Nghiên cứu trích ly và tách caffeine từ trà xanh

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 d

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 TẠITRƯỜNG ĐẠI HỌC BÁCH KHOAĐẠI 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

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

rắn-Khảo sát sự hấp phụ caffeine khi cho dịch trích chảy qua cột hấp phụ với bốnloại 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 giảihấp 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

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 enthusiasmthroughout the project

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

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 of1/20, 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 extractsthrough the columns XAD-4 was found to have the highest adsorptionaffinity for caffeine while IR-120H has the highest adsorption ability forEGCG

• Caffeine was desorbed from the columns by different solvents: ethanol,acetone, ethyl acetate, chloroform and hexane Acetone showed the bestdesorption capability for caffeine compared to other solvents EGCG wasnot found in the desorption solutions from XAD-4, XAD-7, activatedcarbon but was detected in the desorption solutions by ethanol, acetoneand ethyl acetate from IR-120H column

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

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 6

2.1.4.Tea production in the world 11

2.1.5.Tea in Vietnam 13

2.2.1.Extraction 13

2.2.2.Extraction of caffeine from green tea 16

2.2.2.1.Extraction by organic solvents 16

2.3 Adsorption and adsorption in caffeine isolation 18

2.3.1Adsorption 18

2.3.2Adsorbents 20

3.CHAPTER 3: EXPERIMENTAL PROCEDURES 25

3.3 Sample preparation 26

3.4 Apparatus 27

3.5 Description of extraction and purification procedure 27

4.2.Caffeine and EGCG extraction from green tea leaves by pure water: 34

4.2.1.Comparison between HPLC and UV-VIS method for determination of caffeine amount 34

4.2.2.Comparison between HPLC and UV-VIS method for determination of EGCG amount 36 4.2.3.Effect of extraction time to extracted caffeine and EGCG amount (solid/liquid 1/20; 50oC) 38

iii

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

min) 41

4.2.5.Effect of solid-liquid (tea-water) ratio to extracted caffeine and EGCG amount (75oC, 10min) 42

4.2.6.Effect of number of extraction times to extracted caffeine and EGCG amount (75oC, 10min, 1/20) : 44

4.3.Caffeine isolation by column adsorption and desorption 47

4.3.1.XAD-4 column 47

4.3.2.Adsorption affinity of different adsorbents for caffeine 53

4.3.3.XAD-7 column: 54

4.3.4.Activated carbon column 56

4.3.5.IR-120H column 58

REFERENCES 62

APPENDICES 65

1.Tables of data 65

2.Calculation formulas 83

3.Typical HPLC and UV-VIS spectra 85

iv

LIST OF TABLES

v

LIST OF FIGURES

vi

1 CHAPTER 1: INTRODUCTION

Caffeine is one of the most popular compounds which are taken everyday by millions ofpeople all around the world Due to its pleasant flavor and stimulating effect, caffeine ismore common than any chemicals and has been consumed for hundreds of years It isalso 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 stimulatethe central nervous system and make people more alert, less drowsy and improvecoordination With its unique properties, caffeine has been combined with certain painrelievers or medicines for treating headaches because it makes those drugs work morequickly and effectively Therefore, caffeine is becoming more and more important tofood 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 theworld Caffeine was discovered in green tea in the 1820s Caffeine content in green tealeaves was found to be 3-4 %, which is higher than that in coffee bean (1.1-2.2%) Teaplants 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 beverageproduction So, it is necessary to develop a method to extract and isolate caffeine fromVietnamese 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 Whensailors 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 inprocessing is to stop oxidation at a predetermined stage by heating, whichdeactivates the enzymes responsible Without careful moisture and temperaturecontrol during manufacture and packaging, the tea will grow fungi The funguscauses real fermentation that will contaminate the tea with toxic and sometimescarcinogenic substances, as well as off-flavors Tea is traditionally classifiedbased 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 tealeaves Therefore, green tea’s chemical composition is very similar to that of freshleaf and presented in table 2.1 [1-8]

Green tea contains catechins, a type of antioxidant with EGCG as the maincomponent, 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 containstheobromine, theophylline, amino acids, vitamins, minerals, etc

3

Table 2 1 Green tea’s chemical composition

Flavonol and flavonol glucoside 3-4

Volatile substances 0.01 – 0.02

5

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 onthem [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 bytea leaves (3-4%), maté tea leaves (0.89–1.73%), coffee beans (1.1–2.2%), colanuts (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/100ml(100oC)

Caffeine is a legal drug which is taken everyday by millions of people all aroundthe world It is more common than any medicine The average daily caffeineintake 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 asRed Bull can start at 80 milligrams of caffeine per serving The caffeine in thesedrinks either originates from the ingredients used or is an additive derived fromthe product of decaffeination or from chemical synthesis Guarana, a primeingredient of energy drinks, contains large amounts of caffeine with smallamounts of theobromine and theophylline Chocolate derived from cocoa beanscontains 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 Atypical 28-gram serving of a milk chocolate bar has about as much caffeine as acup of decaffeinated coffee, although some dark chocolate currently in productioncontains 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, puddingsand soft candy [4].

Caffeine is a substance that can stimulate the central nervous system It makespeople more alert, less drowsy and improves coordination Combined with certainpain relievers or medicines for treating migraine headache, caffeine makes thosedrugs work more quickly and effectively Caffeine alone can also help to relieveheadaches Antihistamines are sometimes combined with caffeine to weaken thedrowsiness that those drugs cause Caffeine is also used to treat breathingproblems 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, variousmanufacturers have begun putting caffeine into shower products such as shampooand soap, claiming that caffeine can be absorbed through the skin However, theeffectiveness of such products has not been proven, and they are likely to havelittle stimulatory effect on the central nervous system because caffeine is notreadily absorbed through the skin

Table 2 2 Caffeine in some commercial products

(mg)

Caffeine tablet (regular-strength) 1 tablet 100

Midol Menstrual Maximum

Strength

8

Pain Reliever Tablets 1 tablet 65

2.1.3.3 Amino acid

Amino acid is another important constituent of green tea and there are about 20different types of amino acids found in green tea Theanine is the major form ofamino acid, which is unique to green tea because the steaming process does noteliminate it It gives the elegant taste and sweetness to green tea As a naturalprocess, tea plant converts some amino acids into catechins This means that thetheanine content of green tea varies greatly according to the harvesting season oftea leaves [1, 2]

2.1.3.4 Vitamins, minerals and other components

Green tea contains several B vitamins and C vitamin These vitamins are leftintact in the tea-making process Other green tea ingredients include 6% to 8% of

9

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

organic acids such as gallic and quinic acids, and 10% to 15% of carbohydrateand 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 thatproduces green tea, oolong tea and black tea Other than China, tea is alsoproduced 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 producedcommercially since the beginning of the 20th century Tea plantations are most plentiful inthe north but are also found in central Vietnam Vietnam has traditionally been anexporter 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 areputation as some of the finest green tea available

There are many different types of Vietnam tea Black tea is the leader in exports, but ithas a reputation as being a “cheap tea” that can only be used for blending Vietnam alsoproduces oolong tea and white tea The best Vietnam tea, however, is green tea Vietnamhas been producing green tea for thousands of years and this long history shows in thequality of the tea The climate and soil are ideal for growing tea, and there are manyregional variations and methods of production Since 1995 tea production in Vietnam hasdoubled and exports have increased almost 300% Taiwan and Japan are the biggestAsian 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

Gas, liquid Liquid or solid

stationary phase Partitioning or adsorption

Soxhlet extraction solid liquid Partitioning (with applied heat)

ultrasound energy)Accelerated solvent

Microwave-assisted

Partitioning (with applied microwave irradiation)Supercritical fluid

extraction

Solid, liquid Supercritical fluid Partitioning (with applied heat)

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 thesample matrix, which may contain interfering species Prior to choosing an extractionmethod, 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 twophases

• Ability to recover the extracted material Thus the formation of emulsions and otherdeleterious 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 soluteconcentration increases In liquid-liquid extraction low interfacial tension allows thedisruption 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 inthe extracting fluid is used In addition to being nonreactive with the feed, the solventshould be nonreactive with the extraction system (e.g., noncorrosive) and should bestable

• Inexpensive Cost considerations should emphasize the energy costs of an extractionprocedure, 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 performedusing a number of different solvents Benzene, chloroform, trichloroethylene anddichloromethane have all been used over the years [6, 19-25]

In Misra et al.’s study, different organic solvents and aqueous mixtures of varying naturewere used for the screening of caffeine extraction from tea granules Order of recovery ofcaffeine with different organic solvents and aqueous mixtures was: n-hexane < ethylacetate < methylene dichloride< chloroform < methanol < water < 5% sulphuric acid inwater < 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 thanmost of organic solvents In Kim et al.’s work, caffeine and EGCG (epigallocatechingallate) were extracted from green tea using supercritical carbon dioxide (SCCO2) withwater as a cosolvent [26] Various experimental conditions were explored includingtemperatures ranging 40–80oC, pressure ranging 200–400 bar, and water contents ranging4–7 wt% At 40oC, 400 bar and the water content of 7 wt%, the caffeine extraction yieldwas 54% while the EGCG extraction yield was 21%, resulting in caffeine/EGCGextraction 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 teapolyphenols (TP) and tea caffeine from green tea leaves Various experimentalconditions, 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 anddifferent 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 theextract The extraction of tea polyphenols and tea caffeine with MAE for 4 min (30 and4%) were higher than those of extraction at room temperature for 20 h, ultrasonicextraction 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 teapolyphenols or tea caffeine in extracts, MAE was more effective than the conventionalextraction 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 caffeinefrom green tea leaves The effect of different parameters such as high hydrostaticpressure (100–600 MPa), different solvents (acetone, methanol, ethanol and water),ethanol concentration (0–100% ml/ml), pressure holding time (1–10 min) and liquid/solidratio (10:1 to 25:1 ml/g) were studied for the optimal caffeine extraction from green tealeaves The highest yields (4.0 ± 0.22%.) were obtained at 50% (ml/ml) ethanolconcentration, 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 thatextraction using high pressure processing possessed higher yields, shorter extractiontimes 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 statisticallysignificant 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 wasdecreased from 23.7 to 4.0 mg/g, while total tea catechins decreased from 134.5 to 127.6mg/g; 83% of caffeine was removed and 95% of total catechins was retained in thedecaffeinated leaf It was found that the hot water treatment is a safe, effective andinexpensive 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 amonolayer but sometimes as a multilayer - at the surface of a solid material This is afundamental property of matter, having its origin in the attractive forces betweenmolecules The solid material is called the adsorbent and the material adsorbed at thesurface of the adsorbent is the adsorbate

There are two kinds of adsorption: chemisorption and physisorption, depending on thenature of the surface forces Physisorption is caused mainly by Van der Waals forces and

electrostatic forces between adsorbate molecules and the atoms which compose theadsorbent surface In chemisorption, there is significant electron transfer, equivalent tothe formation of a chemical bond between the sorbate and the solid surface Theseinteractions are both stronger and more specific than the forces of physical adsorptionand 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

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

monolayer only, mayinvolve dissociation

Temperature range only significant at relatively

low temperatures

possible over a wide range

of temperatureForces of adsorption no electron transfer, although

polarization of sorbate mayoccur

electron transfer leading tobond formation betweensorbate 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 activatedcarbons, macroporous polymeric adsorbents are more attractive alternatives because

of their wide range of pore structures and physic-chemical characteristics Because ofits high chemically stability and excellent selectivity towards aromatic solutes,Amberlite XAD-4 polymeric resin, a macroporous styrene-divinylbenzenecopolymer, is found to be a good polymeric adsorbent for organic compounds.Amberlite XAD-7 is a nonionic aliphatic acrylic polymer, which derives itsadsorptive properties from its macroreticular structure (containing both a continuouspolymer phase and a continuous pore phase), high surface area and the aliphaticnature of its surface It is characterized as a hydrophobic adsorbent having asomewhat more hydrophilic structure comparing to XAD-4 Its macroreticularstructure also gives to it excellent physical and thermal stability and it is also stable atall pH range in aqueous solution The typical properties of both resins are listed inTable 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 forcaffeine 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

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

Polystyrene-divinylbenzene

Methylacrylate ester

• Amberlite IR-120H

Amberlite IR-120H resin is a strongly acidic cation exchange resin of the sulfonatedpolystyrene 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 ≤ 1.8Harmonic 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 dateback to the 19th century Its usefulness derives mainly from its large micropore andmesopore volumes and the resulting high surface area Compared with several othersorbents, it is important to consider the charge of the surface because it determinesthe capacity of the carbon for ion exchange AC is dominantly used for purposes ofadsorption, a task for which it is well designed AC is often used for adsorption oforganic solutes covers a wide spectrum of systems such as drinking water andwastewater treatments, and applications in the food, beverage, pharmaceutical andchemical 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 arestill uncertain Adsorption of organic compounds and of aromatics in particular, is acomplex interplay of electrostatic and dispersive interactions This is particularly truefor phenolic compounds [40-42]

2.4 Scope of the project

As listed in previous sections, there have been some studies on extraction and isolation ofcaffeine 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 isolationcaffeine from Vietnamese green tea, which is cheaper and easier to be scaled up forindustrial application

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

• Extracting caffeine from green tea by distilled water and investigating the effect oftime, temperature, solid-liquid ratio and number of extraction times.

• Investigating adsorption step for caffeine in the tea extract by using columnadsorption method with different adsorbents

• Investigating desorption step for caffeine from the adsorbent columns with differentdesorption solvents

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

3 CHAPTER 3: EXPERIMENTAL PROCEDURES

3.1 Chemicals and reagents

Anhydrous caffeine and EGCG used for preparation of the standard solutions were purchasedfrom Sigma (St Louis, MO, USA) Methanol for the mobile phase was HPLC grade (FisherScientific, Pittsburgh, PA, USA) Deionized water was obtained from a water purificationsystem XAD-4, XAD-7, IR-120H and activated carbon were purchased from Merck.Ethanol, methanol, ethyl acetate, chloroform, hexane and acetone used in the experimentalwork were all of analytical reagent grade chemicals Green tea (Kim Tuyen) was collectedfrom Bao Loc-Lam Dong

3.2 Preparation of standard solutions

The caffeine determination was accomplished by utilizing high performance liquidchromatography (HPLC) equipped with a UV/Visible detector and by UV-VIS method withHACH (DR-5000) UV/VIS spectrophotometer The mobile phase for HPLC consists of30:70 (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 mLvolumetric 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, fivestandard solutions of 1, 5, 10, 15 and 20ppm were prepared The five standard solutionswere stored at room temperature These solutions were analyzed to prepare theappropriate standard curve

• Preparation of EGCG standard solutions

EGCG (10 mg) was weighed and transferred into a 100 mL volumetric flask Deionizedwater was added to get a 100 mL bulk standard solution Shake was applied tocompletely 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 of10ml Keep them in dark for 1 hour Then these five standard solutions were analyzed toprepare 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 wasfiltered by vacuum filtration 2 ml of each tea extract, 10 ml of 0.01M HCl and 2 ml oflead acetate basic solution (50 g of Pb(CH3COO)2 Pb(OH)2 were mixed in 100 ml waterand then were collected to stand at least for 12 h) were mixed with water in a 100-mlvolumetric 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 with49.8 ml water in a 100-ml volumetric flask The mixture was stand for 30 min and thenwas filtered The filtered solution was measured by HACH (DR-5000) UV/visspectrophotometer 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 ml20% Na2CO3 were added with deionized water to get 10 ml The mixtures were kept indark for 1 hour Then these solutions were analyzed by HACH (DR-5000) UV/visspectrophotometer 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 byvacuum 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 highperformance liquid chromatography (HPLC) system equipped with a UV/Visibledetector The injector with a 1 μL volume introduced a known sample volume into thesystem The chromatographic separation occurred on a Prodigy 250-mm x 4.6-mm C-18column (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 andthe 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 ofcaffeine Spectra were recorded at a wave-length range of 190 to 800 nm fordetermination of EGCG

(DR-3.5 Description of extraction and purification procedure

Green tea leaves were collected from Bao Loc, Lam Dong province Then these tea leaveswere ground in 5 min at 100oC, and put into an oven at 80oC for 24 hours These dried tealeaves 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 wereinvestigated in this study These tea extracts were filtered to remove residue Then thesesamples 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 fouradsorbent 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) wereevaporated to remove solvent Then these samples were analyzed to determine caffeine andEGCG content

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

Green tea, dried and ground

Extraction (with pure water)

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