Study on the extraction and isolation of caffeine from green tea camellia sinensis (l )

18 Effect of different desorption solvents to desorbed caffeine amount using XAD-4 7g, 125 ml solvent .... 21 Effect of different desorption solvents to desorbed caffeine amount using XA

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.)

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

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

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

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

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

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

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; 50 o C) 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 (75 o C, 10min) 41

4.2.6 Effect of number of extraction times to extracted caffeine and EGCG amount (75 o C, 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 

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

Table 4 11 Effect of temperature to extracted EGCG amount (solid/liquid 1/20; 10 min) 39

10min) 41

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 Table 4 21 Effect of different desorption solvents to desorbed caffeine amount using XAD-7 (7g), 125 ml solvent 53 Table 4 22 Effect of different desorption solvents to desorbed caffeine amount using 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

Table 4 24 Effect of different desorption solvents to desorbed EGCG amount using 120H (7g), 125 ml solvent 56

IR-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

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 Figure 4 17 Effect of different desorption solvents to desorption yield of caffeine, using 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

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

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]

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]:

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

Table 2 1 Green tea’s chemical composition

2.1.3 Main components in green tea

2.1.3.1 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)

Some basic information about caffeine is displayed as below:

• Molar mass: 194.19 g/mol

• Appearance: odorless in liquid, white needles or powder

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 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

Product Serving size Caffeine per serving

(mg)

Midol Menstrual Maximum

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

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]

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]

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

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

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

Leaching solid liquid Partitioning

ultrasound energy) Accelerated solvent

Microwave-assisted

Partitioning (with applied microwave irradiation) Supercritical fluid

extraction

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

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

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

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

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

monolayer only, may involve dissociation

low temperatures

possible over a wide range

of temperature

polarization of sorbate may occur

electron transfer leading to bond formation between sorbate and surface

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

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

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

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,

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

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 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

(DR-determination of EGCG

3.5 Description of extraction and purification procedure

Green tea leaves were collected from Bao Loc, Lam Dong province Then these tea leaves

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)

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