Tài liệu 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 7 5 tháng 0 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 rắnlỏng 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 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-todate, 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: 227oC • Boiling point: 178oC • Solubility in water: 2.17 g/100 ml (25oC), 18.0 g/100ml (80oC), 67.0g/100 ml(100oC) Caffeine is a legal drug which is taken everyday by millions of people all around the world. It is more common than any medicine. The average daily caffeine intake in the United States is about 200 mg per individual [12]. Caffeine is widely used in beverage industry. Soft drinks typically contain about 10 to 50 milligrams of caffeine per serving. By contrast, energy drinks such as Red Bull can start at 80 milligrams of caffeine per serving. The caffeine in these drinks either originates from the ingredients used or is an additive derived from the product of decaffeination or from chemical synthesis. Guarana, a prime ingredient of energy drinks, contains large amounts of caffeine with small amounts of theobromine and theophylline. Chocolate derived from cocoa beans contains a small amount of caffeine. The weak stimulant effect of chocolate may be due to a combination of theobromine and theophylline as well as caffeine. A typical 28-gram serving of a milk chocolate bar has about as much caffeine as a cup of decaffeinated coffee, although some dark chocolate currently in production contains as much as 160 mg per 100g. It is also used as a flavor enhancer in food 7 and as a flavoring agent in baked goods, frozen dairy desserts, gelatins, puddings and soft candy [4]. Caffeine is a substance that can stimulate the central nervous system. It makes people more alert, less drowsy and improves coordination. Combined with certain pain relievers or medicines for treating migraine headache, caffeine makes those drugs work more quickly and effectively. Caffeine alone can also help to relieve headaches. Antihistamines are sometimes combined with caffeine to weaken the drowsiness that those drugs cause. Caffeine is also used to treat breathing problems in newborns and in young babies after surgery [1, 12]. Caffeine content in some commercial products is shown in table 2.2. In recent years, various manufacturers have begun putting caffeine into shower products such as shampoo and soap, claiming that caffeine can be absorbed through the skin. However, the effectiveness of such products has not been proven, and they are likely to have little stimulatory effect on the central nervous system because caffeine is not readily absorbed through the skin. Table 2. 2 Caffeine in some commercial products Serving size Product Caffeine per serving (mg) Caffeine tablet (regular-strength) Caffeine tablet (extra-strength) 1 tablet 1 tablet 100 200 Excedrin tablet 1 tablet 65 Excedrin 1 tablet 65 1 tablet Bayer Select Maximum Strength Midol Menstrual Maximum 65.4 1 tablet 60 1 tablet 32.4 Strength NoDoz 100 mg 8 1 tablet Pain Reliever Tablets 1 tablet Vivarin Panadol 500mg 2.1.3.2. 1 tablet 65 200 65 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