Nghiên cứu trích ly và tách caffeine từ trà xanh
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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
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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 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: C
8
H
10
N
4
O
2
•
Molar mass: 194.19 g/mol
•
Appearance: odorless in liquid, white needles or powder.
•
Density: 1.23 g/cm
3
•
Melting point: 227
o
C
•
Boiling point: 178
o
C
•
Solubility in water: 2.17 g/100 ml (25
o
C), 18.0 g/100ml (80
o
C), 67.0g/100
ml(100
o
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 20
th
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.
