Laboratory Experiment pubs.acs.org/jchemeduc
Identification of Onion Dye Chromophores in the Dye Bath and Dyed Wool by HPLC-DAD: An Educational Approach Cristina Barrocas Dias,*,†,‡,§ Marco Miranda,§ Ana Manhita,†,‡,§ António Candeias,†,‡,§,∥ Teresa Ferreira,†,‡,§ and Dora Teixeira†,§,⊥ HERCULES Laboratory, Largo do Marquês de Marialva, 8, 7000-809 Évora, Portugal Évora Chemistry Centre, Rua Romão Ramalho 59, 7000-671 Évora, Portugal § Chemistry Department, Universidade de Évora, Rua Romão Ramalho 59, 7000-671 Évora, Portugal ∥ José de Figueiredo Conservation and Restoration Laboratory, Institute of Museums and Conservation, Rua das Janelas Verdes 37, 1249-018 Lisboa, Portugal ⊥ ICAAM, Institute of Mediterranean Agricultural and Environmental Sciences, Núcleo da Mitra Apartado 94, 7002-774 Évora, Portugal † ‡
S Supporting Information *
ABSTRACT: Onion skins (Allium cepa L.) and hydrated potassium aluminum sulfate were used to dye wool samples. The main chromophores associated with this natural dye source, quercetin and quercetin-4′-O-glucoside, were identified in the dye bath and in wool extracts by high-pressure liquid chromatography (HPLC) equipped with a diode array detector (DAD) with the help of standards. Two procedures were used to extract dye molecules from dyed wool prior to HPLCDAD qualitative analysis and the analytical methodology used was discussed in terms of the analysis of historical textile pieces dyed with natural sources. KEYWORDS: Second-Year Undergraduate, Analytical Chemistry, History/Philosophy, Laboratory Instruction, Hands-On Learning/Manipulatives, Chromatography, Dyes/Pigments, Natural Products
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atural dyes have been used since ancient times to dye vegetable and animal fibers, and nowadays, they are becoming more significant because they are safer and more ecofriendly than synthetic materials. Several reviews in the literature discuss in detail the natural sources of the different colors and the chemical nature of dyes.1−3 Although some dyes are of animal origin (e.g., red cochineal), the vast majority comes from plants, and all dye molecules possess highly conjugated systems that absorb electromagnetic radiation between 400 and 800 nm, therefore, appearing colored (Figure 1). Recent papers published in this Journal describe the use of natural dyes to dye silk4 and the effect of different mordants on the color and hue obtained with the same dye5,6 (for more information on mordants and mordant−wool bonds, please see the Supporting Information). A survey of interesting Internet sites dealing with the use of natural dyes was also published in this Journal in 2008.7 The laboratory described here makes students aware of the use of natural dyes and of their importance to the study of cultural heritage and to the conservation of museum pieces. Chromatographic analysis of dyes extracted from textiles of historical interest can give valuable information as to where, when, and how the textiles were produced. Students use highpressure liquid chromatography (HPLC) equipped with a diode array detector (DAD), a common analytical methodology. The protocol was initially used in a workshop for the © 2013 American Chemical Society and Division of Chemical Education, Inc.
Figure 1. Chemical structures of some natural dyes.
general public with interest in conservation science and later in an undergraduate second-year course on natural product chemistry. A general introduction on the use of natural dyes was presented because students were generally not familiar with this subject. Information was given on the different classes and Published: October 25, 2013 1498
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sources of natural dyes1−3 and also on the analytical methodology that can be used to extract and identify the natural dyes in historical textiles and their contribution to cultural heritage studies.8−16
Laboratory Experiment
HAZARDS AND MATERIALS DISPOSAL Formic acid and HCl solutions are corrosive to eyes, skin, and mucous membranes. Methanol and dimethylformamide are toxic and the former is highly flammable. Flavonoid compounds can be toxic if swallowed. EDTA may irritate the lungs if inhaled. Students should wear chemical safety goggles and compatible chemical-resistant gloves. The solvents should be handled in a well-ventilated hood. Solution disposal should take into consideration the toxicity of the materials.
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EXPERIMENTAL SUMMARY The experimental procedure is briefly summarized here, and student directions and information for instructors are provided in the Supporting Information. The experiment was performed by a class of 16 second-year undergraduate chemistry and biochemistry students divided in four groups, during two 3-h lab periods. In the first lab period, the dyeing bath was prepared, and wool was mordanted and dyed. In the second lab period, the chromophores from wool were extracted and analyzed with HPLC-DAD. To mordant the wool, hydrated potassium aluminum sulfate (alum), 0.50 g, was dissolved in 30.0 mL of water and 1.0 g of white wool was added. The mixture was heated at 90 °C for about 15 min. The wool was removed, washed with distilled water, and allowed to air-dry. The dye bath was prepared by boiling 2.0 g of yellow onion skins in 50.0 mL of water for 15 min, after which the solids were removed by filtration and the supernatant saved. The mordanted wool was introduced into the dye bath, and the mixture was heated to 90 °C for 15 min. The dark-yellow dyed wool was removed, washed with distilled water, and allowed to air-dry. Two methods were used to extract the dyes from wool: the hydrochloric acid (HCl) method16 and the ethylenediaminetetracetic acid disodium salt/dimethylformamide (EDTA/ DMF) method.11 For both methods, 20.0 mg samples of dyed wool were weighted and transferred to two test tubes equipped with a small magnetic stir bar. A small volume, 4 mL, of concentrated HCl/methanol/H2O, 2:1:1 (v/v/v) or 10.0 mL of 0.1% EDTA in H2O/DMF 1:1 (v/v) was added to the test tubes and the mixtures were heated at 100 °C for 10 min for the HCl method and 15 min for the EDTA/DMF method. After cooling to room temperature, the mixtures were filtered through a 0.45 μm filter and saved for chromatographic analysis. The HPLC analyses of the dye bath, the wool extracts, and the quercetin and quercetin-4′-O-glucose standards were accomplished with a 150 × 4.6 mm 3.5 μm C18 column with a diode array detector (DAD) from 200 to 450 nm; chromatograms were recorded at 350 nm. A mobile phase consisting of two solvent systems (A, methanol and B, water with 0.1% (v/v) formic acid) was employed using the following gradient program: 0−100% A, 0−15 min; 100% A, 15−20 min. The flow rate was 0.5 mL min−1, and the injection volume was 20 μL. Equilibration for 5 min between samples was accomplished with 100% B. Because of the length of the analyses and the use of manual injection, students could not perform all aspects of the analyses. UV spectra of quercetin and quercetin-4′-O-glucoside, used to identify both analytes in the chromatograms, were provided by the instructor. The waiting periods were used to read and review literature reports dealing with the identification and use of natural dyes as well as their study in historical textile artifacts (see Supporting Information for suggestions). The final hue of the wool is strongly dependent on the dyeing conditions.5 Instructors can introduce differences in the proposed protocol and encourage the students to look and discuss the HPLC data taking in consideration the wool final hue.
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RESULTS AND DISCUSSION The HPLC chromatograms of the dye bath and the wool extracts obtained with HCl and EDTA/DMF are shown in Figure 2. Identification of the analytes was easily achieved by
Figure 2. HPLC-DAD chromatograms recorded at 350 nm of (A) onionskin dye bath, (B) HCl wool extract, and (C) EDTA/DMF wool extract. Identification of the peaks: I, quercetin-4′-O-glucoside; II, quercetin.
individually injecting the two standard solutions and recording their retention times under the same conditions used to separate the extracts. Identification of dyes in historical pieces requires the identification of the major chromophores obtained from a certain dye source; therefore, the number of compounds and the amounts extracted are important factors when choosing an extraction procedure. Acid hydrolysis with heated HCl is still a widely reported extraction method despite the fact these 1499
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conditions lead to the hydrolysis of flavonoid glycosides common when studying yellow dyed textiles. HCl methodology is normally perceived as the most aggressive and therefore the most efficient methodology. With this project, students easily understand that data were lost when HCl method was applied as only the quercetin peak was observed in the chromatogram (Figure 2B). The EDTA/DMF procedure allowed the extraction of both flavonoids (Figure 2C) yielding more information. Quercetin is a widely distributed flavonoid in plants. If the dye source was unknown, it would have been difficult to attribute the onion skins as dye source based solely on chromatogram in Figure 2B. Instructors with some experience with HPLC method development can easily perform this experiment using the same chromatographic system with small alterations in the gradient and other common dye sources, namely, weld (Reseda luteola L.) whose major chromophore is the flavonoid luteolin,2,3 madder (Rubia tinctorum L.) that has the anthraquinones purpurine and alizarin as their major red colorants,2,3 or cochineal that has the carminic acid derivatives as their red chromophores.2,3 Indigo (Indigofera tinctorum L.) can also be used, but the procedure to dye the textile fibers in blue is different because indigo is a vat dye and does not require mordants17,18 Mass spectrometry detection is a much more powerful technique than UV detection and whenever available, instructors are encouraged to use it. A mass detector with an ESI (electrospray ionization) interface can be used to identify the major phenolic compounds in the onion skins19,20 without the need of standards.
REFERENCES
(1) Kumar, J. K.; Sinha, A. K. Nat. Prod. Lett. 2003, 18, 59−84. (2) Ferreira, E. S. B.; Hulme, A. N.; MacNab, H.; Quye, A. Chem. Soc. Rev. 2004, 33, 329−336. (3) Séquim-Frey, M. J. Chem. Educ. 1981, 58 (4), 301−305. (4) Paixão, M. F.; Pereira, M. M.; Cachapuz, A. F. J. Chem. Educ. 2006, 83, 1546−1549. (5) Mihalick, J. E.; Donnelly, K. M. J. Chem. Educ. 2006, 83, 1550− 1551. (6) Mihalick, J. E.; Donnelly, K. M. J. Chem. Educ. 2007, 84, 96A− 96B. (7) Fanis, L. N. J. J. Chem. Educ. 2008, 85 (9), 1172−1174. (8) Zhang, X.; Good, I.; Laursen, R. J. Archeol. Sci. 2008, 35, 1095− 1103. (9) Surowiec, I.; Quye, A.; Trojanowicz, M. J. Chromatogr., A 2006, 1112, 209−217. (10) Zhang, X.; Laursen, R. A. Anal. Chem. 2005, 77, 2022−2025. (11) Tiedemann, E. J.; Yang, Y. J. Am. Inst. Conserv. 1995, 34, 195− 206. (12) Orska-Gawrýs, J.; Surowiec, I.; Kehl, J.; Rejmiak, H.; UrbaniakWalczak, K.; Trajanowicz, M. J. Chromatogr., A 2003, 989, 239−248. (13) L. Rafaëlly, L.; Héron, S.; Nowik, W.; Tchapla, A. Dyes Pigm. 2008, 77, 91−203. (14) Karapanagiotis, I.; Lakka, A.; Valianou, L.; Chryssoulakis, Y. Microchim, Acta 2008, 160, 477−483. (15) Zhang, X.; Laursen, R. Int. J. Mass Spectrom. 2009, 284 (2009), 108−114. (16) Wouters. J. Stud. Conserv. 1985, 30, 119. (17) Park, J. H.; Gatwood, B. M.; Ramaswamy, G. N. J. Appl. Polym. Sci. 2005, 98 (1), 322−328. (18) Fernelius, C. W.; Renfrew, E. E. J. Chem. Educ. 1983, 60 (8), 633−634. (19) Bonaccorsi, P.; Caristi, C.; Gargiulli, C.; Leuzzi, U. J. Agric. Food Chem. 2005, 53, 2733−2740. (20) Ly, T. N.; Hazama, C.; Shimoyamada, M.; Ando, H.; Kato, K.; Yamauchi, R. J. Agric. Food Chem. 2005, 53, 8183−8189.
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CONCLUSIONS This laboratory experiment was performed by chemistry and biochemistry students in a natural product chemistry undergraduate course where the information on the sources of natural dyes provided interesting discussions on the origins of the color and how color is perceived by humans and animals. Apart from the discussion on the role of the flavonoids as products of the secondary metabolism of the plants and their biosynthesis, this laboratory project enabled the students to understand that worldwide plants were the main sources of color before the synthetic dyes became available in the second half of the 19th century.
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Laboratory Experiment
ASSOCIATED CONTENT
S Supporting Information *
Student directions and information for instructors. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the financial support of Project REMATAR, FCOMP-01-0124-FEDER-010482 (FCT PTDC/ HAH/64045/2006) from the Portuguese Foundation for Science and Technology, FCT. A. Manhita also acknowledges FCT for the Ph.D. fellowship (SFRH/BD/22411/2005). 1500
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