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

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 HỒN THÀNH TẠI TRƯỜNG ĐẠI HỌC BÁCH KHOA ĐẠI HỌC QUỐC GIA TP HỒ CHÍ MINH Cán hướng dẫn khoa học: TS PHẠM THÀNH QUÂN Cán chấm nhận xét 1: TS PHẠM S Cán chấm nhận xét 2: TS NGUYỄN THỊ LAN PHI Luận văn thạc sĩ bảo vệ 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: PGS.TS Trần Thi Việt Hoa TS Phạm Thành Quân TS Trần Thị Kiều Anh TS Phạm S TS Trần Lê Quan Xác nhận Chủ tịch Hội đồng đánh giá LV Bộ môn quản lý chuyên ngành sau luận văn 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 Độc Lập - Tự Do - Hạnh Phúc PHÒNG ĐÀO TẠO SAU ĐẠI HỌC Tp.HCM, ngày tháng năm 2010 NHIỆM VỤ LUẬN VĂN THẠC SĨ Họ 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 tách caffeine từ trà xanh II NHIỆM VỤ VÀ NỘI DUNG Khảo sát ảnh hưởng yếu tố nhiệt độ, thời gian, tỷ lệ rắnlỏng số lần trích đến lượng caffeine dịch trích từ trà nước Khảo sát hấp phụ caffeine cho dịch trích chảy qua cột hấp phụ với bốn loại chất hấp phụ khác nhau: XAD-4, XAD-7, IR 120H than hoạt tính Khảo sát giải hấp caffeine từ cột hấp phụ nói với dung mơi giải hấp khác nhau: ethanol, acetone, ethyl acetate, chloroform 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 ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES viii CHAPTER 1: INTRODUCTION CHAPTER 2: LITERATURE REVIEW 2.1 2.1.1 Overview 2.1.2 Green tea’s composition 2.1.3 Main components in green tea 2.1.4 Tea production in the world 2.1.5 Tea in Vietnam 11 2.2 Extraction and extraction of caffeine from green tea 11 2.2.1 Extraction 11 2.2.2 Extraction of caffeine from green tea 14 2.2.2.1 Extraction by organic solvents 14 2.3 Green tea and caffeine Adsorption and adsorption in caffeine isolation 16 2.3.1 Adsorption 16 2.3.2 Adsorbents 18 CHAPTER 3: EXPERIMENTAL PROCEDURES 23 3.1 Chemicals and reagents 23 3.2 Preparation of standard solutions 23 3.3 Sample preparation 24 3.4 Apparatus 25 3.5 Description of extraction and purification procedure 25 CHAPTER 4: RESULTS & DISCUSSION 27 iii 4.1 Standard curves 27 4.2 Caffeine and EGCG extraction from green tea leaves by pure water: 32 4.2.1 Comparison between HPLC and UV-VIS method for determination of caffeine amount …………………………………………………………………………………….32 4.2.2 Comparison between HPLC and UV-VIS method for determination of EGCG amount …………………………………………………………………………………….34 4.2.3 Effect of extraction time to extracted caffeine and EGCG amount (solid/liquid 1/20; 50oC) 36 4.2.4 Effect of temperature to extracted caffeine and EGCG amount ( solid/liquid 1/20; 10 min) …………………………………………………………………………………….39 4.2.5 Effect of solid-liquid (tea-water) ratio to extracted caffeine and EGCG amount (75oC, 10min) 41 4.2.6 Effect of number of extraction times to extracted caffeine and EGCG amount o (75 C, 10min, 1/20) : 43 4.3 Caffeine isolation by column adsorption and desorption 45 4.3.1 XAD-4 column 45 4.3.2 Adsorption affinity of different adsorbents for caffeine 51 4.3.3 XAD-7 column: 53 4.3.4 Activated carbon column 54 4.3.5 IR-120H column 56 CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 58 REFERENCES 60 APPENDICES 63 Tables of data 63 Calculation formulas 81 Typical HPLC and UV-VIS spectra 83  iv   LIST OF TABLES Table Green tea’s chemical composition Table 2 Caffeine in some commercial products Table Tea production in the world (tons) 10 Table Summary of selected extraction techniques by phases involved and the basic for separation 12 Table Parameters of physisorption and chemisorptions 17 Table Typical properties of Amberlites XAD-4 and XAD-7 19 Table Equations of standard curves 29 Table Data of caffeine determination by UV-VIS 32 Table Data of caffeine determination by HPLC method 33 Table 4 Comparison of UV-VIS and HPLC results 33 Table Data of EGCG determination by UV-VIS 34 Table Data of EGCG determination by HPLC 34 Table Comparison of UV-VIS and HPLC results 35 Table Effect of extraction time to extracted caffeine amount (solid/liquid 1/20; 50oC) 36 Table Effect of extraction time to extracted EGCG amount (solid/liquid 1/20; 50oC) 37 Table 10 Effect of temperature to extracted caffeine amount (solid/liquid 1/20; 10 min) 39 v Table 11 Effect of temperature to extracted EGCG amount (solid/liquid 1/20; 10 min) 39 Table 12 Effect of solid-liquid (tea-water) ratio to extracted caffeine amount (75oC, 10min) 41 Table 13 Effect of solid-liquid (tea-water) ratio to extracted EGCG amount (75oC, 10min) 41 Table 14 Effect of number of extraction times to extracted caffeine amount 43 Table 15 Effect of number of extraction times to extracted EGCG amount 43 Table 16 Effect of XAD-4 polymer mass to caffeine amount left in the mother solution 46 Table 17 Effect of desorption solvent volume to desorbed caffeine amount with ethanol as desorption solvent, 7g XAD-4 47 Table 18 Effect of different desorption solvents to desorbed caffeine amount using XAD-4 (7g), 125 ml solvent 49 Table 19 Caffeine contents left in the mother solutions after passing adsorbent columns 51 Table 20 EGCG contents left in the mother solutions after passing adsorbent columns 51 Table 21 Effect of different desorption solvents to desorbed caffeine amount using XAD-7 (7g), 125 ml solvent 53 Table 22 Effect of different desorption solvents to desorbed caffeine amount using activated carbon (7g), 125 ml solvent 54 Table 23 Effect of different desorption solvents to desorbed caffeine amount using IR120H (7g), 125 ml solvent 56 vi Table 24 Effect of different desorption solvents to desorbed EGCG amount using IR120H (7g), 125 ml solvent 56 vii 1.16 0.076 0.82 8.21 1.64 17.91 0.075 0.80 8.05 1.61 17.56 IR-120 column Effect of different desorption solvents to desorbed caffeine and EGCG amount using IR-120 (7g), 125 ml solvent Solvent Ethanol Ethyl acetate Acetone C (ppm) mdesorbed caf.(mg) mdesorbed caf.mg (mg/g tea) 0.149 2.00 20.00 4.00 59.36 0.143 1.90 19.03 3.81 56.49 0.149 2.00 20.00 4.00 59.36 0.197 2.78 27.75 5.55 82.38 0.192 2.69 26.95 5.39 79.98 0.184 2.57 25.65 5.13 76.15 0.208 2.95 29.53 5.91 87.66 0.214 3.05 30.50 6.10 90.53 0.202 2.86 28.56 5.71 84.78 0.72 7.24 1.45 21.48 0.075 0.80 8.05 1.61 23.88 0.074 0.79 7.88 1.58 23.40 0.065 0.64 6.43 1.29 19.08 0.061 0.58 5.78 1.16 17.17 0.057 0.51 5.14 1.03 15.25 A Chloroform 0.07 Hexane 78 Desorption yield (%) Overall mean+ SD 58.41±1.66 79.50±3.14 87.66±2.88 22.92±1.27 17.17±1.92 mdesorbed Desorption yield (%) A C (ppm) mdesorbed EGCG.(mg) 0.017 1.25 12.50 2.50 41.67 0.017 1.25 12.50 2.50 41.67 0.017 1.25 12.50 2.50 41.67 0.020 2.00 20.00 4.00 66.67 0.020 2.00 20.00 4.00 66.67 0.020 2.00 20.00 4.00 66.67 0.020 2.00 20.00 4.00 66.67 0.022 2.50 25.00 5.00 83.33 0.021 2.25 22.50 4.50 75.00 Chloroform 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Solvent Ethanol Ethyl acetate Acetone Hexane 79 EGCG (mg/g tea) Overall mean+ SD 41.67±0.00 66.67±0.00 75.00±8.33 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 (%) 0.178 2.47 24.68 4.94 57.54 0.173 2.39 23.88 4.78 55.66 0.174 2.40 24.04 4.81 56.03 0.223 3.20 31.95 6.39 61.68 0.227 3.26 32.60 6.52 63.19 0.219 3.13 31.31 6.26 60.18 0.223 3.20 31.95 6.39 74.49 0.228 3.28 32.76 6.55 76.37 0.218 3.11 31.15 6.23 72.60 Chloroform 0.093 1.10 10.95 2.19 25.53 0.095 1.13 11.28 2.26 26.28 0.091 1.06 10.63 2.13 24.78 0.064 0.63 6.27 1.25 14.61 0.057 0.51 5.14 1.03 11.98 0.071 0.74 7.40 1.48 17.25 Ethanol Ethyl acetate Acetone Hexane 80 Overall mean+ SD 56.41±1.00 61.68±1.51 74.49±1.88 25.53±0.75 14.61±2.64 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) 81 • 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) 82 Typical HPLC and UV-VIS spectra HPLC chromatogram of the tea extract (10 min, solid/liquid ratio of 1/20; 75oC, 5g tea) before passing absorbent column HPLC chromatogram of the mother solution after passing XAD-4 adsorbent column 83 HPLC chromatograms of the mother solution after passing XAD-7 adsorbent column HPLC chromatogram of the mother solution after passing activated carbon adsorbent column 84 HPLC chromatogram of the mother solution after passing IR-120H adsorbent column HPLC chromatogram of the desorption solution by acetone (XAD-4 column) 85 HPLC chromatogram of the desorption solution by acetone (XAD-7 column) HPLC chromatogram of the desorption solution by acetone (Activated carbon column) 86 HPLC chromatogram of the desorption solution by ethanol (IR-120H column) UV spectrum of caffeine in tea extract  87 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 88 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  89 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  90 UV spectrum of EGCG in desorption solution by acetone from IR-120H column 91 LÝ LỊCH TRÍCH NGANG Họ tên: VÕ THỊ KIM NGÂN Ngày tháng năm sinh: 06/04/1982 Nơi sinh: Tiền Giang Địa liên lạc: 22/15 Hà Huy Giáp Phường Thạnh Lộc, Q12 QUÁ TRÌNH ĐÀO TẠO: 2000-2005: Học đại học trường Đại học Bách Khoa, Đại học quốc gia Thành phố Hồ Chí Minh 2008-2010: Học cao học trường Đại học Bách Khoa, Đại học quốc gia Thành phố Hồ Chí Minh Q TRÌNH CƠNG TÁC: 2005-2010: cơng tác cơng ty Technopia-Việt Nam, khu cơng nghiệp Biên Hịa 2, Đồng Nai 92 ... 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... Effect of extraction time on the extracted amount of caffeine and EGCG 37 • 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... 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