VIS method)

A=absorption

C=concentration of ECGC (ppm = mg/l)

Standard curves of caffeine (using HPLC method)

A= Peak area

C= concentration of caffeine (ppm)

Standard curves of EGCG (using HPLC method)

A= Peak area

C= concentration of EGCG (ppm)

3. Typical HPLC and UV-VIS spectra

HPLC chromatogram of the tea extract (10 min, solid/liquid ratio of 1/20; 75oC, 5g tea) before passing absorbent column

HPLC chromatogram of the mother solution after passing XAD-4 adsorbent column

HPLC chromatograms of the mother solution after passing XAD-7 adsorbent column

HPLC chromatogram of the mother solution after passing activated carbon adsorbent column

HPLC chromatogram of the mother solution after passing IR-120H adsorbent column

HPLC chromatogram of the desorption solution by acetone (XAD-4 column)

HPLC chromatogram of the desorption solution by acetone (XAD-7 column)

HPLC chromatogram of the desorption solution by acetone (Activated carbon column)

HPLC chromatogram of the desorption solution by ethanol (IR-120H column)

UV spectrum of caffeine in tea extract

UV spectrum of caffeine in desorption solution by ethanol from XAD-4 column

UV spectrum of caffeine in desorption solution by ethanol from XAD-7 column

UV spectrum of caffeine in desorption solution by ethanol from activated carbon column

UV spectrum of caffeine in desorption solution by ethanol from IR-120H column

UV spectrum of EGCG in desorption solution by ethanol from IR-120H column

UV spectrum of EGCG in desorption solution by ethyl acetate from IR-120H column

UV spectrum of EGCG in desorption solution by acetone from IR-120H column

LÝ LỊCH TRÍCH NGANG

Họ và tên: VÕ THỊ KIM NGÂN

Ngày tháng năm sinh: 06/04/1982 Nơi sinh: Tiền Giang

Địa chỉ liên lạc: 22/15 Hà Huy Giáp. Phường Thạnh Lộc, Q12

QUÁ TRÌNH ĐÀO TẠO:

2000-2005: Học đại học tại trường Đại học Bách Khoa, Đại học quốc gia Thành phố Hồ

Chí Minh.

2008-2010: Học cao học tại trường Đại học Bách Khoa, Đại học quốc gia Thành phố Hồ

Chí Minh.

QUÁ TRÌNH CÔNG TÁC:

2005-2010: công tác tại công ty Technopia-Việt Nam, khu công nghiệp Biên Hòa 2,

Đồng Nai.

[Luan van] trích ly và cô lập caffeine từ trà xanh

Healthcare