[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
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
iii

4.2.3.Effect of extraction time to extracted caffeine and EGCG amount (solid/liquid 1/20;
50oC).....................................................................................................................................38
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) : ......................................................................................................................45
4.3.Caffeine isolation by column adsorption and desorption...................................................48
4.3.1.XAD-4 column .............................................................................................................48
4.3.2.Adsorption affinity of different adsorbents for caffeine..............................................54
4.3.3.XAD-7 column:.............................................................................................................55
4.3.4.Activated carbon column.............................................................................................57
4.3.5.IR-120H column...........................................................................................................59
REFERENCES..................................................................................................................................63
APPENDICES..................................................................................................................................66
1.Tables of data........................................................................................................................66
2.Calculation formulas..............................................................................................................91
3.Typical HPLC and UV-VIS spectra...........................................................................................93
iv

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 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)
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: 227o
C
• Boiling point: 178o
C
• Solubility in water: 2.17 g/100 ml (25o
C), 18.0 g/100ml (80o
C), 67.0g/100
ml(100o
C)
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
Product
Serving 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 Strength
1 tablet
65.4
Midol Menstrual Maximum
Strength
1 tablet 60
NoDoz 100 mg 1 tablet 32.4
8

Pain Reliever Tablets
1 tablet
65
Vivarin
1 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
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
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 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 phase
Extracting phase Basis for separation
Liquid-liquid
extraction
liquid liquid Partitioning
Solid-phase
extraction ( and
microextraction)
Gas, liquid
Liquid or solid
stationary phase
Partitioning or adsorption
Leaching solid liquid Partitioning
Soxhlet extraction solid liquid Partitioning (with applied heat)
Sonication solid liquid
Partitioning (with applied
ultrasound energy)
Accelerated solvent
extraction
solid liquid Partitioning (with applied heat)
Microwave-assisted
extraction
solid 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 40–80o
C, pressure ranging 200–400 bar, and water contents ranging
4–7 wt%. At 40o
C, 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 100o
C 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 procedure
Green tea leaves were collected from Bao Loc, Lam Dong province. Then these tea leaves
were ground in 5 min at 100o
C, and put into an oven at 80o
C 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 EGCG
UV-VIS y=0.0619x + 0.0252
R2
=0.9978
y=0.004x + 0.012
R2
=0.997
HPLC y=24530x + 15986
R2
=0.997
Y=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 50o
C 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 method
 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.
Extraction 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

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
 Using HPLC method to determine EGCG amount
Table 4. 6 Data of EGCG determination by HPLC
 Comparison:
Table 4. 7 Comparison of UV-VIS and HPLC results
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
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

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; 50o
C)
Table 4. 8 Effect of extraction time to extracted caffeine amount (solid/liquid 1/20; 50o
C)
Table 4. 9 Effect of extraction time to extracted EGCG amount (solid/liquid 1/20; 50o
C)
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(o
C) 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
(o
C) 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 100o
C 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 75o
C because the solubility of caffeine
increases with increasing temperature, and then the caffeine amount slightly
increases at 100o
C. Despite of the temperature increase, the extracted content
of EGCG just goes up a bit due to its limited solubility. This behavior
indicates that 75o
C is an appropriate temperature for caffeine extraction
process.
4.2.5. Effect of solid-liquid (tea-water) ratio to extracted caffeine and
EGCG amount (75o
C, 10min)
Table 4. 12 Effect of solid-liquid (tea-water) ratio to extracted caffeine amount (75o
C, 10min)

Table 4. 13 Effect of solid-liquid (tea-water) ratio to extracted EGCG amount (75o
C, 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
(75o
C, 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 (75o
C, 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 (75o
C, 10min, 1/20) :
Table 4. 14 Effect of number of extraction times to extracted caffeine amount
(75o
C, 10min, 1/20)
Table 4. 15 Effect of number of extraction times to extracted EGCG amount
(75o
C, 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

Figure 4. 10 Effect of number of extraction times to extracted caffeine and EGCG amount
(75o
C, 10min, 1/20)
• In these experiments (table 4.14-15, figure 4.10), the extraction temperature,
time and solid-liquid ratio were kept constant at 75o
C, 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
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

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, 75o
C, 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; 75o
C, 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
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
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

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
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

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
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

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
Table 4. 20 EGCG 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
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:
using XAD-7 (7g), 125 ml solvent

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.
(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

4.3.4. Activated carbon column
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
(mg/g tea)
Desorption
yield (%)
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

Figure 4. 17 Effect of different desorption solvents to desorption yield of caffeine, using
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 column
4.3.5.1. 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
Table 4. 24 Effect of different desorption solvents to desorbed EGCG amount using IR-120H
(mg/g tea)
Desorption
yield (%)
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
Solvent A C (ppm) mdesorbed EGCG(mg) mdesorbed EGCG
(mg/g tea)
Desorption
yield (%)
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, 75o
C, 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.

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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
0.07
2
0.33
1
0.66
9
0.97
5 1.238
Absorbance A
0.01
9
0.04
9
0.09
6
0.13
1 0.176
Peak area 2937
6
14620
3
27098
6
38417
0
500228

1.5. Comparison between HPLC and UV-VIS method for determination of caffeine amount
• Using UV-VIS method to determine caffeine amount
Peak area
48531.
9
24658
4
45289
9
65921
4
905529
Extraction time
(min) A C (ppm) mcaf (mg) mcaf (mg/g dry tea leaves)
Overall mean+ SD
1
0.50
8 7.80 66.30 13.26
13.36±0.09
1
0.51
3 7.88 66.98 13.40
1
0.51
4 7.90 67.12 13.42
5 0.74
7 11.66 99.12 19.82
19.83±0.07
5 0.74
5 11.63 98.84 19.77
5 0.75
0 11.71 99.53 19.91
10 0.88
0 13.81 117.38 23.48
23.48±0.16
10 0.88
6 13.91 118.20 23.64
10 0.87
4 13.71 116.56 23.31

• Using HPLC method to determine caffeine amount
Extraction time
(min) Area C (ppm) mcaf (mg) mcaf (mg/g dry tea leaves)
Overall mean+ SD
1
20026
5 7.51 63.86 12.77
12.78±0.02
1
20044
7 7.52 63.92 12.78
1
20068
5 7.53 64.00 12.80
5 28927
9 11.14 94.70 18.94
5 29378
5 11.32 96.26 19.25
19.14±0.17
5 29345
6 11.31 96.15 19.23
10 34220
9 13.30 113.04 22.61
22.67±0.24
10 34015
0 13.22 112.33 22.47
10 34697
9 13.49 114.69 22.94

1.6. Comparison between HPLC and UV-VIS method for determination of EGCG amount
• UV-VIS method
Extraction time
(min)
Extraction time
(min) A C (ppm) MEGCG (mg)
MEGCG (mg/g dry tea leaves)
1
0.022
2.5
0 21.25 4.25
4.25±0.431
0.020
2.0
0 17.00 3.40
1
0.024
3.0
0 25.50 5.10
5
0.025
3.2
5 27.63 5.53
5.53±0.745
0.023
2.7
5 23.38 4.68
5
0.027
3.7
5 31.88 6.38
10
0.027
3.7
5 31.88 6.38
7.23±0.43
10
0.032
5.0
0 42.50 8.50
10
0.028
4.0
0 34.00 6.80

• HPLC method
1.7. Effect of extraction time to extracted caffeine and EGCG amount (solid/liquid 1/20; 50o
C)
Extraction
time
(min) A C (ppm) mcaf (mg) mcaf (mg/g dry tea leaves)
Overall mean+ SD
1
0.50
8
7.80 66.30 13.26
13.36±0.09
1
0.51
3
7.88 66.98 13.40
1
0.51
4
7.90 67.12 13.42
3
0.64
7
10.05 85.38 17.08
17.09±0.34
Extraction time
(min)
Extraction time
(min) Area C (ppm) MEGCG (mg)
MEGCG (mg/g dry tea leaves)
1
116385 2.38 20.24 4.05
4.02±0.03
1
114635 2.34 19.91 3.98
1
115945 2.37 20.16 4.03
5 147434 3.08 26.21 5.24
5.23±0.04
5 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

3
0.63
5
9.85 83.74 16.75
3
0.66
0
10.26 87.17 17.43
5
0.74
7
11.66 99.12 19.82
19.83±0.07
5
0.74
5
11.63 98.84 19.77
5
0.75
0
11.71 99.53 19.91
10
0.88
0
13.81 117.38 23.48
23.48±0.16
10
0.88
6 13.91 118.20 23.64
10
0.87
4 13.71 116.56 23.31
15
0.86
6 13.58 115.46 23.09 23.08±0.07
15
0.86
3 13.53 115.05 23.01
15
0.86
8 13.62 115.73 23.15
20
0.89
0 13.97 118.75 23.75 23.74±0.04
20
0.89
1 13.99 118.89 23.78
20
0.88
8 13.94 118.48 23.70
30
0.86
1 13.50 114.77 22.95 22.94±0.12
30
0.86
5 13.57 115.32 23.06
30
0.85
6 13.42 114.08 22.82
60
0.87
6 13.74 116.83 23.37
23.61±0.22
60
0.88
8
13.94 118.48 23.70

60
0.89
1 13.99 118.89 23.78
90
0.88
5 13.89 118.07 23.61 23.56±0.20
90
0.88
9 13.95 118.62 23.72
90
0.87
5 13.73 116.69 23.34
Extraction
time
(min) A
C
(ppm)
MEGCG
(mg)
MEGCG (mg/g dry tea
leaves) Overall mean+
SD
1
0.02
2 2.50 21.25 4.25
4.25±0.43
1
0.02
0 2.00 17.00 3.40
1
0.02
4 3.00 25.50 5.10
3
0.02
4 3.00 25.50 5.10
5.10±1.283
0.02
7 3.75 31.88 6.38
3
0.02
1 2.25 19.13 3.83
5 0.02
5 3.25 27.63 5.53
5.53±0.745 0.02
3 2.75 23.38 4.68
5 0.02
7 3.75 31.88 6.38
10 0.02
7
3.75 31.88 6.38 7.23±0.43

10 0.03
2 5.00 42.50 8.50
10 0.02
8 4.00 34.00 6.80
15
0.03
1 4.75 40.38 8.08 6.80±1.12
15
0.02
7 3.75 31.88 6.38
15
0.02
6 3.50 29.75 5.95
20
0.02
8 4.00 34.00 6.80
7.65±0.74
20
0.03
1 4.75 40.38 8.08
20
0.03
1 4.75 40.38 8.08
30
0.02
9 4.25 36.13 7.23
7.23±0.43
30
0.02
8 4.00 34.00 6.80
30
0.03
0 4.50 38.25 7.65
60
0.03
1 4.75 40.38 8.08
6.80±1.12
60
0.02
7 3.75 31.88 6.38
60
0.02
6 3.50 29.75 5.95
90
0.02
9 4.25 36.13 7.23
7.23±0.43
90
0.02
8 4.00 34.00 6.80
90
0.03
0 4.50 38.25 7.65

1.8. Effect of temperature to extracted caffeine and EGCG amount ( solid/liquid 1/20; 10 min)
Temperature
(o
C) 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
1.07
1
16.89 143.61 28.72
28.83±0.22
1.08
1
17.06 144.98 29.00
1.07
3
16.93 143.88 28.78
100
1.07
6
16.98 144.29 28.86
29.13±0.26
1.09
5
17.28 146.90 29.38
1.08
7
17.15 145.80 29.16

Temperature
(o
C) A C (ppm) mEGCG (mg)
mEGCG
(mg/g tea)
Overall
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 8.50
8.50±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 (75o
C, 10min)
Solid-liquid
ratio A C (ppm) mcaf (mg)
mcaf
(mg/g tea)
Overall mean+ SD
1:10
1.47
8 23.47 93.88 18.78
18.84±0.061.48
6 23.60 94.40 18.88
1.48
5 23.58 94.33 18.87
1:15
1.34
3 21.29 127.74 25.55
25.62±0.211.33
8 21.21 127.25 25.45
1.35
9 21.55 129.29 25.86
1:20
1.07
5 16.96 144.16 28.83
28.83±0.22
1.08
3 17.09 145.26 29.05
1.06
7 16.83 143.06 28.61
1:25
0.83
1 13.02 143.20 28.64
28.60±0.160.82
5 12.92 142.13 28.43
0.83
4 13.07 143.73 28.75
1:30
0.68
6 10.68 144.12 28.82
29.00±0.20
0.68
9 10.72 144.77 28.95
0.69
5 10.82 146.08 29.22

Solid-liquid ratio A C (ppm) m EGCG (mg)
m EGCG
(mg/g tea)
Overall mean+ SD
1:10
0.02
8 4.00 16.00 3.20
3.20±0.20
0.02
7 3.75 15.00 3.00
0.02
9 4.25 17.00 3.40
1:15
0.03
8 6.50 39.00 7.80
7.50±0.79
0.03
9 6.75 40.50 8.10
0.03
4 5.50 33.00 6.60
1:20
0.03
2 5.00 42.50 8.50
8.50±0.85
0.03
4 5.50 46.75 9.35
0.03
0 4.50 38.25 7.65
1:25
0.03
1 4.75 52.25 10.45
8.80±1.46
0.02
7 3.75 41.25 8.25
0.02
6 3.50 38.50 7.70
1:30
0.02
5 3.25 43.88 8.78
8.78±0.68
0.02
6 3.50 47.25 9.45
0.02
4 3.00 40.50 8.10

1.10. Effect of number of extraction times to extracted caffeine amount (75o
C, 10min,
1/20) :
Extraction
times A C (ppm) m caf (mg)
mcaf
(mg/g tea)
Overall mean+ SD
1st
1.09
5
17.28 146.90 29.38
28.83±0.481.06
3
16.77 142.51 28.50
1.06
7
16.83 143.06 28.61
2nd
0.16
1
2.19 18.65 3.73
3.78±0.130.16
8
2.31 19.61 3.92
0.15
9
2.16 18.37 3.67
3rd
0.07
8
0.85 7.25 1.45 1.18±0.28
3rd
0.06
9
0.71 6.01 1.20
1.18±0.28
0.05
8
0.53 4.50 0.90
Extraction
times A C (ppm) m EGCG (mg)
m EGCG
(mg/g tea)
Overall mean+ SD
1st
0.03
5 5.75 48.88 9.78
8.50±1.28
0.03
2 5.00 42.50 8.50
0.02
9 4.25 36.13 7.23

2nd
0.02
0 2.00 17.00 3.40
2.55±1.120.01
9 1.75 14.88 2.98
0.01
5 0.75 6.38 1.28
3rd
0.01
7 1.25 10.63 2.13
1.28±0.74
0.01
4 0.50 4.25 0.85
0.01
4 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 10.50 104.98
104.17±1.13
0.662 10.29 102.88
0.673 10.47 104.65
3
0.426 6.47 64.75
63.30±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±0.58
0.131 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
Ethanol
volume
(ml)
A C (ppm) mdesorbed caf.(mg)
mdesorbed caf.
(mg/g tea)
Overall mean+
SD
75
0.289 4.26 42.62 8.52
8.43±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 10.27
10.11±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 14.53
14.89±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
Solvent A
C
(ppm)
mdesorbed caf.
(mg)
mdesorbed
caf.
(mg/g
tea)
Desorption
yield %
Overall mean+
SD
Ethanol
0.47
4 7.25 72.50 14.50 56.99
58.64±1.44
0.49
2 7.54 75.41 15.08 59.27
0.49
5 7.59 75.90 15.18 59.65
Ethyl
acetate
0.63
6 9.87 98.68 19.74 77.56
77.05±0.51
0.62
8 9.74 97.38 19.48 76.54
0.63
2 9.80 98.03 19.61 77.05
Acetone 0.71
3 11.11 111.11 22.22 87.33
87.97±0.79
0.72
5 11.31 113.05 22.61 88.86
0.71
6 11.16 111.60 22.32 87.71
Chlorofor
m
0.25
9 3.78 37.77 7.55 29.69
29.43±1.41
0.26
7 3.91 39.06 7.81 30.70
0.24
5 3.55 35.51 7.10 27.91
Hexane 0.12
8 1.66 16.61 3.32 13.05
12.67±0.77
0.11 1.50 14.99 3.00 11.78

8
0.12
9 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±0.030.028 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
Solvent A C (ppm)
mdesorbed caf.
(mg)
mdesorbed
caf.mg (mg/g
tea)
Desorption
yield (%)
Overall
mean+ SD
Ethanol
0.187 2.61 26.14 5.23 57.06
56.35±0.54
0.184 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 76.81
75.75±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
Chlorofor
m
0.124 1.60 15.96 3.19 34.84
34.84±1.76
0.119 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±2.150.076 0.82 8.21 1.64 17.91
0.075 0.80 8.05 1.61 17.56
1.16. IR-120 column

Effect of different desorption solvents to desorbed caffeine and EGCG amount using
IR-120 (7g), 125 ml solvent
Solvent A C (ppm)
mdesorbed caf.
(mg)
mdesorbed
caf.mg (mg/g
tea)
Desorption
yield (%)
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
Chlorofor
m
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)
Desorption
yield (%)
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
Chlorofor
m
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
Solvent A C (ppm)
mdesorbed caf.
(mg)
mdesorbed caf.
mg/g tea
Desorption
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
Chlorofor
m
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 formulas
All 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; 75o
C, 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

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