臺灣博碩士論文加值系統

本研究使用高效陰離子交換層析-脈衝安培流檢測法High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection(HPAEC-PAD)對於單醣和雙醣進行分析，醣類在高鹼性(pH12-14)的環境中易於離子化，利用每一種醣分子有不同的解離常數(pKa)，調整流洗液的鹼性濃度，利用陰離子交換樹脂有不同的滯留時間可分離不同的醣分子，再利用脈衝安培流檢測法(Pulsed Amperometric Detection) (PAD)的氧化電位偵測濃度，並判斷分析物成分。
由於脈衝安培偵測器的靈敏度與鹼性環境密切相關，當鹼性溶液濃度增加時，醣類的解離度增加，解離常數小的醣類，需要在流洗液中加入比較強的流洗離子增加靈敏度，醣類組成複雜的樣品，如同時含有單醣、雙醣以及含有組成結構相似的多種醣樣品的分離則需要採用梯度流洗方式。所以當使用較低鹼性流洗液進行梯度流洗方式分離時，可在管柱後加入一較高濃度鹼性溶液即柱後反應(post-column reaction,PCR)以得到較佳的靈敏度，可同時分離多種醣類和得到最佳的靈敏度，並降低偵測極限。並針對實際樣品分析（如牛奶、優酪乳和奶粉），以乳糖和半乳糖分析結果之回收率、再現性作為此研究方法之確認。

This study utilizes the “High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)” to analyze the monosaccharides and disaccharides. In high alkaline (pH12-14) environment, the carbohydrates were easily ionized. Since carbohydrates have different pKa, the concentration of alkaline eluent was investigated. The result showed that changing the concentration of alkaline eluent changes the retention time and could separate different carbohydrates.
The carbohydrate with lower pKa was washed with high ion concentration of NaOH to achieve higher sensitivity. For complicated carbohydrate samples, monosaccharides, disaccharides or similar structure carbohydrate was separated by gradient washing using NaOH. The result showed that the addition of NaOH could separate various carbohydrates with good sensitivity and detection limit. The proposed method was successfully applied in milk and yogurt samples and found the presence of lactose and galactose.

目錄

摘 要 I
Abstract II
目錄 III
圖目錄 VII
表目錄 X
第一章 緒論 1
1.1醣類物質的定義 1
1.2 醣類物質分類和特性 1
1.2.1單醣類 1
1.2.2 雙醣類 1
1.2.3 寡醣 1
1.2.4多醣 1
1.3 研究動機 2
第二章 文獻回顧 3
2.1 醣類之分析方法 3
2.2陰離子交換層析(Anion-Exchange Chromatography) 14
2.2.1 離子交換的反應機制 14
2.2.2 靜相的選擇 15
2.2.3 醣類分子在動相的過程 19
2.3 醣類的脈衝式電流偵測法偵測(pulsed amperometric detection, PAD) 21
2.3.1 脈衝電化學偵測法的基本原理 22
2.4 牛乳去蛋白的機制 23
第三章 實驗設備與方法 25
3.1離子層析儀儀器設備 25
3.2 操作條件 25
3.3 試劑藥品 26
3.3.1 阿拉伯醣 26
3.3.2 半乳醣 26
3.3.3葡萄醣 27
3.3.4 木醣 27
3.3.5甘露醣 27
3.3.6蔗醣 28
3.3.7果醣 28
3.3.8乳醣 28
3.3.9纖維二醣 29
3.3.10超純水系統 29
3.3.11 氫氧化鈉 29
3.3.12 過氯酸 29
第四章 結果與討論 30
4.1 分析物進樣量的影響 30
4.1.1 操作條件 30
4.1.2 不同進樣體積的結果 30
4.2分析物和流洗液濃度的關係 35
4.2.1單一醣類滯留時間之再現性和線性關係 35
4.2.2降低流洗液濃度的分離效果 46
4.3 流洗液的NaOH濃度對醣分離之滯留時間關係 52
4.4 檢測器訊號強度放大效應 54
4.5 方法之確認 65
4.5.1 樣品製備 66
4.5.2 操作條件 66
4.5.3 乳製品中醣類的分析結果 66
第五章 結論 70
第六章 參考文獻 71


圖目錄
圖 2.1-1 文獻學科類別分佈 4
圖 2.2-1 Ion-exchange chromatography 15
圖 2.2-2 Structures of styrene-divinylbenzene cross-linked ion-exchange resins. 16
圖 2.2-3 Pellicular anion-exchange resin bead 18
圖 2.2-4 CarboPac PA( non-porous pellicular resins) and CarboPac MA (macroporus resins) 18
圖 2.2-5 葡萄醣在pH 12 – 13解離為陰離子 20
圖 2.3-1 Diagram of the pulse sequence for carbohydrate detection 22
圖 2.3-2 Standard Quadruple Waveform for Carbohydrates 23
圖 3.3-1 阿拉伯醣 26
圖 3.3-2 半乳醣 26
圖 3.3-3 葡萄醣 27
圖 3.3-4 木醣 27
圖 3.3-5 甘露醣 27
圖 3.3-6 蔗醣 28
圖 3.3-7 果醣 28
圖 3.3-8 乳醣 28
圖 3.3-9 纖維二醣 29
圖 4.1-1 葡萄醣濃度0.1~10 ug/mL進樣量分別為200 uL和50 uL 30
圖 4.1-2 果醣濃度0.1~10 ug/mL進樣量分別為200 uL和50 uL 31
圖 4.1-3 蔗醣濃度0.1~10 ug/mL進樣量分別為200 uL和50 uL 31
圖 4.1-4 甘露醣濃度0.1~10 ug/mL進樣量分別為200 uL和50 uL 32
圖 4.1-5 木醣濃度0.1~10 ug/mL進樣量分別為200 uL和50 uL 32
圖 4.1-6 半乳醣濃度0.1~10 ug/mL進樣量分別為200 uL和50 uL 33
圖 4.1-7 阿拉伯醣濃度0.1~10 ug/mL進樣量分別為200 uL和50 uL 33
圖 4.2-1 葡萄醣濃度0.1~10 ug/mL波峰重疊的層析圖和檢量線 36
圖 4.2-2 果醣濃度0.1 ~10 ug/mL波峰重疊的層析圖和檢量線 37
圖 4.2-3 蔗醣濃度0.1 ~10 ug/mL波峰重疊的層析圖和檢量線 38
圖 4.2-4 甘露醣濃度0.1 ~10 ug/mL波峰重疊的層析圖和檢量線 39
圖 4.2-5 木醣濃度0.1~10 ug/mL波峰重疊的層析圖和檢量線 40
圖 4.2-6 半乳醣濃度0.1 ~10 ug/mL波峰重疊的層析圖和檢量線 41
圖 4.2-7 阿拉伯醣濃度0.1 ~10 ug/mL波峰重疊的層析圖和檢量線 42
圖 4.2-8 乳醣濃度0.1 ~10 ug/mL波峰重疊的層析圖和檢量線 43
圖 4.2-9 纖維二醣濃度0.1 ~10 ug/mL波峰重疊的層析圖和檢量線 44
圖 4.2-10 醣類10 ug/mL在流洗液100 mM NaOH波峰重疊的層析圖 45
圖 4.2-11葡萄醣D-(+)-Glucose ：100 ug/mL，RT：11.60 min 47
圖 4.2-12果醣D-(−)-Fructose：100 ug/mL，RT：13.90 min 47
圖 4.2-13蔗醣Sucrose：100 ug/mL，RT：18.73 min 47
圖 4.2-14甘露醣D-(+)-Mannose：100 ug/mL，RT：12.03 min 48
圖 4.2-15木醣D-(+)-Xylose：100 ug/mL，RT：12.30 min 48
圖 4.2-16半乳醣D-(+)-Galactose：100ug/mL，RT：10.00 min 48
圖 4.2-17阿拉伯醣D-(−)-Arabinose：100 ug/mL，RT：7.73 min 49
圖 4.2-18纖維二醣D-(+)-Cellobiose：100 ug/mL，RT：43.30 min 49
圖 4.2-19分析物重疊的層析圖 50
圖 4.2-20低濃度流洗液配合梯度流洗使混合醣有效分離 51
圖 4.3-1 分析物10 ug/mL在流洗液100 mM NaOH之層析圖 53
圖 4.3-2 分析物10 ug/mL在流洗液20 mM NaOH之層析圖 53
圖 4.3-3 分析物10 ug/mL在流洗液10 mM NaOH之層析圖 54
圖 4.4-1 PCR的方法示意圖 54
圖 4.4-2 Isocratic層析圖 55
圖 4.4-3 Gradient層析圖 56
圖 4.4-4 Isocratic+PCR層析圖 57
圖 4.4-5 Gradient+PCR層析圖 58
圖 4.4-6 阿拉伯醣濃度0.1 ~10 ug/mL Gradient +PCR的檢量線 59
圖 4.4-7 半乳醣濃度0.1 ~10 ug/mL Gradient +PCR的檢量線 60
圖 4.4-8 葡萄醣濃度0.1 ~10 ug/mL Gradient +PCR的檢量線 60
圖 4.4-9 木醣濃度0.1 ~10 ug/mL Gradient +PCR的檢量線 61
圖 4.4-10蔗醣濃度0.1 ~10 ug/mL Gradient +PCR的檢量線 61
圖 4.4-11果醣濃度0.1 ~10 ug/mL Gradient +PCR的檢量線 62
圖 4.4-12乳醣濃度0.1 ~10 ug/mL Gradient +PCR的檢量線 62
圖 4.4-13纖維二醣濃度0.1 ~10 ug/mL Gradient +PCR的檢量線 63
圖 4.5-1 九種單雙醣標準液的層析圖 67
圖 4.5-2 全脂鮮乳未添加與添加標準液的層析比較圖 68
圖 4.5-3 低脂鮮乳未添加與添加標準液的層析比較圖 68
圖 4.5-4 優酪乳未添加與添加標準液的層析比較圖 69
圖 4.5-5 奶粉未添加與添加標準液的層析比較圖 69
 
表目錄
表 2.1-1 各年度的來源出版物中文獻數量 4
表 2.1-2 各年度的每篇文章中來源出版物的標準化影響 5
表 2.1-3 醣類以不同方法分析之整理 6
表 2.2-1 離子交換樹脂的性質 17
表 2.2-2 一般常見醣類用於高效陰離子交換層析-脈衝安培檢測法的層析管柱 19
表 2.2-3 一些常見醣類的解離常數(在 25 °C的水中) 21
表 4.1-1 流洗液對分析物濃度0.1~10 ug/mL進樣量分別為200 uL和50 uL檢量線之線性相關係數（r2） 34
表 4.2-2 分析物濃度滯留時間與檢量線之線性相關係數（r2） 46
表 4.2-3 流洗液濃度與滯留時間和訊號強度的關係 50
表 4.4-1 混合分析物10 ug/mL在梯度流洗加入後管柱流洗液之檢測器訊號強度放大效應倍率 59
表 4.4-2 分析物濃度0.1~10 ug/mL加入PCR後的檢量線之線性相關係數（r2） 63
表4.4-3 分析物濃度重覆分析3次之滯留時間與波峰面積誤差值 64
表4.5-1 乳製品中醣類的分析結果和回收率 66
表4.5-2 乳製品中醣類的方法偵測極限 67

1.Amanda Terol, Eduardo Paredes, Salvador E Maestre, Soledad Prats, José L Todolí, “Rapid and Sensitive Determination of Carbohydrates in Foods Using High Temperature Liquid Chromatography with Evaporative Light Scattering Detection.,” Journal of Separation Science 35, no. 8 (April 2012): 929–936.

2.Ayman A Ghfar, Saikh M Wabaidur, A Yacine Badjah, Hadj Ahmed, Zeid A Alothman, Mohammad R Khan, Nora H Al-shaalan, “Simultaneous Determination of Monosaccharides and Oligosaccharides in Dates Using Liquid Chromatography-Electrospray Ionization Mass Spectrometry.,” Food Chemistry 176 (2015): 487–492.

3.Beizhen Hu, Wenhong Dong, Biqi Xia, Weihua Song, Chao Han, “Simultaneous Determination of Monosaccharide and Oligosaccharide in Rice Wine by High Performance Anion Exchange Chromatography with Integrated Pulsed Amperometric Detection and the Establishment of Their Fingerprints,” Chinese Journal of Chromatography (Se Pu) 33, no. 6 (2015): 662–666.

4.Broder Rühmann, Jochen Schmid, and Volker Sieber, “Fast Carbohydrate Analysis via Liquid Chromatography Coupled with Ultra Violet and Electrospray Ionization Ion Trap Detection in 96-Well Format.,” Journal of Chromatography. A 1350 (2014): 44–50.

5.Chia-Wei Wang, Wen-Tsen Chen, and Huan-Tsung Chang, “Quantification of Saccharides in Honey Samples through Surface-Assisted Laser Desorption/ionization Mass Spectrometry Using HgTe Nanostructures.,” Journal of the American Society for Mass Spectrometry 25, no. 7 (2014): 1247–1252.

6.Claudio Corradini, Antonella Cavazza, and Chiara Bignardi, “High-Performance Anion-Exchange Chromatography Coupled with Pulsed Electrochemical Detection as a Powerful Tool to Evaluate Carbohydrates of Food Interest: Principles and Applications,” International Journal of Carbohydrate Chemistry 2012 (2012): 1–13.

7.Claudio Corradini, Claudia Lantano, and Antonella Cavazza, “Innovative Analytical Tools to Characterize Prebiotic Carbohydrates of Functional Food Interest.,” Analytical and Bioanalytical Chemistry 405, no. 13 (May 2013): 4591–4605.
8.Daniel C. Harris, Quantitative Chemical Analysis, 2007.

9.Dionex, “Optimal Settings for Pulsed Amperometric Detection of Carbohydrates Using the Dionex ED40 Electrochemical Detector,” Dionex.com, 1–4.

10.Dionex, “Technical Note 20: Analysis of Carbohydrates by High-Performance-Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD).,” 2004.

11.Elbert A Peterson and Herbert A Sober, “Chromatography of Proteins. I. Cellulose Ion-Exchange Adsorbents.,” Journal of the American Chemical Society 78 (1956): 751–755.

12.Giuseppe Arfelli and Elisa Sartini, “Characterisation of Brewpub Beer Carbohydrates Using High Performance Anion Exchange Chromatography Coupled with Pulsed Amperometric Detection,” Food Chemistry 142 (2014): 152–158.

13.He Zhu, Xuan Chen, Xiao Zhang, Lili Liu, Dapeng Cong, Xia Zhao, Guangli Yu, “Acidolysis-Based Component Mapping of Glycosaminoglycans by Reversed-Phase High-Performance Liquid Chromatography with off-Line Electrospray Ionization–tandem Mass Spectrometry: Evidence and Tags to Distinguish Different Glycosaminoglycans,” Analytical Biochemistry 465 (2014): 63–69.

14.Hongliu Ding , Can Li, Ping Jin, Lihong Yuan, Yongqing Yao, Ying Chen, Pei Li, “Simultaneous Determination of Monosaccharides, Disaccharides, Oligosaccharides and Sugar Alcohols in Foods by High Performance Liquid Chromatography with Evaporative Light-Scattering Detection,” Se Pu = Chinese Journal of Chromatography / Zhongguo Hua Xue Hui 31, no. 8 (2013): 804–808.

15.Hong-xu Wang, Jun Zhao, Dong-mei Li, Shuang Song, Liang Song, Ying-huan Fu, Li-peng Zhang, “Structural Investigation of a Uronic Acid-Containing Polysaccharide from Abalone by Graded Acid Hydrolysis Followed by PMP-HPLC-MSn and NMR Analysis.,” Carbohydrate Research 402 (2015): 95–101.


16.J. A. Polta and D. C. Johnson, “DIRECT ELECTROCHEMICAL DETECTION OF AMINO ACIDS AT A PLATINUM ELECTRODE IN AN ALKALINE CHROMATOGRAPHIC EFFLUENT.,” Journal of Liquid Chromatography Volume: 6, Issue: 10, (1983): 1727-1743

17.James D. Oliver, Marianne Gaborieau, Emily F. Hilder, Patrice Castignolles, “Simple and Robust Determination of Monosaccharides in Plant Fibers in Complex Mixtures by Capillary Electrophoresis and High Performance Liquid Chromatography.,” Journal of Chromatography. A 1291 (2013): 179–186.

18.James D. Oliver, Adam A. Rosser, Christopher M. Fellows, Yohann Guillaneuf, Jean Louis Clement, Marianne Gaborieau, Patrice Castignolles, “Understanding and Improving Direct UV Detection of Monosaccharides and Disaccharides in Free Solution Capillary Electrophoresis.,” Analytica Chimica Acta 809 (2014): 183–193.

19.Jennifer L. Behan and Kevin D. Smith, “The Analysis of Glycosylation: A Continued Need for High pH Anion Exchange Chromatography,” Biomedical Chromatography 25, no. 1 (2011): 39–46.

20.Jing Tang,Peng Fei He,Mao Guang Li,Hong Li,Ya Nan Li,Li Liang,Cui Ping Chen,Qiang Ye,, “Determination of Polysaccharide Content in Tetravalent Meningococcal Polysaccharide Conjugate Vaccine by High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection,” Chinese Journal of Biologicals 26, no. 9 (2013): 1313–1317.

21.Joachim Weiss, “Ion Chromatography,” Trends in Food Science &; Technology 1 (1990): 162.

22.J S Rohrer, L Basumallick, and D Hurum, “High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection for Carbohydrate Analysis of Glycoproteins.,” Biochemistry. Biokhimii͡a 78, no. 7 (2013): 697–709.

23.Leon Coulier, Ying Zha, Richard Bas, Peter J. Punt, “Analysis of Oligosaccharides in Lignocellulosic Biomass Hydrolysates by High-Performance Anion-Exchange Chromatography Coupled with Mass Spectrometry (HPAEC-MS).,” Bioresource Technology 133 (2013): 221–231.
24.Lijuan Geng, Junrong Huang, Feng Feng, Pingping Jiang, Xiaogang Chu, Feng Zhang, Yun Ling, “Determination of Fructooligosaccharides in Milk Powder Using High Performance Anion-Exchange Chromatography Coupled with Pulsed Amperometric Detection,” Chinese Journal of Chromatography (Se Pu) 32, no. 12 (2014): 1380–1384.

25.Manu P. Gangola, Sarita Jaiswal, Yogendra P. Khedikar, Ravindra N. Chibbar, “A Reliable and Rapid Method for Soluble Sugars and RFO Analysis in Chickpea Using HPAEC-PAD and Its Comparison with HPLC-RI.,” Food Chemistry 154 (2014): 127–133.

26.Michael Raessler, “Sample Preparation and Current Applications of Liquid Chromatography for the Determination of Non-Structural Carbohydrates in Plants,” TrAC Trends in Analytical Chemistry 30, no. 11 (2011): 1833–1843.

27.Michelle R. Toutounji, Matthew P. Van Leeuwen, James D. Oliver, Ashok K. Shrestha, Patrice Castignolles, Marianne Gaborieau, “Quantification of Sugars in Breakfast Cereals Using Capillary Electrophoresis.,” Carbohydrate Research 408 (2015): 134–141.

28.Na Lange, Handbook of Chemistry., Soil Science, 1999.

29.Özlem Bahadir Acikara, “Ion-Exchange Chromatography and Its Applications,” in Column Chromatography, 2013, 31–56.

30.Philip M Cummins, Oonagh Dowling, and Brendan F O’Connor, “Ion-Exchange Chromatography: Basic Principles and Application to the Partial Purification of Soluble Mammalian Prolyl Oligopeptidase.,” Methods in Molecular Biology (Clifton, N.J.) 681 (2011): 215–228.

31.P.R. Haddad and P.E. Jackson, “Ion Chromatography: Principles and Applications,” Journal of Chromatography Library, 1990.

32.Roy D. Rocklin, Alan P. Clarke, and Michael Weitzhandler, “Improved Long-Term Reproducibility for Pulsed Amperometric Detection of Carbohydrates via a New Quadruple-Potential Waveform,” Analytical Chemistry 70, no. 8 (1998): 1496–1501.

33.R. Rodríguez-Gómez, I. Jiménez-Díaz, A. Zafra-Gómez, J.C. Morales, “Improved Sample Treatment for the Determination of Fructooligosaccharides in Milk Related Products by Liquid Chromatography with Electrochemical and Refractive Index Detection,” Talanta 144 (2015): 883–889.

34.Shifen Mou, Hong Yu, and Yaqi Cai, “Analysis of Sugars by High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection,” Chinese Journal of Chromatography (Se Pu) 27, no. 5 (2009): 667–674.

35.Waka Kamichatani, Yoshinori Inoue, and Atsushi Yamamoto, “Separation Properties of Saccharides on a Hydrophilic Stationary Phase Having Hydration Layer Formed Zwitterionic Copolymer.,” Analytica Chimica Acta 853 (2015): 602–607.

36.Warangkana Suksom, Wannilak Wannachai, Suthasinee Boonchiangma, Saksit Chanthai, Supalax Srijaranai, “Ion Chromatographic Analysis of Monosaccharides and Disaccharides in Raw Sugar,” Chromatographia 78, no. 13–14 (2015): 873–879.

37.Wilbur Widmer, “An Improved Method for Analysis of Biomass Sugars and Galacturonic Acid by Anion Exchange Chromatography.,” Biotechnology Letters 32, no. 3 (2010): 435–438.

38.Miao Yuqing, “Electrochemical Thin Layer Flow Measuring Pool,” CN101306468 A

39.Yu Zhao, Bin Guo, Jinyun Liu, Huawu Yang, Xinqiang Yin, Suxing Tuo, Qiugen Zhang, “Simultaneous Determination of Seven Carbohydrates in Tobacco by Ultrasonic Extraction-Ion Chromatography,” Asian Journal of Chemistry 26, no. 16 (2014): 5149–5155.