本研究使用基質輔助雷射脫附游離飛行時間質譜法和拉曼光譜技術，探討茜素與媒染劑（包括醋酸鋁，硫酸鋁鉀，醋酸鋁與醋酸鐵）反應時，形成「茜素-金屬離子」有機金屬錯合物的可能結構。此反應一旦形成有機金屬錯合物，其沉澱物的顏色分佈為紅色到褐色。該沉澱物可使用基質輔助雷射脫附飛行時間型質譜儀加以測量，並以CHCA (alpha-cyano-4-hydroxycinnamic acid)作為基質。實驗結果發現在正電模式時，質譜圖上有一明顯的訊號在m/z=504.043，符合兩個茜素分子與醋酸鋁中的鋁離子錯合的荷質比。此外在測量茜素與明礬溶液的訊號時，還偵測到了茜素與鋁離子和鉀離子的中間產物之訊號在m/z=316.049、m/z=331.069、m/z=375.075、m/z=392.082，符合茜素分子與鋁離子和鉀離子錯合時配位了羥基和甲氧基的荷質比。
利用拉曼光譜觀察到特徵峰值分別為1298 cm-1、1332 cm-1、1454 cm-1、1480 cm-1代表了C-OH和C-H的彎曲振動，以及C-C的伸縮振動，還觀察到了1630 cm-1 的C=O伸縮振動和1162 cm-1及1193 cm-1的 C-H彎曲振動和C-C的伸縮振動，基於這些特徵峰值最後推測出化學結構，並比對文獻參考進行驗證。
為了探討媒染後的顏色變化，利用LabVIEW程式中RGB數值轉換為波長的程式，探討該方法取代反射式吸收光譜法的可行性。

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Raman spectroscopy techniques were used to investigate the potential structures of "Alizarin -Metal Ion" organic-metal complexes formed during the reaction between alizarin and mordants (including aluminum acetate and alum). Once these organic-metal complexes are formed, the precipitate exhibits a color range from red to brown. The precipitate can be measured using MALDI-TOF MS with CHCA (alpha-cyano-4-hydroxycinnamic acid ) as the matrix. Experimental results revealed a prominent signal at m/z=504.043 in positive ion mode on the mass spectrum, matching the mass-to-charge ratio of two alizarin molecules complexed with aluminum ions in aluminum acetate. Furthermore, when measuring signals from the alizarin and alum solution, intermediate products of alizarin with aluminum and potassium ions were detected at m/z=316.049, m/z=331.069, m/z=375.075, and m/z=392.082, corresponding to the mass- to-charge ratios expected when alizarin molecules coordinate with hydroxyl and methoxy groups in aluminum and potassium ions.
Using Raman spectroscopy, characteristic peaks at 1298 cm-1, 1332 cm-1, 1454 cm-1, and 1480 cm-1 were observed, representing the bending vibrations of C-OH and C-H, as well as t he stretching vibrations of C-C. Additionally, peaks at 1630 cm-1 for C=O stretching vibration and 1162 cm-1 and 1193 cm-1 for C-H bending vibration and C-C stretching vibration were observed. Based on these characteristic peaks, the chemical structure was inferred and validated against literature references.
In order to explore the color changes after mordant dyeing, the program for converting RGB values into wavelengths in the LabVIEW program was used to explore the feasibility of this method instead of reflection absorption spectroscopy

[1] Benkhaya, S.; M’ rabet, S.; El Harfi, A. A Review on Classifications, Recent Synthesis and Applications of Textile Dyes. Inorganic Chemistry Communications, 2020, 115, 107891.
[2] Irshad, S.; Sultana, H.; Usman, M.; Saeed, M.; Akram, N.; Yusaf, A.; Rehman, A. Solubilization of Direct Dyes in Single and Mixed Surfactant System: A Comparative Study. Journal of Molecular Liquids, 2021, 321, 114201.
[3] HARLEY, R. D. Field’s Manuscripts: Early Nineteenth Century Colour Samples and Fading Tests. Studies in Conservation, 1979, 24 (2), 75–84.
[4] Kiel, E.; Heertjes, P. Metal Complexes of Alizarin II—The Structure of Some Metal Complexes of Alizarin Other than Turkey Red. Journal of the Society of Dyers and Colourists, 2008, 79, 61–64.
[5] Baykuş, O.; Tugce Celik, I.; Dogan, S. D.; Davulcu, A.; Dogan, M. Enhancing the Dyeability of Poly(Lactic Acid) Fiber with Natural Dyes of Alizarin, Lawsone, and Indigo. Fibers Polym, 2017, 18 (10), 1906–1914.
[6] Chemchame, Y.; Moudden, M. E.; Mansar, A. Dyeing Wool Fiber with Natural Alizarin in a Vat System. Am. J. Appl. Chem., 2016, 4 (5), 170–173.
[7] Ding, Y.; Freeman, H. S. Mordant Dye Application on Cotton: Optimisation and Combination with Natural Dyes. Coloration Technology, 2017, 133 (5), 369–375.
[8] De Santis, D.; Moresi, M. Production of Alizarin Extracts from Rubia Tinctorum and Assessment of Their Dyeing Properties. Industrial Crops and Products, 2007, 26 (2), 151–162.
[9] Van Elslande, E.; Guérineau, V.; Thirioux, V.; Richard, G.; Richardin, P.; Laprévote, O.; Hussler, G.; Walter, P. Analysis of Ancient Greco-Roman Cosmetic Materials Using Laser Desorption Ionization and Electrospray Ionization Mass Spectrometry. Anal Bioanal Chem, 2008, 390 (7), 1873–1879.
[10] Capeletti, L. B.; Dos Santos, J. H. Z.; Moncada, E.; Da Rocha, Z. N.; Pepe, I. M. Encapsulated Alizarin Red Species: The Role of the Sol–Gel Route on the Interaction with Silica Matrix. Powder Technology, 2013, 237, 117–124.
[11] Derksen, G. C. H.; Van Beek, T. A.; De Groot, Æ.; Capelle, A. High-Performance Liquid Chromatographic Method for the Analysis of Anthraquinone Glycosides and Aglycones in Madder Root (Rubia Tinctorum L.). Journal of Chromatography A, 1998, 816 (2), 277–281.
[12] Boldizsár, I.; Szűcs, Z.; Füzfai, Zs.; Molnár-Perl, I. Identification and Quantification of the Constituents of Madder Root by Gas Chromatography and High-Performance Liquid Chromatography. Journal of Chromatography A, 2006, 1133 (1), 259–274.
[13] Krizsán, K.; Szókán, Gy.; Toth, Z. A.; Hollósy, F.; László, M.; Khlafulla, A. HPLC Analysis of Anthraquinone Derivatives in Madder Root (Rubia Tinctorum) and Its Cell Cultures. Journal of Liquid Chromatography & Related Technologies, 1996, 19 (14), 2295–2314.
[14] Karapanagiotis, I.; Chryssoulakis, Y. Investigation of Red Natural Dyes Used in Historical Objects by HPLC-DAD-MS. Annali di Chimica, 2006, 96 (1–2), 75–84.
[15] Sun, R.; Lou, J.; Fan, X.; Gao, W.; Gu, Z. Dyeing and Functionalization of Wool Fabric with Alizarin Red S via Covalent Combination Catalyzed by Horseradish Peroxidase and Hydrogen Peroxide. Fibers and Polymers, 2023, 24 (12), 4311–4321.
[16] Ren, C.; Chen, C.; Dong, S.; Wang, R.; Xian, B.; Liu, T.; Xi, Z.; Pei, J.; Chen, J. Integrated Metabolomics and Transcriptome Analysis on Flavonoid Biosynthesis in Flowers of Safflower (Carthamus Tinctorius L.) during Colour-Transition. PeerJ, 2022, 10, e13591.
[17] Ebeid, H.; Di Gianvincenzo, F.; Kralj Cigić, I.; Strlič, M. Chromatographic Analysis of Natural Dyes in Mediaeval Islamic Paper. Heritage Science, 2024, 12 (1), 13.
[18] Rehman, F. U.; Adeel, S.; Haddar, W.; Bibi, R.; Azeem, M.; Mia, R.; Ahmed, B. Microwave-Assisted Exploration of Yellow Natural Dyes for Nylon Fabric. Sustainability, 2022, 14 (9), 5599.
[19] Jen, M.; Lee, S.; Jeon, K.; Hussain, S.; Pang, Y. Ultrafast Intramolecular Proton Transfer of Alizarin Investigated by Femtosecond Stimulated Raman Spectroscopy. J. Phys. Chem. B, 2017, 121 (16), 4129–4136.
[20] Li, J.; Zhang, M. Physics and Applications of Raman Distributed Optical Fiber Sensing. Light Sci Appl, 2022, 11 (1), 128.
[21] Dueñas, M. E.; Trost, M. MALDI-TOF Mass Spectrometry in the 21st Century. The Biochemist, 2022, 44 (5), 2–4.
[22] Blades, A. T.; Ikonomou, M. G.; Kebarle, Paul. Mechanism of Electrospray Mass Spectrometry. Electrospray as an Electrolysis Cell. Anal. Chem., 1991, 63 (19), 2109–2114.
[23] Snyder, E. M.; Buzza, S. A.; Castleman, Jr., A. W. Intense Field-Matter Interactions: Multiple Ionization of Clusters. Phys. Rev. Lett., 1996, 77 (16), 3347–3350.
[24] Wabnitz, H.; Bittner, L.; de Castro, A. R. B.; Döhrmann, R.; Gürtler, P.; Laarmann, T.; Laasch, W.; Schulz, J.; Swiderski, A.; von Haeften, K.; et al. Multiple Ionization of Atom Clusters by Intense Soft X-Rays from a Free-Electron Laser. Nature, 2002, 420 (6915), 482–485.
[25] Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. Molecular Beams of Macroions. The Journal of Chemical Physics, 1968, 49 (5), 2240–2249.
[26] Iribarne, J. V.; Thomson, B. A. On the Evaporation of Small Ions from Charged Droplets. The Journal of Chemical Physics, 1976, 64 (6), 2287–2294.
[27] Cao, Y.; Guan, J.; Yang, J.; Guan, R.; Liy, O.; Jin, J.; Llu, J. Nondestructive Identification of Natural Dyes by Fiber Optic Reflectance Spectroscopy. Journal of Silk, 2023, 60 (12), 51–58.
[28] Shahid, M.; Wertz, J.; Degano, I.; Aceto, M.; Khan, M. I.; Quye, A. Analytical Methods for Determination of Anthraquinone Dyes in Historical Textiles: A Review. Analytica Chimica Acta, 2019, 1083, 58–87.
[29] Whitney, A. V.; Casadio, F.; Van Duyne, R. P. Identification and Characterization of Artists’ Red Dyes and Their Mixtures by Surface-Enhanced Raman Spectroscopy. Appl Spectrosc, 2007, 61 (9), 994–1000.
[30] Yang, B.; Cao, X.; Lang, H.; Wang, S.; Sun, C. Study on Hydrogen Bonding Network in Aqueous Methanol Solution by Raman Spectroscopy. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2020, 225, 117488.
[31] Puglieri, T. S.; Madden, O.; Andrade, G. F. S. SHINERS in Cultural Heritage: Can SHINERS Spectra Always Be Compared with Normal Raman Spectra? A Study of Alizarin and Its Adsorption in the Silicon Dioxide Shell. Journal of Raman Spectroscopy, 2021, 52 (8), 1406–1417.
[32] Nekoei, A.-R.; Vakili, M.; Hakimi-Tabar, M.; Tayyari, S. F.; Afzali, R.; Kjaergaard, H. G. Theoretical Study, and Infrared and Raman Spectra of Copper(II) Chelated Complex with Dibenzoylmethane. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2014, 128, 272–279.
[33] Zhang, J.; Frankevich, V.; Knochenmuss, R.; Friess, S. D.; Zenobi, R. Reduction of Cu(II) in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Journal of the American Society for Mass Spectrometry, 2003, 14 (1), 42–50.
[34] Kiel, E. G.; Heertjes, P. M. Metal Complexes of Alizarin I—The Structure of the Calcium–Aluminium Lake of Alizarin. Journal of the Society of Dyers and Colourists, 1963, 79 (1), 21–27.
[35] Jeliński, T.; Cysewski, P. Structure and Properties of Alizarin Complex Formed with Alkali Metal Hydroxides in Methanol Solution. J Mol Model, 2016, 22 (6), 126.
[36] Blackburn, R. S. Natural Dyes in Madder (Rubia Spp.) and Their Extraction and Analysis in Historical Textiles. Coloration Technology, 2017, 133 (6), 449–462.
[37] Pazalja, M.; Salihović, M. Spectrophotometric Determination of Cysteine Based on Complex Reaction Alizarin Red with Cooper. In CMBEBIH 2021; Badnjevic, A., Gurbeta Pokvić, L., Eds.; Springer International Publishing: Cham, 2021; pp 474–480.
[38] Soubayrol, P.; Dana, G.; Man, P. P. Aluminium-27 Solid-State NMR Study of Aluminium Coordination Complexes of Alizarin. Magnetic Resonance in Chemistry, 1996, 34 (8), 638–645.
[39] James, A. l.; Perry, J. d.; Chilvers, K.; Robson, I. s.; Armstrong, L.; Orr, K. e. Alizarin-β- d-Galactoside: A New Substrate for the Detection of Bacterial β-Galactosidase. Letters in Applied Microbiology, 2000, 30 (4), 336–340.
[40] Gao, S.; Zhao, L.; Fan, Z.; Kodibagkar, V. D.; Liu, L.; Wang, H.; Xu, H.; Tu, M.; Hu, B.; Cao, C.; et al. In Situ Generated Novel 1H MRI Reporter for β-Galactosidase Activity Detection and Visualization in Living Tumor Cells. Front Chem, 2021, 9, 709581.