薄層層析法（Thin Layer Chromatography, TLC）是一種已廣泛應用於有機、無機和生化混合物的分離技術，具有操作簡單、成本低廉、樣品需求量少及分析快速等優點，其中包括一維薄層層析法（One-Dimensional Thin Layer Chromatography, 1D-TLC）及二維薄層層析法（Two-Dimensional Thin Layer Chromatography, 2D-TLC），而2D-TLC為1D-TLC分離方法的延伸，其操造方式是將分析物經由一維薄層層析分離後，TLC片轉九十度角，在不需移動樣品情況下，直接改變溶劑組成，繼續進行另一維的分離。電噴灑輔助雷射脫附游離質譜法 (Electrospray-assisted Laser Desorption Ionization Mass Spectrometry, ELDI-MS) 是一種可在一大氣壓下，且不需添加任何基質可以直接對樣品進行快速分析的新質譜游離方法，其游離的原理是利用雷射直接對固態樣品進行脫附產生中性氣態分子，再與電噴灑所產生的帶電液滴融合後，再進入質譜儀內進行分析，即可獲得分析物的離子訊號。本篇論文的研究目的為開發薄層層析分離技術與ELDI-MS偵測技術介面，藉由ELDI-MS來分析經TLC分離後的樣品，可以直接定性與定量該分析物。
在本研究中的實驗設計將1D-TLC分離分為兩部份，第一部分為使用商業化的TLC片(如silica gel 60 TLC plate、C18 TLC plate)，來分離FD& C染劑混合物、胺基化合物及市售藥物中的有效成分。當分析物經由TLC片分離後，透過UV雷射(337 nm)從TLC片上被脫附出來，再由電噴灑產生的帶電液滴提供氫離子使其游離進入質譜內，就可以得到分子離子訊號。一個完整的TLC連結ELDI-MS的分析，則是將分析物以TLC分離後再以ELDI-MS進行快速掃描偵測，就可以在不同時間點獲得不同離子訊號。此外研究中以紅色染劑(erythrosine B [M-H]-=m/z 835)來進行ELDI-MS偵測極限、定量及結構鑑定的探討，其實驗結果顯示偵測極限可達10-6~10-7 M間，其定量範圍從10-6~10-4 M ，R2=0.9886，由此我們証實透過TLC/ELDI-MS可對分析物種進行定性、定量測定。第二部份為以自製之monolithic TLC片來分離生化混合物種(如蛋白質混合物)為樣品，使用IR雷射(1064 nm)作為ELDI-MS之脫附雷射，此部份的實驗也成功得到各個生化物種的離子訊號。
至於2D-TLC的研究則以胺基酸混合物為樣品，分析物透過IR雷射(1064 nm)從TLC片上被脫附出來，再與電噴灑產生的帶電荷液滴產生游離後得到分子離子訊號，並以ELDI-MS進行整個TLC片的快速掃描分析，就可以在2D-TLC片上所對應不同時間點比對出分析物的離子訊號。
本研究藉由TLC分離方法與ELDI-MS偵測技術的結合，可針對TLC片上的樣品進行掃描偵測並且可同時鑑定其結構，此操作過程不僅簡單且快速，相信此技術對混合物種快速鑑定上必有很大的助益。

none

論文摘要………………………….…………………………….………Ⅰ
目錄…………………………………………………………..…………Ⅲ
圖目錄………………………………………………..…………………Ⅳ
表目錄…………………………………………………..………………Ⅸ
第一章 緒論…………………………………………………………….1
一、 前言………………………………………………….……1
二、 簡介…………………………………………………….....2
三、 研究目的………………………………………….……..14
第二章 實驗……………………………………………………………18
一、 TLC結合ELDI-MS介面之設計………………….……..18
二、 儀器裝置…………………………………………….…….25
三、 化學藥劑及配製……………………………….………….30
四、 Monolithic TLC製作過程……………………..………….38
第三章 結果與討論……………………………..……………………..39
第四章 結論………………………………………….………….……..65
第五章 參考資料………………………………………………………67

1. York, H.; Funk, W.; Fischer, W.; Wimmer, H. “Thin-Layer Chromatography; Reagents and Detection Methods”; VCH: New York, 1990, p 144.
2. Geiss, F. “Fundamentals of Thin Layer Chromatography (Planar Chromatography)”; Huethig: Heidelberg, 1987, p 283.
3. Sherma, J. “Planar Chromatography”, Anal. Chem. 2000, 72, 9R.
4. Fried, B.; Sherma, J. “Thin-Layer Chromatography”, 4th ed.; Marcel Dekker: New York, 1999.
5. Sherma, J. “Planar Chromatography”, Anal. Chem. 2002, 74, 2653.
6. Poole C. “Thin-layer chromatography: challenges and opportunities” J. Chromatogr. A 2003, 1000, 963.
7. Poole C. “Planar chromatography at the turn of the century”, J. Chromatogr. A 2000, 856, 399.
8. Busch K. L. “Mass spectrometric detectors for samples separated by planar electrophoresis”, J. Chromatogr. A 1995, 692, 275.
9. Wilson I. D.; Morden W. “The Detection and Characterization of Analgesics and Anti-Inflammatory Drugs on High Performance Thin-Layer Chromatography Plates Using Tandem Mass Spectrometry: Application to Drugs and Metabolites in Urine”, Rapid Commun. Mass Spectrom. 1996, 10, 1951.
10. Wilson I. D. “The state of the art in thin-layer chromatography–mass spectrometry: a critical appraisal”, J. Chromatogr. A 1999, 856, 429.
11. Gusev A. I. Fresenius J. Anal. Chem. 2000, 366, 691.
12. Touchstone, J. C.; Rogers, D. “Thin-layer chromatography: Quantitative environmental and clinications”, John Wiley & Sons, New York, 1980.
13. Lawrence, J. F.; Frei, R. W. “Chemical derivatization in liquid chromatography”, 3rd ed., Elsevier, 1985.
14. Kuksis, A. “Chromatography of lipids in biomedical research and clinical diagnosis”, Elsevier, 1987.
15. Busch, K. L. in Sherma, J.; Fried, B. (Eds) Handbook of Thin-Layer Chromatography Marcel Dekker, New York, 1990, p 241.
16. Kaiser, R. “Thin-Layer Chromatography in Direct Coupling with Gas Chromatography and Mass Spectrometry”, Chem. Br. 1969, 5, 54
17. Didonato, G. C.; Busch, K. L. “Phase-Transition Matrix for Chromatography/Secondary Ion Mass Spectrometry”, Anal. Chem. 1986, 58, 3231.
18. Monaghan, J. J.; Morden, W. E.; Johnson, T.; Wilson, I. D.; Martin, P. “Thin Layer Chromatography/Mass Spectrometry: The Advantages of Tandem Mass Spectrometry”, Rapid Commun. Mass Spectrom. 1992, 6, 608.
19. Danigel, H.; Schmidt, L.; Jungclas, H.; Pfluger, K. H. Biomed. Mass Spectrom.1985, 12, 542.
20. Novak, F.P.; Hercules, D.M. “Thin Layer Chromatography-Laser Mass Spectrometry (TLC-LMS) of Triphenylmethane Dyes: Initial Results”, Anal. Lett., 1985, 8, 503.
21. Kubis, A. J.; Somayajula, K. V.; Sharkey, A. G.; Hercules, D. M. “Laser Mass Spectrometric Analysis of Compounds Separated by Thin-Layer Chromatography”, Anal. Chem.1989, 61, 2516.
22. Rogers, K.; Milnes, J.; Gormally, J. “The Laser Desorption/Laser Ionization Mass Spectra of some Methylated Xanthines and the Laser Desorption of Caffeine and Theophylline from Thin Layer Chromatography Plates” Int. J. Mass Spectrom. Ion Processes 1993, 123, 125.
23. Fanibanda, T.; Milnes, J.; Gormally, J. “Thin Layer Chromatography-Mass Spectrometry Using Infrared Laser Desorption” Int. J. Mass Spectrom. Ion Processes 1994, 140, 127
24. Gusev, A. L.; Proctor, A.; Rabinovich, Y. I.; Hercules, D. M. “Thin-Layer Chromatography Combined with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry”, Anal. Chem. 1995, 67, 1805.
25. Mehl, J. T.; Hercules, D. M. “Direct TLC-MALDI Coupling Using a Hybrid Plate”, Anal. Chem. 2000, 72, 68.
26. Gusev A. I. Proctor A, Rabinovich Y. I.; Hercules D. M. “Imaging of Thin-Layer Chromatograms Using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry”, Anal. Chem. 1995, 67, 4565.
27. Mehl, J. T.; Gusev, A. I.; Hercules, D. M. “Coupling Protocol for Thin Layer Chromatography Matrix-Assisted Laser Desorption Ionization”, Chromatographia 1997, 46, 358.
28. Isbell D. T.; Gusev A. I.; Taranenko N. I.; Chen C. H.; Hercules D. M. “Analysis of Nucleotides Directly from TLC Plates Using MALDI-MS Detection”, Fresenius J. Anal. Chem. 1999, 365, 625.
29. Mowthorpe S.; Clench M. R.; Cricelius A.; Richards D. S.; Parr V, Tetler L. W. “Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight/Thin-Layer Chromatography/Mass Spectrometry - a Rapid Method for Impurity Testing”, Rapid Commun. Mass Spectrom. 1999, 13, 264.
30. Crecelius A.; Clench M. R.; Richards D. S.; Mather J; Parr V. “Analysis of UK-224,671 and Related Substances by Thin-Layer Chromatography-Matrix-Assisted Laser-Desorption Ionization-Time of Flight Mass Spectrometry”, J. Planar Chromatogr. 2000, 13, 76.
31. Crecelius A,; Clench M. R.; Richards D. S.; Evason D, Parr V. “Thin-Layer Chromatography - Postsource-Decay Matrix-Assisted Laser Desorption/Ionization Time-of Flight Mass Spectrometry of Small Drug Molecules”, J. Chromatogr. Sci. 2002, 40, 614.
32. Crecelius A.; Clench M. R.; Richards D. S.; Parr V. “Thin-layer Chromatography–Matrix Assisted Laser Desorption Iionisation–Time-of-Flight Mass Spectrometry Using Particle Suspension Matrices”, J. Chromatogr. A 2002, 958, 249.
33. Crecelius A.; Clench M. R.; Richards D. S. “TLC-MALDI in Pharmaceutical Analysis”, LC GC Eur. 2003, 16, 225.
34. Doroshenko V. M.; Laiko V. V.; Taranenko N. I.; Berkout V. D.; Lee H. S. “Recent Developments in Atmospheric Pressure MALDI Mass Spectrometry”, Int. J. Mass Spectrom. 2002, 221, 39.
35. Moyer S. C.; Cotter R. J. “Atmospheric Pressure MALDI”, Anal. Chem. A-Page 2002, 74, 469A.
36. Cole R. B. “Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications”, John Wiley, New York, 1997.
37. Chai W.; Leteux C,; Lawson A. M.; Stoll M. S. “On-Line Overpressure Thin-Layer Chromatographic Separation and Electrospray Mass Spectrometric Detection of Glycolipids”, Anal. Chem. 2003, 75, 118.
38. Chen, Y. C.; Shiea, J.; Sunner, J. “Thin-Layer Chromatography-Mass Spectrometry Using Activated Carbon, Surface-Assisted Laser Desorption/Ionization”, J. Chromatogr. A 1998, 826, 77.
39. Van Berkel, G. J.; Sanchez, A. D.; Quirke, J. M. E. “Thin-Layer Chromatography and Electrospray Mass Spectrometry Coupled Using a Surface Sampling Probe”, Anal. Chem. 2002, 74, 6216.
40. Hsu F. L.; Chen C. H.; Yuan C. H.; Shiea J. “Interfaces To Connect Thin-Layer Chromatography with Electrospray Ionization Mass Spectrometry “, Anal. Chem. 2003, 75, 2493.
41. Van Berkel G. J.; Llave J. L.; De Apadoca M. F.; Ford M. J. “Rotation Planar Chromatography Coupled On-Line with Atmospheric Pressure Chemical Ionization Mass Spectrometry”, Anal. Chem. 2004, 76, 479.
42. Ford, M. J.; Van Berkel, G. J. “An Improved Thin-Layer Chromatography/Mass Spectrometry Coupling Using a Surface Sampling Probe Electrospray Ion Trap System”, Rapid. Commun. Mass Spectrom. 2004, 18, 1303.
43. Ford, M. J.; Kertesz, V.; Van Berkel, G. J. “Thin-Layer Chromatography/Electrospray Ionization Triple-Quadrupole Linear Ion Trap Mass Spectrometry System: Analysis of Rhodamine Dyes Separated on Reversed-Phase C-8 Plates”, J. Mass Spectrom. 2005, 40, 866.
44. Van Berkel, G. J.; Ford, M. J.; Deibel, M. A. “Thin-Layer Chromatography and Mass Spectrometry Coupled Using Desorption Electrospray Ionization”, Anal. Chem. 2005, 77, 1207.
45. Van Berkel, G. J.; Tomkins, B. A.; Kertesz, V. “Automated Sampling and Imaging of Analytes Separated on Thin-Layer Chromatography Plates Using Desorption Electrospray Ionization Mass Spectrometry”, Anal. Chem. 2006, 78, 4938.
46. Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J. “Electrospray-Assisted Laser Desorption/Ionization Mass Spectrometry for Direct Ambient Analysis of Solids”, Rapid. Commun. Mass Spectrom. 2005, 19, 3701.
47. Huang, M. Z.; Hsu, H. J.; Lee, J. Y.; Jeng. J.; Shiea, J. “Direct Protein Detection from Biological Media through Electrospray-Assisted Laser Desorption Ionization/Mass Spectrometry”, J. Protemoe Res. 2006, 5, 1107.
48. Kalasz, H. “Spot and Mobile-Phase Front Anomalies in Planar (Thin-Layer) Chromatography”, Chromatographia Supplement, 2005, 62, S57.
49. Joseph W. Le Fevre “Complete Analysis of a Biologically Active Tetrapeptide:A Project Utilizing Thin-Layer Chromatography and Mass Spectrometry”, J. Chem. Educ.2000, 77, 504.
50. Kalasz, H.; Hunyadi A.; Bathori M. “Novel Results of Two-Dimensional Thin-Layer Chromatography”, J. Liq. Chromatogr. &Relat. Technol. 2005, 28, 2489.
51. Tuzimski, T.; Wojtowicz, J. “Separation of a Mixture of Pesticides by 2D-TLC on Two-Adsorbent-Layer Multi-K SC5 Plate”, Liq. Chromatogr. &Relat. Technol. 2005, 28, 277.
52. Bodennec J.; Pelled, D.; Futerman, A. H. “Aminopropyl Solid Phase Extraction and 2D- TLC of Neutral Glycosphingolipids and Neutral Lysoglycosphingolipids J. Lipid Res. 2003, 44, 218.
53. Bakry R.; Bonn G. K.; Mair D.; Svec F. Anal. Chem. 2007, 79, 486.