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Non-toxic hybrid pigments: Sequestering betanidin chromophores on inorganic matrices
Applied Clay Science 42 (2009) 478–482
Contents lists available at ScienceDirect
Applied Clay Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l a y
Non-toxic hybrid pigments: Sequestering betanidin chromophores on inorganic matrices Enrique Lima a,⁎, Pedro Bosch b, Sandra Loera a, Ilich A. Ibarra a, Humberto Laguna a, Victor Lara a a b
Universidad Autónoma Metropolitana, Iztapalapa, Av. San Rafael Atlixco No. 186 Col. Vicentina, 09340 México D.F., Mexico Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, 04510 México D.F., Mexico
A R T I C L E
I N F O
Article history: Received 26 February 2008 Received in revised form 4 June 2008 Accepted 14 June 2008 Available online 22 June 2008 Keywords: Alumina Dye Chromophores Adsorption Pigment Betanidin
A B S T R A C T Betalain was extracted from bougainvillea flower. Alumina, layered double hydroxides (LDHs) and zeolites were tested as adsorbents of the chromophore. Gamma alumina stabilized betalains over a long period. The adsorption sites which stabilized the chromophores were elucidated by nuclear magnetic resonance. Although the oxides obtained by calcination of LDHs retain betalains, they were oxidized within 20 h or react with Zn2+ of the calcined LDH. No betalain was adsorbed in zeolite X because of the too small pores. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In flowers, colour can be as ephemeral as perfume. Both are due to chemical groups which in contact with air degrade and fade out. Goethe (1970) defined chemical colours as “the colours which we can produce, and more or less fix, in certain bodies; which we can render more intense, which we can again take away and communicate to other bodies, and to which, therefore, we ascribe a certain permanency: duration is their prevailing characteristic”. Nowadays colours are known to be the result of light absorption by chemical groups. The main chromophore molecules, i.e. the “chemical colours” of Goethe, present in fruits and flowers are, on the one hand, the anthocyanins (cherries, Atropa belladonna…) and on the other the betalains (bougainvillea, flower of Christmas cactus, beet…) (Gonzalez-San-Jose et al., 1990; Steglich and Strack, 1991). Betalains, a group of reddish and yellowish chromophores, are important markers and have never been found jointly with anthocyanins in the same plant (Clement and Mabry, 1996). Still, betalains not only provide colour, they are a family of compounds which play a major role in the high antioxidant activity levels observed in red fruits and vegetables
⁎ Corresponding author. Tel.: +52 5 55804 4667; fax: +52 5 55804 4666. E-mail address: [email protected] (E. Lima). 0169-1317/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2008.06.005
(Kanner et al., 2001). Furthermore, betalains may be used for food colouring and their antioxidant and radical scavenging properties for protection against certain oxidative stress-related disorders. Today, colourants free of toxic elements, particularly nature colourants are leaving synthetic colourants behind as they are safe and eco-friendly in nature (Ibarra et al., 2005; Simoncic et al., 2004; Jansen and Letschert, 2000; Hradil et al., 2003). Betalain isolation and stabilization is a hard task. Betalains are commonly extracted from plants in aqueous or water–methanol solutions and purified by chromatography. A major difficulty is that purified betalains decompose easily. Hence, their stabilization on supports is recommended. Usually organic materials are proposed, dyes are, then, obtained. Stabilization of betalains as well as other organic molecules, should also be possible by immobilization in inorganic matrices (Ibarra et al., 2005; Lima et al., 2007; Murata et al., 2001; Lopes et al., 2007; Baskaralingam et al., 2007). A well known chromophore–inorganic matrix system is the Maya blue pigment where the indigo is incorporated in the pores of palygorskite clay (Gettens, 1962; Giustetto et al., 2005; Domenech et al., 2006). Inspired by the Maya technique and the beautiful colours of the Mexican bougainvilleas (Heuer et al., 1994), we have tested three materials well known as supports in catalysis: layered double hydroxides (LDHs), gamma alumina and zeolites. Alumina is a spinel with octahedrally and tetrahedrally coordinated aluminium ions in an oxygen lattice, which adsorbs organic species such as carboxylic acids or polychlorobiphenyls (Goyne et al., 2005; Fuoco et al., 2005).
E. Lima et al. / Applied Clay Science 42 (2009) 478–482
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Table 1 Chemical composition of the adsorbents Inorganic matrix
Label
Chemical composition
Sodium X zeolite Protonated X zeolite Mixed oxide Mixed oxide γ-Alumina
NaX HX Mg–Al–O Mg,Zn–Al–O γ-Al2O3
Na+86(SiO2)106(AlO2)86·xH2O H+86(SiO2)106(AlO2)86·xH2O Mg0.744Al0.256[OH]2(CO3)0.128·0.850H2O Mg0.614Zn0.093Al0.293[OH]2(CO3)0.146·0.731H2O Al2O3
The specific surface area of γ-Al2O3 (250–300 m2/g) is mainly due to macropores. γ-Alumina is prepared at 500 °C and is stable up to 850 °C, then transforms to δ-Al2O3. Instead, the two other chosen oxides, although also constituted by aluminium, present very different features. 2. Experimental section
Fig. 2. Structure of the betanidin chromophore, betalain which is responsible of the purple colour. R can be 9 different groups.
2.1. Materials 2.1.1. Alumina (γ-Al2O3) Alumina was prepared by the sol–gel technique using aluminium tri-sec-butoxide as aluminium source, ethanol as solvent and sulfuric acid as catalyst in the hydrolysis step. The gel was aged for 30 days, dried at 80 °C and calcined at 550 °C. 2.1.2. Zeolite Zeolite NaX was supplied by Union Carbide. This zeolite was used in sodium (NaX) or protonated (HX) form. The protonated zeolite was prepared by exchanging NaX zeolite in 1 N NH4OH solution. Then, the ammonium ions were decomposed at 300 °C. 2.1.3. LDHs The Mg–Al LDH was prepared by coprecipitation of magnesium and aluminium nitrate at pH 9–10 in the presence of carbonate. The precipitate was filtered, washed with deionized water and dried at 100 °C for 24 h. (HTMgAl). Another LDH containing Mg, Al and Zn was prepared by coprecipitation. The ratio divalent on trivalent metal was maintained close to 3. (HTMgZnAl). Mixed oxides were obtained by calcining HTMgAl and HTMgZnAl, at 600 °C for 8 h. These samples were labeled Mg–Al–O and Mg–Zn– Al–O, respectively.
The chemical compositions of the adsorbents are presented in Table 1. 2.2. Extraction of the chromophore from the flowers and their adsorption 1 g of bougainvillea flowers, Fig. 1, was slightly ground and then added to 40 ml of water. The mixture was agitated for 20 min. A transparent and coloured solution was obtained containing the betalain (Fig. 2). A small amount of solid was easily removed by centrifugation. 10 ml of an aqueous solution containing the betalain was put in contact with 100 mg of the adsorbent for 30 min. The solid was separated by centrifugation, washed and dried at room temperature. 2.3. Characterization 2.3.1. X-ray diffraction X-ray diffraction patterns were obtained with a Siemens D500 diffractometer (Kα radiation). Compounds were identified conventionally with the JCPDS files (Joint Committee of Powder Diffraction Standards). 2.3.2. Ultraviolet–Visible spectroscopy Spectra UV–Vis of the samples, as pellets, were acquired in a Perkin Elmer Lambda 40 UV–Vis spectrometer in the spectral window from 800 to 300 nm. 2.3.3. Infrared spectroscopy The attenuated total reflectance Fourier infrared (ATR/FTIR) spectra were obtained in a GX Perkin Elmer instrument at 2 cm− 1 resolution.
Table 2 Average pore size and specific surface area of the adsorbents
Fig. 1. Fresh bougainvillea flowers used in this report.
Sample
Specific surface area (m2/g)⁎
Average pore diameter (Å)⁎⁎
NaX HX Mg–Al–O Mg,Zn–Al–O γ-Al2O3
388 409 187 201 320
9 11 41 49 98
⁎Determined from nitrogen isotherms (at 77 K) and applying the BET equation. ⁎⁎Determined from nitrogen isotherms data and BJH method. All samples were outgassed at 400 °C before nitrogen adsorption.
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Table 3 Colour of the adsorbents after betalain adsorption Solid
γ-Al2O3 Mg–Al–O Mg,Zn–Al–O
Colour Solid suspended in the betalain solution
Solid separated from the solution and dried
Solid after 20 months
Purple Purple Red
Purple Purple Yellow
Purple Dark brown Yellow
2.3.4. Solid state nuclear magnetic resonance Nuclear magnetic resonance (NMR) measurements were performed in a Bruker ASX300 Spectrometer. The spectrometer was operated at a resonance frequency of 78.2 MHz for 27Al MAS NMR spectroscopy. Spectra were acquired using short single excitation pulses (π/12) with repetition times of 500 ms. Samples were spun at 10 kHz and the chemical shifts were referenced to 1 N aqueous solution of AlCl3. Spectra of 13C CP MAS NMR were recorded at 75.4 MHz with a contact time of 1 ms, a 5 kHz spinning rate, and 90° pulses of 4 μs. 51,500 scans were accumulated. Chemical shifts were referenced to solid CH2 adamantane shift at 38.2 ppm relative to TMS. 3. Results and discussion A small amount (1 g) of fresh bougainvillea flowers, Fig. 1, was enough to obtain a transparent aqueous coloured solution (0.250 l) containing the betalain responsible of the purple colour (Heuer et al., 1994; Strack et al., 1988), Fig. 2. After drying, the zeolites did not show any colour, they were white, and therefore they were discarded. The chromophore was not retained due to the small size of the pores (Table 2). Alumina and LDH without Zn, both originally white, turned out to be as purple as the fresh bougainvillea showing that the betalain was sequestered. A chemical analyses revealed that the percentage (by mass) of adsorbed betalain was close to 2.7 (alumina) and 3.1% (LDH). The LDH containing Zn was red (in suspension) but became yellow after drying.
In room atmosphere, the stability of the coloured adsorbents varied significantly with time. Only on alumina, the initial purple colour was maintained for more than 20 months (Table 3). The purple colour on Mg–Al–O became dark brown within 20 h. The presence of zinc ions in the mixed oxide (Mg,Zn–Al–O sample) changed the colour, from red in the suspended solid to yellow in separated solid, this colour was maintained after 20 months. The XRD patterns of the white and coloured samples did not show any modification of the structure (Fig. 3). The mixed oxides (Mg–Al–O and Mg,Zn–Al–O) did not recover the layered structure of LDHs precursors when they were put in contact with the containing betalain solutions. It should be concluded that the adsorption was at external surface of the mixed oxides and no intercalation occurred. In the FTIR spectra of γ-Al2O3, Mg–Al–O and Mg,Zn–Al–O with betalain new absorption bands appeared at 1088 and 2942 cm− 1 due to CN and aromatic CH bonds (Nakamoto, 1986) of the chromophore. The strong band due to CO absorption was also observed. If the 27Al MAS NMR spectra of white (original inorganic matrices) and coloured solids (dried matrices after contact with the solution) are compared, Fig. 4, in mixed oxides, the typical signals due to tetrahedral and octahedral aluminium, at 64 and 1 ppm, respectively, were observed, but their relative intensities varied. The spectrum of alumina presented 3 signals, two due to octahedral (AlVI) and tetrahedral (AlIV) aluminium (3.4 and 62 ppm) and a third one at 33 ppm attributed to five-fold aluminium (AlV) (Lippmaa et al., 1986; Lima et al., 2005; Coster and Fripiat, 1993). The intensity ratio (AlIV/AlVI) diminishes in the mixed oxides as the samples adsorbed the chromophore. As well, in γ-alumina, the intensity ratio AlV/AlVI was changed as a consequence of the betalain adsorption. This result indicates that betalain was preferentially adsorbed at the coordinately unsaturated sites (CUS) of aluminium. Unfortunately, the 13C CP/MAS NMR spectrum of betanidin adsorbed on γ-alumina does not provide more details about the interaction between betalain and the adsorbent because the signal/ noise ratio was very low. The main resonances of the molecule are resolved at 175 and 135 ppm, Fig. 5, and they may be attributed to carbonyl groups and aromatic carbons present in the betanidin structure.
Fig. 3. X-ray diffraction patterns of the adsorbents, before and after betalain adsorption. Mg–Al–O, Mg,Zn–Al–O and γ-Al2O3; (a), (b) and (c) respectively.
E. Lima et al. / Applied Clay Science 42 (2009) 478–482
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Fig. 4. 27Al MAS NMR spectra of the adsorbents, before and after betalain adsorption.
The chemical nature of betalain suggests that the molecule was stabilized on the surface through chemisorption. Acid–base pairs present in oxides interact with the carbonyl and ammonium groups of the betanidin. Acid–base pairs present in the channels and cavities of the zeolites are not accessible to betanidin as the entry into the large cavity in zeolite X is 0.74 nm; this value is smaller than the smallest dimension of the molecule (0.99 nm). Thus, betalain is adsorbed at the external surfaces of zeolite X and the chromophore concentration is not enough to produce colour for the human eye. The adsorption sites in γ-alumina, as shown by NMR, were identified as AlV–O pairs. Pentahedral aluminium is considered a strong Lewis acid site (Blumenfeld and Fripiat, 1997). In fact, aluminium atoms interacting with betanidin are unsaturatedly coordinated. This interaction stabilized the chromophore. Due to
Fig. 5. 13C CP/MAS NMR spectrum of betanidin adsorbed on γ-alumina.
this successful immobilization of chromophore, the dye becomes a pigment, Fig. 6. Adsorption of betanidin on mixed oxides does not cause the reconstruction of the layered precursors, i.e., betanidin in solution is not deprotonated to promote anion intercalation. Betanidin, then, interacts also with the metal–oxygen pairs as schematized in Fig. 7. Although adsorption sites on the mixed oxides could be either Mg–O, Zn–O or Al–O, the Al–O pairs are preferentially occupied by the betanidin, as they are easily accessible. Probably the active sites are AlIV–O because of the decreasing of the Al (IV) NMR signal after chromophore adsorption, Fig. 4. Thus, betanidin is selectively adsorbed on the unsaturated coordinated Al–O sites (AlV and AlIV). Although the nature of adsorption cationic sites seems to be similar in alumina or the mixed oxides, the colour and the stability of the colour differ. Such variations may be explained in terms of the basicity of the oxygen atoms. The acidic and basic properties of the solids used are well known from the literature. Basicity increases as follows: MgAlZnO≈ MgAlO ≫ γ-alumina (Lima et al., 2003). The solid with the weakest basicity (γ-alumina) stabilizes and retains the betanidin because the interaction between nitrogen and oxygen is weak. Instead, in mixed oxides, oxygen is attracted strongly to the amine group and induces breaking of the molecule which leads to partial oxidation of the double bond, changing thus the colour. This reaction is typical of many organic compounds in the presence of molecular oxygen. Often, an initiator is required. This initiator could be ozone or radicals emerged from NO2, NO, which are pollutants commonly present in the air of Mexico City (Pine et al., 1988). To confirm that atmospheric air degradates the organic molecules, a sample of Mg–Al–O containing betanidin was stored in vacuum immediately after preparation and the purple colour was maintained as long as the sample was isolated from air. In contact with air, it became dark brown. Surprisingly, the mixed oxide with zinc did not sequester the purple colour of the betanidin, instead a yellow colour was acquired which was stable in air. It seems that betanidin, typically purple, reacted on the surface of Mg,Zn–Al–O to produce another
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Fig. 6. Two ways to use the betanidin chromophore dissolved (as dye) or immobilized/dispersed (as pigment).
chromophore. Another possibility could be that the acidic sites changed the colour of the chromophore. In order to discard or confirm this last hypothesis, we have carried out additional experiments where the pH of an aqueous betanidin solution was varied from 1.7 to 11. The yellow colour was not observed in this wide range of pH. Then, the persistent yellow colour is not due to differences of acidity. As this phenomenon is only observed in the zinc containing materials, another experiment was performed to settle this point. Zinc nitrate was dissolved in the chromophore containing solution. A yellow precipitate was obtained which was identified as a mixture of ZnCO3 and Zn5(OH)6(CO3)2. ZnAlO dispersed in water did not lead to a yellow suspension. Hence it has to be concluded that betalains react with the zinc ions of ZnAlO. Zinc carbonates are not found in the coloured Mg, Zn–Al–O. Most probably, zinc carbonates are present as small particles on the mixed oxide and beyond the X-ray diffraction detection range (particles smaller than 3 nm). 4. Conclusions Betalains may be sequestered in solids of adequate basicity/ acidity. Gamma alumina retains betalain for periods as long as 20 months. The betanidin chromophore behaves as a chemical colour defined by Goethe. The chromophores were adsorbed and stabilized by acid–base pairs of the oxides. In gamma alumina betalain is immobilized by the coordinately unsaturated aluminium sites. These materials are hybrid pigments with antioxidant properties and without toxic chromophores.
Fig. 7. Possible interaction between betanidin with the surface of mixed oxide.
References Baskaralingam, P., Pulikesi, M., Ramamurthi, V., Sivanesan, S., 2007. Modified hectorites and adsorption studies of a reactive dye. Appl. Clay Sci. 37 (1–2), 207–214. Blumenfeld, A.L., Fripiat, J.J., 1997. Acid sites topology in aluminas and zeolites from high-resolution solid-state NMR. Top. Catal. 11 (4), 119–129. Clement, J.S., Mabry, T.J., 1996. Pigment evolution in the caryophyllales: a systematic overview. Bot. Acta 109 (5), 360–367. Coster, D., Fripiat, J.J., 1993. Memory effects in gel–solid transformations: coordinately unsaturated Al sites in nanosized aluminas. Chem. Mater. 5 (9), 1204–1210. Domenech, A., Domenech-Carbo, M.T., Pascual, M.L.V.d.A., 2006. Dehydroindigo: a new piece into the Maya Blue puzzle from the voltammetry of microparticles approach. J. Phys. Chem., B 110 (12), 6027–6039. Fuoco, R., Giannarelli, S., Onor, M., Ceccarini, A., Carli, V., 2005. Optimized cleanup methods of organic extracts for the determination of organic pollutants in biological samples. Microchem. J. 79 (1–2), 69–76. Gettens, R.J.,1962. Maya Blue: an unsolved problem in ancient pigments. Am. Antiq. 27, 557. Giustetto, R., Llabre, F.X., Ricchiardi, G., Bordiga, S., Damin, A., Gobetto, R., Chierotti, M.R., 2005. Maya Blue: a computational and spectroscopic study. J. Phys. Chem., B 109 (41), 19360–19368. Goethe, J.W., 1970. Farbenlehre (Theory of Colours). The MIT Press, London, pp. 202–242 (Original work published in 1810). Gonzalez-San-Jose, M.L., Bron, L., Diez, C., 1990. Evolution of anthocyanins during maturation of Tempranillo grape variety (Vitis vinifera) using polynomial regression models. J. Sci. Food Agric. 51 (3), 337–343. Goyne, K.W., Chorover, J., Kubicki, J.D., Zimmerman, A.R., Brantley, S.L., 2005. Sorption of the antibiotic ofloxacin to mesoporous and nonporous alumina and silica. J. Col. Interface Sci. 283 (1), 160–170. Heuer, S., Richter, S., Metzger, J.W., Wray, V., Nimtz, M., Strack, D., 1994. Betacyanins from bracts of Bougainvillea glabra. Phytochem. 37 (3), 761–767. Hradil, D., Grygar, T., Hradilov, J., Bezdika, P., 2003. Clay and iron oxide pigments in the history of painting. Appl. Clay Sci. 22 (5), 223–236. Ibarra, I.A., Loera, S., Laguna, H., Lima, E., Lara, V., 2005. Irreversible adsorption of an Aztec dye on fractal surfaces. Chem. Mater. 17 (23), 5763–5769. Jansen, M., Letschert, H.P., 2000. Inorganic yellow-red pigments without toxic metals. Nature 404, 980–982. Kanner, J., Jarel, S., Granit, R., 2001. Betalains — a new class of dietary cationized antioxidants. J. Agric. Food Chem. 49 (11), 5178–5185. Lima, E., Martínez-Ortiz, M.J., Fregoso, E., Méndez-Vivar, J., 2007. Capturing natural chromophores on natural and synthetic aluminosilicates. Stud. Surf. Sci. Catal. 170B, 2110–2115. Lima, E., Menorval, L.-C.d., Tichit, D., Lasperas, M., Graffin, P., Fajula, F., 2003. Characterization of the acid–base properties of oxide surfaces by 13C CP/MAS NMR using adsorption of nitromethane. J. Phys. Chem., B 107 (17), 4070–4073. Lima, E., Valente, J., Bosch, P., Lara, V., 2005. Structural evolution of phosphated alumina during sol–gel synthesis. J. Phys. Chem., B 109 (37), 17435–17439. Lippmaa, E., Samoson, A., Magi, M., 1986. High-resolution 27Al NMR of aluminosilicates. J. Am. Chem. Soc. 108 (8), 1730–1735. Lopes, T.J., Quadri, M.G.N., Quadri, M.B., 2007. Recovery of anthocyanins from red cabbage using sandy porous medium enriched with clay. Appl. Clay Sci. 37 (1–2), 97–106. Murata, S., Furukawa, H., Kuroda, K., 2001. Effective inclusion of chlorophyllous pigments into mesoporous silica modified with diols. Chem. Mater. 13 (8), 2722–2729. Nakamoto, K., 1986. Infrared and Raman Spectra Inorganic and Coordination Compounds. John Wiley & Sons, New York. Pine, S.H., Hendrickson, J.B., Hammond, D.J., 1988. Organic Chemistry. McGraw-Hill, New York, p. 98. Simoncic, P., Armbruster, T., Pattison, P., 2004. Cationic thionin blue in the channels of zeolite mordenite: a single-crystal X-ray study. J. Phys. Chem., B 108 (45),17352–17360. Steglich, W., Strack, D., 1991. Betalains the Alkaloids, Chemistry and Pharmacology. Academic Press, London, pp. 1–62. Strack, D., Bokern, M., Marxen, N., Wray, V., 1988. Feruloylbetanin from petals of Lampranthus and feruloylamaranthin from cell suspension cultures of Chenopodium rubrum. Phytochem. 27 (11), 3529–3531.
Contents lists available at ScienceDirect
Applied Clay Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l a y
Non-toxic hybrid pigments: Sequestering betanidin chromophores on inorganic matrices Enrique Lima a,⁎, Pedro Bosch b, Sandra Loera a, Ilich A. Ibarra a, Humberto Laguna a, Victor Lara a a b
Universidad Autónoma Metropolitana, Iztapalapa, Av. San Rafael Atlixco No. 186 Col. Vicentina, 09340 México D.F., Mexico Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, 04510 México D.F., Mexico
A R T I C L E
I N F O
Article history: Received 26 February 2008 Received in revised form 4 June 2008 Accepted 14 June 2008 Available online 22 June 2008 Keywords: Alumina Dye Chromophores Adsorption Pigment Betanidin
A B S T R A C T Betalain was extracted from bougainvillea flower. Alumina, layered double hydroxides (LDHs) and zeolites were tested as adsorbents of the chromophore. Gamma alumina stabilized betalains over a long period. The adsorption sites which stabilized the chromophores were elucidated by nuclear magnetic resonance. Although the oxides obtained by calcination of LDHs retain betalains, they were oxidized within 20 h or react with Zn2+ of the calcined LDH. No betalain was adsorbed in zeolite X because of the too small pores. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In flowers, colour can be as ephemeral as perfume. Both are due to chemical groups which in contact with air degrade and fade out. Goethe (1970) defined chemical colours as “the colours which we can produce, and more or less fix, in certain bodies; which we can render more intense, which we can again take away and communicate to other bodies, and to which, therefore, we ascribe a certain permanency: duration is their prevailing characteristic”. Nowadays colours are known to be the result of light absorption by chemical groups. The main chromophore molecules, i.e. the “chemical colours” of Goethe, present in fruits and flowers are, on the one hand, the anthocyanins (cherries, Atropa belladonna…) and on the other the betalains (bougainvillea, flower of Christmas cactus, beet…) (Gonzalez-San-Jose et al., 1990; Steglich and Strack, 1991). Betalains, a group of reddish and yellowish chromophores, are important markers and have never been found jointly with anthocyanins in the same plant (Clement and Mabry, 1996). Still, betalains not only provide colour, they are a family of compounds which play a major role in the high antioxidant activity levels observed in red fruits and vegetables
⁎ Corresponding author. Tel.: +52 5 55804 4667; fax: +52 5 55804 4666. E-mail address: [email protected] (E. Lima). 0169-1317/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2008.06.005
(Kanner et al., 2001). Furthermore, betalains may be used for food colouring and their antioxidant and radical scavenging properties for protection against certain oxidative stress-related disorders. Today, colourants free of toxic elements, particularly nature colourants are leaving synthetic colourants behind as they are safe and eco-friendly in nature (Ibarra et al., 2005; Simoncic et al., 2004; Jansen and Letschert, 2000; Hradil et al., 2003). Betalain isolation and stabilization is a hard task. Betalains are commonly extracted from plants in aqueous or water–methanol solutions and purified by chromatography. A major difficulty is that purified betalains decompose easily. Hence, their stabilization on supports is recommended. Usually organic materials are proposed, dyes are, then, obtained. Stabilization of betalains as well as other organic molecules, should also be possible by immobilization in inorganic matrices (Ibarra et al., 2005; Lima et al., 2007; Murata et al., 2001; Lopes et al., 2007; Baskaralingam et al., 2007). A well known chromophore–inorganic matrix system is the Maya blue pigment where the indigo is incorporated in the pores of palygorskite clay (Gettens, 1962; Giustetto et al., 2005; Domenech et al., 2006). Inspired by the Maya technique and the beautiful colours of the Mexican bougainvilleas (Heuer et al., 1994), we have tested three materials well known as supports in catalysis: layered double hydroxides (LDHs), gamma alumina and zeolites. Alumina is a spinel with octahedrally and tetrahedrally coordinated aluminium ions in an oxygen lattice, which adsorbs organic species such as carboxylic acids or polychlorobiphenyls (Goyne et al., 2005; Fuoco et al., 2005).
E. Lima et al. / Applied Clay Science 42 (2009) 478–482
479
Table 1 Chemical composition of the adsorbents Inorganic matrix
Label
Chemical composition
Sodium X zeolite Protonated X zeolite Mixed oxide Mixed oxide γ-Alumina
NaX HX Mg–Al–O Mg,Zn–Al–O γ-Al2O3
Na+86(SiO2)106(AlO2)86·xH2O H+86(SiO2)106(AlO2)86·xH2O Mg0.744Al0.256[OH]2(CO3)0.128·0.850H2O Mg0.614Zn0.093Al0.293[OH]2(CO3)0.146·0.731H2O Al2O3
The specific surface area of γ-Al2O3 (250–300 m2/g) is mainly due to macropores. γ-Alumina is prepared at 500 °C and is stable up to 850 °C, then transforms to δ-Al2O3. Instead, the two other chosen oxides, although also constituted by aluminium, present very different features. 2. Experimental section
Fig. 2. Structure of the betanidin chromophore, betalain which is responsible of the purple colour. R can be 9 different groups.
2.1. Materials 2.1.1. Alumina (γ-Al2O3) Alumina was prepared by the sol–gel technique using aluminium tri-sec-butoxide as aluminium source, ethanol as solvent and sulfuric acid as catalyst in the hydrolysis step. The gel was aged for 30 days, dried at 80 °C and calcined at 550 °C. 2.1.2. Zeolite Zeolite NaX was supplied by Union Carbide. This zeolite was used in sodium (NaX) or protonated (HX) form. The protonated zeolite was prepared by exchanging NaX zeolite in 1 N NH4OH solution. Then, the ammonium ions were decomposed at 300 °C. 2.1.3. LDHs The Mg–Al LDH was prepared by coprecipitation of magnesium and aluminium nitrate at pH 9–10 in the presence of carbonate. The precipitate was filtered, washed with deionized water and dried at 100 °C for 24 h. (HTMgAl). Another LDH containing Mg, Al and Zn was prepared by coprecipitation. The ratio divalent on trivalent metal was maintained close to 3. (HTMgZnAl). Mixed oxides were obtained by calcining HTMgAl and HTMgZnAl, at 600 °C for 8 h. These samples were labeled Mg–Al–O and Mg–Zn– Al–O, respectively.
The chemical compositions of the adsorbents are presented in Table 1. 2.2. Extraction of the chromophore from the flowers and their adsorption 1 g of bougainvillea flowers, Fig. 1, was slightly ground and then added to 40 ml of water. The mixture was agitated for 20 min. A transparent and coloured solution was obtained containing the betalain (Fig. 2). A small amount of solid was easily removed by centrifugation. 10 ml of an aqueous solution containing the betalain was put in contact with 100 mg of the adsorbent for 30 min. The solid was separated by centrifugation, washed and dried at room temperature. 2.3. Characterization 2.3.1. X-ray diffraction X-ray diffraction patterns were obtained with a Siemens D500 diffractometer (Kα radiation). Compounds were identified conventionally with the JCPDS files (Joint Committee of Powder Diffraction Standards). 2.3.2. Ultraviolet–Visible spectroscopy Spectra UV–Vis of the samples, as pellets, were acquired in a Perkin Elmer Lambda 40 UV–Vis spectrometer in the spectral window from 800 to 300 nm. 2.3.3. Infrared spectroscopy The attenuated total reflectance Fourier infrared (ATR/FTIR) spectra were obtained in a GX Perkin Elmer instrument at 2 cm− 1 resolution.
Table 2 Average pore size and specific surface area of the adsorbents
Fig. 1. Fresh bougainvillea flowers used in this report.
Sample
Specific surface area (m2/g)⁎
Average pore diameter (Å)⁎⁎
NaX HX Mg–Al–O Mg,Zn–Al–O γ-Al2O3
388 409 187 201 320
9 11 41 49 98
⁎Determined from nitrogen isotherms (at 77 K) and applying the BET equation. ⁎⁎Determined from nitrogen isotherms data and BJH method. All samples were outgassed at 400 °C before nitrogen adsorption.
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Table 3 Colour of the adsorbents after betalain adsorption Solid
γ-Al2O3 Mg–Al–O Mg,Zn–Al–O
Colour Solid suspended in the betalain solution
Solid separated from the solution and dried
Solid after 20 months
Purple Purple Red
Purple Purple Yellow
Purple Dark brown Yellow
2.3.4. Solid state nuclear magnetic resonance Nuclear magnetic resonance (NMR) measurements were performed in a Bruker ASX300 Spectrometer. The spectrometer was operated at a resonance frequency of 78.2 MHz for 27Al MAS NMR spectroscopy. Spectra were acquired using short single excitation pulses (π/12) with repetition times of 500 ms. Samples were spun at 10 kHz and the chemical shifts were referenced to 1 N aqueous solution of AlCl3. Spectra of 13C CP MAS NMR were recorded at 75.4 MHz with a contact time of 1 ms, a 5 kHz spinning rate, and 90° pulses of 4 μs. 51,500 scans were accumulated. Chemical shifts were referenced to solid CH2 adamantane shift at 38.2 ppm relative to TMS. 3. Results and discussion A small amount (1 g) of fresh bougainvillea flowers, Fig. 1, was enough to obtain a transparent aqueous coloured solution (0.250 l) containing the betalain responsible of the purple colour (Heuer et al., 1994; Strack et al., 1988), Fig. 2. After drying, the zeolites did not show any colour, they were white, and therefore they were discarded. The chromophore was not retained due to the small size of the pores (Table 2). Alumina and LDH without Zn, both originally white, turned out to be as purple as the fresh bougainvillea showing that the betalain was sequestered. A chemical analyses revealed that the percentage (by mass) of adsorbed betalain was close to 2.7 (alumina) and 3.1% (LDH). The LDH containing Zn was red (in suspension) but became yellow after drying.
In room atmosphere, the stability of the coloured adsorbents varied significantly with time. Only on alumina, the initial purple colour was maintained for more than 20 months (Table 3). The purple colour on Mg–Al–O became dark brown within 20 h. The presence of zinc ions in the mixed oxide (Mg,Zn–Al–O sample) changed the colour, from red in the suspended solid to yellow in separated solid, this colour was maintained after 20 months. The XRD patterns of the white and coloured samples did not show any modification of the structure (Fig. 3). The mixed oxides (Mg–Al–O and Mg,Zn–Al–O) did not recover the layered structure of LDHs precursors when they were put in contact with the containing betalain solutions. It should be concluded that the adsorption was at external surface of the mixed oxides and no intercalation occurred. In the FTIR spectra of γ-Al2O3, Mg–Al–O and Mg,Zn–Al–O with betalain new absorption bands appeared at 1088 and 2942 cm− 1 due to CN and aromatic CH bonds (Nakamoto, 1986) of the chromophore. The strong band due to CO absorption was also observed. If the 27Al MAS NMR spectra of white (original inorganic matrices) and coloured solids (dried matrices after contact with the solution) are compared, Fig. 4, in mixed oxides, the typical signals due to tetrahedral and octahedral aluminium, at 64 and 1 ppm, respectively, were observed, but their relative intensities varied. The spectrum of alumina presented 3 signals, two due to octahedral (AlVI) and tetrahedral (AlIV) aluminium (3.4 and 62 ppm) and a third one at 33 ppm attributed to five-fold aluminium (AlV) (Lippmaa et al., 1986; Lima et al., 2005; Coster and Fripiat, 1993). The intensity ratio (AlIV/AlVI) diminishes in the mixed oxides as the samples adsorbed the chromophore. As well, in γ-alumina, the intensity ratio AlV/AlVI was changed as a consequence of the betalain adsorption. This result indicates that betalain was preferentially adsorbed at the coordinately unsaturated sites (CUS) of aluminium. Unfortunately, the 13C CP/MAS NMR spectrum of betanidin adsorbed on γ-alumina does not provide more details about the interaction between betalain and the adsorbent because the signal/ noise ratio was very low. The main resonances of the molecule are resolved at 175 and 135 ppm, Fig. 5, and they may be attributed to carbonyl groups and aromatic carbons present in the betanidin structure.
Fig. 3. X-ray diffraction patterns of the adsorbents, before and after betalain adsorption. Mg–Al–O, Mg,Zn–Al–O and γ-Al2O3; (a), (b) and (c) respectively.
E. Lima et al. / Applied Clay Science 42 (2009) 478–482
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Fig. 4. 27Al MAS NMR spectra of the adsorbents, before and after betalain adsorption.
The chemical nature of betalain suggests that the molecule was stabilized on the surface through chemisorption. Acid–base pairs present in oxides interact with the carbonyl and ammonium groups of the betanidin. Acid–base pairs present in the channels and cavities of the zeolites are not accessible to betanidin as the entry into the large cavity in zeolite X is 0.74 nm; this value is smaller than the smallest dimension of the molecule (0.99 nm). Thus, betalain is adsorbed at the external surfaces of zeolite X and the chromophore concentration is not enough to produce colour for the human eye. The adsorption sites in γ-alumina, as shown by NMR, were identified as AlV–O pairs. Pentahedral aluminium is considered a strong Lewis acid site (Blumenfeld and Fripiat, 1997). In fact, aluminium atoms interacting with betanidin are unsaturatedly coordinated. This interaction stabilized the chromophore. Due to
Fig. 5. 13C CP/MAS NMR spectrum of betanidin adsorbed on γ-alumina.
this successful immobilization of chromophore, the dye becomes a pigment, Fig. 6. Adsorption of betanidin on mixed oxides does not cause the reconstruction of the layered precursors, i.e., betanidin in solution is not deprotonated to promote anion intercalation. Betanidin, then, interacts also with the metal–oxygen pairs as schematized in Fig. 7. Although adsorption sites on the mixed oxides could be either Mg–O, Zn–O or Al–O, the Al–O pairs are preferentially occupied by the betanidin, as they are easily accessible. Probably the active sites are AlIV–O because of the decreasing of the Al (IV) NMR signal after chromophore adsorption, Fig. 4. Thus, betanidin is selectively adsorbed on the unsaturated coordinated Al–O sites (AlV and AlIV). Although the nature of adsorption cationic sites seems to be similar in alumina or the mixed oxides, the colour and the stability of the colour differ. Such variations may be explained in terms of the basicity of the oxygen atoms. The acidic and basic properties of the solids used are well known from the literature. Basicity increases as follows: MgAlZnO≈ MgAlO ≫ γ-alumina (Lima et al., 2003). The solid with the weakest basicity (γ-alumina) stabilizes and retains the betanidin because the interaction between nitrogen and oxygen is weak. Instead, in mixed oxides, oxygen is attracted strongly to the amine group and induces breaking of the molecule which leads to partial oxidation of the double bond, changing thus the colour. This reaction is typical of many organic compounds in the presence of molecular oxygen. Often, an initiator is required. This initiator could be ozone or radicals emerged from NO2, NO, which are pollutants commonly present in the air of Mexico City (Pine et al., 1988). To confirm that atmospheric air degradates the organic molecules, a sample of Mg–Al–O containing betanidin was stored in vacuum immediately after preparation and the purple colour was maintained as long as the sample was isolated from air. In contact with air, it became dark brown. Surprisingly, the mixed oxide with zinc did not sequester the purple colour of the betanidin, instead a yellow colour was acquired which was stable in air. It seems that betanidin, typically purple, reacted on the surface of Mg,Zn–Al–O to produce another
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Fig. 6. Two ways to use the betanidin chromophore dissolved (as dye) or immobilized/dispersed (as pigment).
chromophore. Another possibility could be that the acidic sites changed the colour of the chromophore. In order to discard or confirm this last hypothesis, we have carried out additional experiments where the pH of an aqueous betanidin solution was varied from 1.7 to 11. The yellow colour was not observed in this wide range of pH. Then, the persistent yellow colour is not due to differences of acidity. As this phenomenon is only observed in the zinc containing materials, another experiment was performed to settle this point. Zinc nitrate was dissolved in the chromophore containing solution. A yellow precipitate was obtained which was identified as a mixture of ZnCO3 and Zn5(OH)6(CO3)2. ZnAlO dispersed in water did not lead to a yellow suspension. Hence it has to be concluded that betalains react with the zinc ions of ZnAlO. Zinc carbonates are not found in the coloured Mg, Zn–Al–O. Most probably, zinc carbonates are present as small particles on the mixed oxide and beyond the X-ray diffraction detection range (particles smaller than 3 nm). 4. Conclusions Betalains may be sequestered in solids of adequate basicity/ acidity. Gamma alumina retains betalain for periods as long as 20 months. The betanidin chromophore behaves as a chemical colour defined by Goethe. The chromophores were adsorbed and stabilized by acid–base pairs of the oxides. In gamma alumina betalain is immobilized by the coordinately unsaturated aluminium sites. These materials are hybrid pigments with antioxidant properties and without toxic chromophores.
Fig. 7. Possible interaction between betanidin with the surface of mixed oxide.
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