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Thermal analysis and structural characterization of chitinous exoskeleton from two marine invertebrates
Thermochimica Acta 610 (2015) 16–22
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Thermochimica Acta journal homepage: www.elsevier.com/locate/tca
Thermal analysis and structural characterization of chitinous exoskeleton from two marine invertebrates B.A. Juárez-de la Rosa a,b, * , J. May-Crespo b , P. Quintana-Owen b , W.S. Gónzalez-Gómez b , J.M. Yañez-Limón c, J.J. Alvarado-Gil b, ** a
Laboratory of Natural Polymers, CIAD – Coordinación Guaymas, Carretera al Varadero Nacional km. 6.6, Col. Las Playitas, 85480 Guaymas, Sonora, Mexico Applied Physics Department, CINVESTAV-IPN Unidad Mérida, Carretera antigua a Progreso, km. 6. Apdo, Postal 73, Cordemex, 97310 Mérida, Yucatan, Mexico c Materials and Engineering Science, CINVESTAV-IPN, Unidad Querétaro, Libramiento Norponiente No. 2000, Fracc. Real de Juriquilla, 76230 Santiago de Querétaro, Querétaro, Mexico b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 August 2014 Received in revised form 10 January 2015 Accepted 7 April 2015 Available online 17 April 2015
Thermomechanical and structural properties of two marine species exoskeletons, Antipathes caribbeana (black coral) and Limulus polyphemus (xiphosure), were studied using dynamic mechanical thermal analysis (DMTA), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). DMTA curves indicate the viscoelastic behavior and glass transition around 255 C, black coral presented a second transition (175 C) associated to the acetamide group attached to the a-chitin chain. DSC measurements showed a endothermic peak around 100 C, with enthalpies of 4.02 and 118.04 J/g, indicating strong differences between exoskeletons respect to their water holding capacity and strength water–polymer interaction. A comparative analysis involving DSC and X-ray diffraction showed that lower values DH in xiphosure correspond to a material with a higher crystallinity (30), in contrast black coral exhibits higher values DH and a lower crystallinity (19). FTIR confirmed a-chitin based structure, at higher temperature diminishes the amide bands and a new one appears, related to C–N groups. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Black coral Horseshoe crab Thermal properties DMTA TGA XRD
1. Introduction Chitin is a linear polysaccharide constituted of b-(1 ! 4)-2deoxy-2-acetamido-D-glucopyranose repeating units [1]. It is similar to cellulose in terms of its structure and abundance in nature. In its native state, this biopolymer is associated with proteins, pigments, lipids, and inorganic substances, and is chiefly found as a fibrillar semicrystalline material [2]. Chitin is an important component of the exoskeleton organic matrix in many invertebrate animals (insects, crustaceans, mollusks, corals, annelids, ectoprocts, brachiopods), fungi and bacteria [3–6]. The analysis of chitin has been done by several research groups [7,8], although there are numerous open problems that remain to be
* Corresponding author at: Laboratory of Natural Polymers, CIAD – Coordinación Guaymas, Carretera al Varadero Nacional km. 6.6, Col. Las Playitas, 85480 Guaymas, Sonora, Mexico. Tel.: +52 6222252829. ** Corresponding author. E-mail addresses: [email protected], [email protected] (B.A. Juárez-de la Rosa), [email protected] (J.J. Alvarado-Gil). http://dx.doi.org/10.1016/j.tca.2015.04.015 0040-6031/ ã 2015 Elsevier B.V. All rights reserved.
investigated, in particular, chitin is only a fraction of the exoskeleton, therefore the properties of those materials are strongly influenced by the remaining components in which proteins and organic components, among many others, are present. In this work the exoskeletons in two species of marine invertebrate, Antipathes caribbeana (black coral) and Limulus polyphemus (horseshoe crab) (Fig. 1) are studied. Both species have exoskeletons in which the basic component is the polysaccharide a-chitin [6,9–11], forming the structures which perform all the functions required for survival in their respective biological environments. Their main function is to stabilize the body, but it also serves as a selective chemical barrier between the organism and its environment [12]. Antipatharians are cnidarians that form coral colonies, which frequently exhibit an arborescent appearance characterized by a spiny, branched skeletal axis based on chitin (Fig. 1a). In black coral, the skeleton consists of growth rings formed by chitin layers (micro-lamellae) segregated by polyps across the epidermis [10–11].
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Fig. 1. Chitinous exoskeletons of marine invertebrates (a) Antipathes caribbeana (black coral) and (b) Limulus polyphemus (xiphosure). AFM micrographs of the skeleton surface (image scan size: 50 mm).
Horseshoe crabs are marine chelicerate arthropods of the order Xiphosura which periodically shed their carapace, or molt, to accommodate body growth (Fig. 1b). To accommodate the larger body size, the new skeleton is initially flexible but then hardens and takes their characteristic color depending on its component tans [13–15]. Black coral A. caribbeana has 12 wt% of the dry weight of chitin [6,10], similar data has been obtained by other authors which reported 10 and 14 wt% for Antipathes salix and Antipathes fiordensis, respectively [8,10]. On the other hand for L. polyphemus, a higher chitin content has been reported, being around 24–28 wt% of the dry weight [5,9,16]. Biopolymers, particularly chitin, are susceptible to structural changes caused by temperature treatments or mechanical influence [17]. Dynamic mechanical thermal analysis (DMTA) is a powerful technique for characterizing polymer viscoelastic properties, because it measures the Young’s modulus (stiffness) and damping (energy-dissipation) properties of materials as they deform under dynamic stress [18]. These measurements provide quantitative information about materials performance [19]. DMTA and conformational transformation comparative study of horseshoe crab and black coral represent a biomimetic model approach, vital to understanding chitin’s crystalline structure, molecular fibrillar arrangement, and thermal behavior. Additional data and analysis on the chitin degradation process will also improve our understanding of its structural role in the skeletons of these species. The present objective of this study was to analyze the exoskeleton of A. caribbeana (black coral) and L. polyphemus (horseshoe crab or xiphosure) using DMTA and thermal characterization. Additional studies were done using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) in order to quantify the heat transfer and weight changes. Also structural effects were monitored during the applied thermal treatments with X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy.
2. Experimental procedure 2.1. Sample preparation L. polyphemus (xiphosure) specimens were collected from the coastal area of the Peninsula of Yucatán, México. A. caribbeana (black coral) colony is native from the deep reefs of Cozumel Island, Quintana Roo México (western Caribbean). The analyzed samples correspond to individual adults, after being collected were washed with fresh water and let dry at room temperature during one week and finally were kept in a desiccator over eight months, before the thermal characterization was carried out. For DMTA measurements flat samples of dried skeleton were cut from dorsal xiphosure carapace and the ones of black coral skeleton were cut from the branches in the longitudinal direction with respect to the axial direction. The specimen dimensions (length, width, thickness) were 16.003 1.682 mm 7.424 0.877 mm 0.793 0.098 mm, and 15.287 0.128 mm 6.775 0.288 mm 0.766 0.089 mm, for black coral and xiphosure, respectively. The effect of the temperature on the structure of the materials evolution was also studied in grounded xiphosure and black coral samples. Measurements at room temperature were followed by thermal treatments during 1 h in an electric furnace at several temperatures: 110 C, 210 C, 270 C and 310 C, afterwards, they were cooled at room temperature. XRD and FT-IR analysis were made immediately after each thermal treatment. 2.2. Thermal analysis DMTA was carried out using a TA Instruments Q-Series DMA (Q800). All the samples were measured in a film clamp intension mode over the temperature ramp of 5–300 C, at a heating rate of 5 C/min and at a frequency of 1 Hz. Measurements of DSC were carried out using DSC 822 Mettler Toledo equipment. Accurately weighed material was placed inside
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an aluminum closed pans of 40 mL capacity and hermetically sealed. Argon atmosphere with a 50 cc/min flux, and a heating rate of 10 C/min was applied. A Mettler Toledo TGA/SDTG 851 apparatus was used to assess the weight loss with heat treatment. Accurately weighed material was placed inside an aluminum cup and hermetically sealed. Also a heating rate of 10 C/min was used in argon atmosphere from 20 C up to 500 C. 2.3. Structural characterization The samples were characterized by X-ray diffraction in Bragg– Brentano geometry using a Siemens D5000 diffractometer with a monochromatic CuKa radiation (l = 1.5418 Å), operating at 34 kV, 25 mA. The powder diffractogram patterns were registered in the range of 5 < 2u < 70 in a step scan mode of 0.02 (2u ) with a counting time of 12 s per step. IR spectra in the transmittance mode were recorded on a Thermo Nicolet Nexus 670 FTIR spectrometer equipped with a DTGS KBr detector in the middle infrared (4000–400 cm1). Powdered samples of xiphosure and black coral skeleton were mixed by grinding the dry blended powders with KBr with a ratio of 5:195 mg, with an agate mortar until homogenization. This mixture was inserted into the sample compartment at the Smart Multi-Bounce HATR of the spectrometer and continuously purged with dry air. The number of scans for each spectrum was 64, and the spectral resolution was 4 cm1. 3. Results and discussion 3.1. Dynamic mechanical thermal analysis (DMTA) The temperature-dependent storage moduli (E) and tan d of the xiphosure and black coral skeleton chitins were recorded within 25–300 C temperature range (Fig. 2). The samples exhibit the typical behavior of a viscoelastic material. Initially the storage modulus of the black coral is stronger and falls about 6000– 3000 MPa before 200 C. Additionally, tan d reaches a maximum around 175 C. This could be associated to the acetamide group attached to the C2 position in a-chitin [8]. On the other hand, this indicates that xiphosure skeleton behaves as a tougher and stable material in the same temperature range (below 200 C).
Above 200 C, the behavior response shows a strong change for both materials, the storage modulus of black coral falls from 3000 MPa up to 100 MPa from 200 C to 300 C and its value of tan d increases from 0.12 up to 0.24, respectively. Whereas in the same temperature range (200–300 C), the storage modulus for xiphosure falls from 4000 to 50 MPa and the value of tan d grows from 0.09 to above 0.25, respectively. This behavior is the clear indication of glass transitions, occurring in the interval 250–260 C, for both black coral and xiphosure skeletons. It is worth to mention that the xiphosure DMTA measurements exhibit steeper changes in the loss moduli, but also display more maxima and minima in tan d, and it also seems to show that in some way the transitions for black coral slightly shifts to a lower temperatures. Coral showed a distinctive maximum at 256 C and xiphosure at 265 C. These results are consistent with the values reported for pure commercial and synthesized a-chitin by Kim et al. [8] in which similar results for the storage modulus and tan d curves are reported. These authors found the glass transition around 236 C, even though the transitions found in our work is higher, since the analysis was made on the exoskeleton which contains chitin, proteins, and others components that can increase the transition temperature. Therefore, the multiple maxima in tan d could be due to the presence of additional non-chitinous compounds. Those effects will be described latter at the DCS and FTIR sections. In the region under 200 C, the polymer is stiff/frozen and therefore a high storage modulus E is observed, the chains are nearly frozen in fixed positions because the available energy is not sufficient for inducing translational and rotational motions of the polymer segments [20]. Above 200 C, the polymer obtains sufficient thermal energy that enables to move its chains more freely, and therefore the storage modules decreases considerably. 3.2. Differential scanning calorimetry (DSC) In the DSC analysis (Fig. 3), both the black coral and xiphosure exhibited several endothermic peaks. The DSC thermograms behavior of both species were similar; however the curve intensity is much smaller for xiphosure than for black coral, also the structure of both species decomposed completely at 350 C, near the temperature reported for other chitinous structures [21–23]. It is important to notice that the first thermal event shows strong
Fig. 2. DMTA results generated on exoskeleton samples of two marine invertebrates: (a) storage moduli as a function of temperature and (b) tan d curves versus temperature.
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which correspond to the observed behavior of DSC measurements (peaks at 262 C for coral and 275 C for xiphosure). The highest temperature peaks at 303 C for coral and 329 C, for xiphosure, were due to the decomposition of the polysaccharide [6,21,24,25]. The final degradation enthalpies of 12.57 J/g and 0.50 J/g for a-chitin suggest that the decomposition efficiency might be different in the mechanism of cleavage of acetyl groups. These results are in accordance with the XRD results since the lower value of DH for xiphosure, which as will be shown later, exhibits a higher crystallinity, on the other hand a higher DH for black coral will reflect a higher free amine groups observed by FTIR analysis [21]. 3.3. Thermogravimetric analysis (TGA)
Fig. 3. DSC thermograms of black coral and xiphosure exoskeleton. The data for xiphosure curve is multiplied by 30 for comparison purposes.
peaks centered at 101 C and 110 C, for xiphosure and black coral respectively, and can be ascribed to the loss of water (dehydration). Chitin is normally a highly hydrophilic polysaccharide; indeed, in a solid state, it depends on the primary and supramolecular structures which can be easily hydrated and dehydrated [6], therefore the endotherm related to evaporation of water is expected to reflect physical and molecular changes during heating especially with hydrophilic hydroxyl groups [21]. Their associated enthalpies were calculated and gave 4.02 J/g and 118.04 J/g for xiphosure and black coral, respectively, indicating strong differences between the black coral and xiphosure macromolecules in relation to their water holding capacity and strength water– polymer interaction [21]. When heating the samples, two endothermic peaks with a reduced area and smaller enthalpies values appear, at 220 C and 262 C (0.75 J/g and 1.06 J/g, respectively) for black coral and 241 C and 275 C for xiphosure (0.08 J/g and 0.18 J/g, respectively). These peaks can be related to the complete removal of hydroxyl groups bound to the polysaccharide rings. These results allows us to identify the transitions observed in the DMTA measurements for the tan d at 256 C for coral and 265 C for xiphosure (Fig. 2b),
TGA and DTG thermograms show mainly two decomposition steps for both species, although the weight loss, in response to the increase of the temperature, was higher in the xiphosure exoskeleton (Fig. 4). The first stage occurs in the range 40–150 C, and is attributed to free water evaporation with a weight loss of 4–5% for both species. In the second stage, relevant thermal transformations occurred within the range 250–370 C, with a higher weight loss for xiphosure (26%) than black coral (13%). This is related to the degradation of the polysaccharide structure of the molecule, in which aliphatic compounds (CH2, CH3, functional groups) were being separated from the chitin structural ring and additionally to the degradation of amides I and II (C¼O, NH) and phosphodiesters groups (COC, C O P, and P O P), (CO, P¼O, and PO2–). Another step was observed in xiphosure between 446 and 550 C which can be related to the complete denaturation of chitin [6,21–23,26]. 3.4. X-ray diffraction (XRD) Both the black coral and xiphosure exoskeleton were analyzed with XRD, and the results compared to standard commercial a-chitin (Fig. 5). Both species exhibited crystalline reflections characteristic of chitin, with peaks mainly at 9 and 19 (2u). However, under thermal treatment, the peaks were more intense in xiphosure than in coral; in particular the intensity of 19 (2u) peak increases, indicating that xiphosure has a more organized
Fig. 4. TGA thermograms and DTG curves of the exoskeleton of (a) black coral and (b) xiphosure. The highest loss weight (13–26%) and the relevant thermal transformations occur at 300 C on black coral exoskeleton and at 330 C on xiphosure exoskeleton.
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Fig. 5. X-ray diffractograms of the marine invertebrate exoskeleton with different thermal treatments (US: untreated sample, 110, 210, 270, and 310 C) respect to the standard a-chitin (inset). (a) Black coral and (b) xiphosure.
structure. XRD patterns became slightly sharper and more intense around 210 C in the coral and at 270 C in the xiphosure. Most of the peaks began to decrease at higher temperatures, and were nearly suppressed at 310 C, at which the polysaccharide crystalline structure is systematically destroyed (Fig. 5a and b). Crystallite size was analyzed for xiphosure exoskeleton with the Scherrer equation [27] obtaining a crystallite size of 3.27 nm at 25 C, very near to the reported previously for black coral extracted a-chitin of 3.34 nm [28]. Also the unit cell parameters were determined considering the orthorhombic P212121 symmetry of chitin using a Gaussian deconvolution of the peaks for planes (0 2 0), (11 0) and (2 11) [28,29]. When compared to standard commercial a-chitin (Table 1), crystallographic parameters showed the xiphosure skeleton to have an a-crystalline structure similar to previous reports for black coral chitin [6,28]. Exoskeleton crystallinity index (CI) was analyzed using the 19 (2u) peak, which corresponds to the hkl (11 0) plane. A method was used that previously developed to calculate the CI of cellulose, as follows [29,30], CI% = [Ic/(Ic + Ia)] 100; where Ic represents the crystalline region at 19 (2u ) corresponding to the hkl (11 0) plane; and Ia is the amorphous region, obtained from the XRD patterns.
Table 1 Crystallographic parameters of xiphosure exoskeleton in comparison to black coral chitina and the standard commercial a-chitin. Species Xiphosure Limulus polyphemus
Black coral chitina Antipathes caribbeana
standard a-chitin
a
2u ( ) (hkl)
d-Spacing (Å) Unit cell parameters (Å)
9.60 19.60 39.25
(020) 9.20 (110) 4.52 (211) 2.29
a = 4.67 b = 18.43 c = 17.13
9.4 12.5 19.6 26.3
(020) (021) (110) (013)
9.51 7.08 4.53 3.38
a = 4.58 b = 19.65 c = 10.56
9.28 (020) 9.53 12.66 (021) 6.99 19.3 (110) 4.59
a = 4.74 b = 19.06 c = 10.29
From Juárez-de la Rosa et al. [6].
CI values were 30% for L. polyphemus and 19% for A. caribbeana exoskeletons (Table 2). Morphologically, the observed XRD patterns suggest that higher temperature (210–270 C) may favor crystalline re-arrangement of the chitin of the exoskeleton (Fig. 5). Due to thermal treatment, this effect is common in sclerotized skeleton in which thermal treatments with specific temperature ranges are used to increase crystallinity [28]. The strong differences in the structure and crystallinity between the coral and xiphosure are consistent with our DSC measurements. Lower values in DH in xiphosure correspond to material with a higher crystallinity. In contrast coral exhibits higher values in DH and a lower crystallinity [21]. 3.5. Infrared analysis (FTIR) Both the xiphosure and black coral samples, exhibited FT-IR spectra distribution pattern similar to that of the standard commercial a-chitin (Fig. 6). The two species show bands at 3448 cm1 related to OH stretching mode of hydroxyl groups and 3290 cm1 associated to NH stretching vibration mode of the amide. The bands at 2886 and 2935 cm1, are correlated to aliphatic compounds, CH2 and CH3 asymmetric and symmetric stretching vibration modes. In the wavelength range from 1600 cm1 up to 1000 cm1, there are bands associated with amides I and II, the saccharide ring, phosphodiester groups and proteinaceous components [4,6,30,31]. These bands were more clearly defined when the temperature increases, intensity and sharpness were highest at 210 C for black coral and 270 C for
Table 2 Crystalline index as a function of thermal treatment in the exoskeleton of two marine invertebrates. Temperature (o C)
Untreated sample 110 210 270 310
Crystalline index (%) Black coral A. caribbeana
Xiphosure L. polyphemus
19.6 20 14.2 16.3 5.4
30.2 26.2 25.5 29.2 9.7
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Fig. 6. FTIR spectra of the marine invertebrate exoskeleton with different thermal treatments (US: untreated sample, 110, 210, 270, and 310 C) respect to the standard a-chitin (inset). (a) Black coral and (b) xiphosure.
xiphosure. The bands at 1646 cm1 and 1666 cm1 for amide I ( CN vibration and C¼O stretching modes), confirms the a-chitin structure [21]. The band at 1557 cm1 corresponding to amide II (NH stretch mode) begins to disappear above 210 C. The main changes were observed in the bands hydroxyl and amides groups after 210 C for black coral and 270 C for xiphosure, respectively. At 310 C, the presence of a new band at 2225 cm1, associated to CN vibrations mode, indicates that deacetylation is smaller in the xiphosure than in the black coral, for this temperature. Therefore, another processes began above 310 C, including degradation, depolymerization, and denaturation of the exoskeleton. The decreasing at high temperature of the FT-IR bands for the amides, are in accordance with the XRD and DSC measurements. In coral all the amide groups are destroyed above 210 C, in this case higher enthalpies and low crystallinity were observed. A strong contrast can be observed for xiphosure, the C N groups (2219 cm1 and 2225 cm1) can stand higher temperatures; in this case we have a material with high crystallinity and low enthalpy changes [21]. 4. Conclusions Thermomechanical, structural and thermal properties of two marine species a-chitin-based exoskeletons, black coral and xiphosure were studied. The mechanical response of both exoskeletons, obtained by DMTA, show a glass transition around 255 C, additionally black coral presented another transition around 175 C, associated to the acetamide group attached to the a-chitin chain. These results are consistent with the reported in the literature for a-chitin, although a shift in the temperature was observed, probably due to the presence of non-chitinous components in the skeleton matrix. DSC measurements revealed that the transition around 100 C, have enthalpies of nearly 30 times larger for black coral than for xiphosure, indicating strong differences between the macromolecular structure in relation to their water holding capacity and strength water–polymer interaction. X-ray diffraction shows that the crystallinity of xiphosure is larger than for coral, which is consistent with DSC and TGA measurements. Lower values in DH in xiphosure correspond to
material with a higher crystallinity. In contrast coral exhibits higher values in DH and a lower crystallinity. FTIR analysis shows clearly that both type skeletons are a-chitin structures, and allows following the thermal transformations of the matrix. At high temperature, the amide bands decrease and are destroyed eventually. However at the higher studied temperature, the C N groups remain, revealing that xiphosure possesses a tougher structure Acknowledgements Support for the XRD, DMA and FTIR analyses at the LANNBIO, Cinvestav Mérida, was provided by the projects FOMIX-Yucatán 2008-108160 and CONACYT LAB-2009-01 No. 123913. We are grateful to MSc. Daniel Aguilar-Treviño for the valuable technical support. References [1] R. Muzzarelli, C. Jeuniaux, G. Gooday, Chitin in Nature and Technology, Plenum Publishing Corporation, New York, USA, 1986. [2] R. Muzzarelli, Chitin: Formation and Diagenesis, 34, Springer, New York, USA, 2011. [3] S.P. Campana-Filho, D. De Britto, E. Curti, F. Abreu, M. Cardoso, M. Battisti, P. Sim, R. Lavall, Extraction, structures and properties of a- and b-chitin, Quim. Nova 30 (2007) 644–650. [4] M. Jang, B. Kong, Y. Jeong, C. Lee, J. Nah, Physicochemical characterization of a-chitin, b-chitin, and a-chitin separated from natural resources, J. Polym. Sci. A 42 (2004) 3423–3432. [5] J. Kumirska, M. Czerwicka, Z. Kaczýnski, A. Bychowska, K. Brzozowski, J. Thöming, P. Stepnowski, Application of spectroscopic methods for structural analysis of chitin and chitosan, Mar. Drugs 8 (2010) 1567–1636. [6] B. Juárez-de la Rosa, P. Quintana, P.-L. Ardisson, J. Yáñez-Limón, J. Alvarado-Gil, Effects of thermal treatments on the structure of two black coral species chitinous exoskeleton, J. Mater. Sci. 47 (2012) 990–998. [7] J.B. Gonzalez-Campos, E. Prokhorov, G. Luna-Barcenas, A. Mendoza-Galvan, I.C. Sanchez, S.M. Nuno-Donlucas, B. Garcia-Gaitan, Y. Kovalenko, Relaxations in chitin: evidence for a glass transition, J. Polym. Sci. B: Polym. Phys. 47 (2009) 932–943. [8] S. Kim, S. Kim, Y. Moon, Y. Lee, Thermal characteristics of chitin and hydroxypropyl chitin, Polymer 35 (1994) 3212–3216. [9] A. Glenn Richards, The Integument of Arthropods: The Chemical Components and Their Properties, the Anatomy and Development, the Permeability, North Central Publishing Co., St. Paul, USA, 1951. [10] W. Goldberg, Chemistry and structure of skeletal growth rings in the black coral Antipathes fiordensis (Cnidaria: Antipatharia), Hydrobiologia 216/217 (1991) 403–409.
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Thermal analysis and structural characterization of chitinous exoskeleton from two marine invertebrates B.A. Juárez-de la Rosa a,b, * , J. May-Crespo b , P. Quintana-Owen b , W.S. Gónzalez-Gómez b , J.M. Yañez-Limón c, J.J. Alvarado-Gil b, ** a
Laboratory of Natural Polymers, CIAD – Coordinación Guaymas, Carretera al Varadero Nacional km. 6.6, Col. Las Playitas, 85480 Guaymas, Sonora, Mexico Applied Physics Department, CINVESTAV-IPN Unidad Mérida, Carretera antigua a Progreso, km. 6. Apdo, Postal 73, Cordemex, 97310 Mérida, Yucatan, Mexico c Materials and Engineering Science, CINVESTAV-IPN, Unidad Querétaro, Libramiento Norponiente No. 2000, Fracc. Real de Juriquilla, 76230 Santiago de Querétaro, Querétaro, Mexico b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 August 2014 Received in revised form 10 January 2015 Accepted 7 April 2015 Available online 17 April 2015
Thermomechanical and structural properties of two marine species exoskeletons, Antipathes caribbeana (black coral) and Limulus polyphemus (xiphosure), were studied using dynamic mechanical thermal analysis (DMTA), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). DMTA curves indicate the viscoelastic behavior and glass transition around 255 C, black coral presented a second transition (175 C) associated to the acetamide group attached to the a-chitin chain. DSC measurements showed a endothermic peak around 100 C, with enthalpies of 4.02 and 118.04 J/g, indicating strong differences between exoskeletons respect to their water holding capacity and strength water–polymer interaction. A comparative analysis involving DSC and X-ray diffraction showed that lower values DH in xiphosure correspond to a material with a higher crystallinity (30), in contrast black coral exhibits higher values DH and a lower crystallinity (19). FTIR confirmed a-chitin based structure, at higher temperature diminishes the amide bands and a new one appears, related to C–N groups. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Black coral Horseshoe crab Thermal properties DMTA TGA XRD
1. Introduction Chitin is a linear polysaccharide constituted of b-(1 ! 4)-2deoxy-2-acetamido-D-glucopyranose repeating units [1]. It is similar to cellulose in terms of its structure and abundance in nature. In its native state, this biopolymer is associated with proteins, pigments, lipids, and inorganic substances, and is chiefly found as a fibrillar semicrystalline material [2]. Chitin is an important component of the exoskeleton organic matrix in many invertebrate animals (insects, crustaceans, mollusks, corals, annelids, ectoprocts, brachiopods), fungi and bacteria [3–6]. The analysis of chitin has been done by several research groups [7,8], although there are numerous open problems that remain to be
* Corresponding author at: Laboratory of Natural Polymers, CIAD – Coordinación Guaymas, Carretera al Varadero Nacional km. 6.6, Col. Las Playitas, 85480 Guaymas, Sonora, Mexico. Tel.: +52 6222252829. ** Corresponding author. E-mail addresses: [email protected], [email protected] (B.A. Juárez-de la Rosa), [email protected] (J.J. Alvarado-Gil). http://dx.doi.org/10.1016/j.tca.2015.04.015 0040-6031/ ã 2015 Elsevier B.V. All rights reserved.
investigated, in particular, chitin is only a fraction of the exoskeleton, therefore the properties of those materials are strongly influenced by the remaining components in which proteins and organic components, among many others, are present. In this work the exoskeletons in two species of marine invertebrate, Antipathes caribbeana (black coral) and Limulus polyphemus (horseshoe crab) (Fig. 1) are studied. Both species have exoskeletons in which the basic component is the polysaccharide a-chitin [6,9–11], forming the structures which perform all the functions required for survival in their respective biological environments. Their main function is to stabilize the body, but it also serves as a selective chemical barrier between the organism and its environment [12]. Antipatharians are cnidarians that form coral colonies, which frequently exhibit an arborescent appearance characterized by a spiny, branched skeletal axis based on chitin (Fig. 1a). In black coral, the skeleton consists of growth rings formed by chitin layers (micro-lamellae) segregated by polyps across the epidermis [10–11].
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Fig. 1. Chitinous exoskeletons of marine invertebrates (a) Antipathes caribbeana (black coral) and (b) Limulus polyphemus (xiphosure). AFM micrographs of the skeleton surface (image scan size: 50 mm).
Horseshoe crabs are marine chelicerate arthropods of the order Xiphosura which periodically shed their carapace, or molt, to accommodate body growth (Fig. 1b). To accommodate the larger body size, the new skeleton is initially flexible but then hardens and takes their characteristic color depending on its component tans [13–15]. Black coral A. caribbeana has 12 wt% of the dry weight of chitin [6,10], similar data has been obtained by other authors which reported 10 and 14 wt% for Antipathes salix and Antipathes fiordensis, respectively [8,10]. On the other hand for L. polyphemus, a higher chitin content has been reported, being around 24–28 wt% of the dry weight [5,9,16]. Biopolymers, particularly chitin, are susceptible to structural changes caused by temperature treatments or mechanical influence [17]. Dynamic mechanical thermal analysis (DMTA) is a powerful technique for characterizing polymer viscoelastic properties, because it measures the Young’s modulus (stiffness) and damping (energy-dissipation) properties of materials as they deform under dynamic stress [18]. These measurements provide quantitative information about materials performance [19]. DMTA and conformational transformation comparative study of horseshoe crab and black coral represent a biomimetic model approach, vital to understanding chitin’s crystalline structure, molecular fibrillar arrangement, and thermal behavior. Additional data and analysis on the chitin degradation process will also improve our understanding of its structural role in the skeletons of these species. The present objective of this study was to analyze the exoskeleton of A. caribbeana (black coral) and L. polyphemus (horseshoe crab or xiphosure) using DMTA and thermal characterization. Additional studies were done using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) in order to quantify the heat transfer and weight changes. Also structural effects were monitored during the applied thermal treatments with X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy.
2. Experimental procedure 2.1. Sample preparation L. polyphemus (xiphosure) specimens were collected from the coastal area of the Peninsula of Yucatán, México. A. caribbeana (black coral) colony is native from the deep reefs of Cozumel Island, Quintana Roo México (western Caribbean). The analyzed samples correspond to individual adults, after being collected were washed with fresh water and let dry at room temperature during one week and finally were kept in a desiccator over eight months, before the thermal characterization was carried out. For DMTA measurements flat samples of dried skeleton were cut from dorsal xiphosure carapace and the ones of black coral skeleton were cut from the branches in the longitudinal direction with respect to the axial direction. The specimen dimensions (length, width, thickness) were 16.003 1.682 mm 7.424 0.877 mm 0.793 0.098 mm, and 15.287 0.128 mm 6.775 0.288 mm 0.766 0.089 mm, for black coral and xiphosure, respectively. The effect of the temperature on the structure of the materials evolution was also studied in grounded xiphosure and black coral samples. Measurements at room temperature were followed by thermal treatments during 1 h in an electric furnace at several temperatures: 110 C, 210 C, 270 C and 310 C, afterwards, they were cooled at room temperature. XRD and FT-IR analysis were made immediately after each thermal treatment. 2.2. Thermal analysis DMTA was carried out using a TA Instruments Q-Series DMA (Q800). All the samples were measured in a film clamp intension mode over the temperature ramp of 5–300 C, at a heating rate of 5 C/min and at a frequency of 1 Hz. Measurements of DSC were carried out using DSC 822 Mettler Toledo equipment. Accurately weighed material was placed inside
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an aluminum closed pans of 40 mL capacity and hermetically sealed. Argon atmosphere with a 50 cc/min flux, and a heating rate of 10 C/min was applied. A Mettler Toledo TGA/SDTG 851 apparatus was used to assess the weight loss with heat treatment. Accurately weighed material was placed inside an aluminum cup and hermetically sealed. Also a heating rate of 10 C/min was used in argon atmosphere from 20 C up to 500 C. 2.3. Structural characterization The samples were characterized by X-ray diffraction in Bragg– Brentano geometry using a Siemens D5000 diffractometer with a monochromatic CuKa radiation (l = 1.5418 Å), operating at 34 kV, 25 mA. The powder diffractogram patterns were registered in the range of 5 < 2u < 70 in a step scan mode of 0.02 (2u ) with a counting time of 12 s per step. IR spectra in the transmittance mode were recorded on a Thermo Nicolet Nexus 670 FTIR spectrometer equipped with a DTGS KBr detector in the middle infrared (4000–400 cm1). Powdered samples of xiphosure and black coral skeleton were mixed by grinding the dry blended powders with KBr with a ratio of 5:195 mg, with an agate mortar until homogenization. This mixture was inserted into the sample compartment at the Smart Multi-Bounce HATR of the spectrometer and continuously purged with dry air. The number of scans for each spectrum was 64, and the spectral resolution was 4 cm1. 3. Results and discussion 3.1. Dynamic mechanical thermal analysis (DMTA) The temperature-dependent storage moduli (E) and tan d of the xiphosure and black coral skeleton chitins were recorded within 25–300 C temperature range (Fig. 2). The samples exhibit the typical behavior of a viscoelastic material. Initially the storage modulus of the black coral is stronger and falls about 6000– 3000 MPa before 200 C. Additionally, tan d reaches a maximum around 175 C. This could be associated to the acetamide group attached to the C2 position in a-chitin [8]. On the other hand, this indicates that xiphosure skeleton behaves as a tougher and stable material in the same temperature range (below 200 C).
Above 200 C, the behavior response shows a strong change for both materials, the storage modulus of black coral falls from 3000 MPa up to 100 MPa from 200 C to 300 C and its value of tan d increases from 0.12 up to 0.24, respectively. Whereas in the same temperature range (200–300 C), the storage modulus for xiphosure falls from 4000 to 50 MPa and the value of tan d grows from 0.09 to above 0.25, respectively. This behavior is the clear indication of glass transitions, occurring in the interval 250–260 C, for both black coral and xiphosure skeletons. It is worth to mention that the xiphosure DMTA measurements exhibit steeper changes in the loss moduli, but also display more maxima and minima in tan d, and it also seems to show that in some way the transitions for black coral slightly shifts to a lower temperatures. Coral showed a distinctive maximum at 256 C and xiphosure at 265 C. These results are consistent with the values reported for pure commercial and synthesized a-chitin by Kim et al. [8] in which similar results for the storage modulus and tan d curves are reported. These authors found the glass transition around 236 C, even though the transitions found in our work is higher, since the analysis was made on the exoskeleton which contains chitin, proteins, and others components that can increase the transition temperature. Therefore, the multiple maxima in tan d could be due to the presence of additional non-chitinous compounds. Those effects will be described latter at the DCS and FTIR sections. In the region under 200 C, the polymer is stiff/frozen and therefore a high storage modulus E is observed, the chains are nearly frozen in fixed positions because the available energy is not sufficient for inducing translational and rotational motions of the polymer segments [20]. Above 200 C, the polymer obtains sufficient thermal energy that enables to move its chains more freely, and therefore the storage modules decreases considerably. 3.2. Differential scanning calorimetry (DSC) In the DSC analysis (Fig. 3), both the black coral and xiphosure exhibited several endothermic peaks. The DSC thermograms behavior of both species were similar; however the curve intensity is much smaller for xiphosure than for black coral, also the structure of both species decomposed completely at 350 C, near the temperature reported for other chitinous structures [21–23]. It is important to notice that the first thermal event shows strong
Fig. 2. DMTA results generated on exoskeleton samples of two marine invertebrates: (a) storage moduli as a function of temperature and (b) tan d curves versus temperature.
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which correspond to the observed behavior of DSC measurements (peaks at 262 C for coral and 275 C for xiphosure). The highest temperature peaks at 303 C for coral and 329 C, for xiphosure, were due to the decomposition of the polysaccharide [6,21,24,25]. The final degradation enthalpies of 12.57 J/g and 0.50 J/g for a-chitin suggest that the decomposition efficiency might be different in the mechanism of cleavage of acetyl groups. These results are in accordance with the XRD results since the lower value of DH for xiphosure, which as will be shown later, exhibits a higher crystallinity, on the other hand a higher DH for black coral will reflect a higher free amine groups observed by FTIR analysis [21]. 3.3. Thermogravimetric analysis (TGA)
Fig. 3. DSC thermograms of black coral and xiphosure exoskeleton. The data for xiphosure curve is multiplied by 30 for comparison purposes.
peaks centered at 101 C and 110 C, for xiphosure and black coral respectively, and can be ascribed to the loss of water (dehydration). Chitin is normally a highly hydrophilic polysaccharide; indeed, in a solid state, it depends on the primary and supramolecular structures which can be easily hydrated and dehydrated [6], therefore the endotherm related to evaporation of water is expected to reflect physical and molecular changes during heating especially with hydrophilic hydroxyl groups [21]. Their associated enthalpies were calculated and gave 4.02 J/g and 118.04 J/g for xiphosure and black coral, respectively, indicating strong differences between the black coral and xiphosure macromolecules in relation to their water holding capacity and strength water– polymer interaction [21]. When heating the samples, two endothermic peaks with a reduced area and smaller enthalpies values appear, at 220 C and 262 C (0.75 J/g and 1.06 J/g, respectively) for black coral and 241 C and 275 C for xiphosure (0.08 J/g and 0.18 J/g, respectively). These peaks can be related to the complete removal of hydroxyl groups bound to the polysaccharide rings. These results allows us to identify the transitions observed in the DMTA measurements for the tan d at 256 C for coral and 265 C for xiphosure (Fig. 2b),
TGA and DTG thermograms show mainly two decomposition steps for both species, although the weight loss, in response to the increase of the temperature, was higher in the xiphosure exoskeleton (Fig. 4). The first stage occurs in the range 40–150 C, and is attributed to free water evaporation with a weight loss of 4–5% for both species. In the second stage, relevant thermal transformations occurred within the range 250–370 C, with a higher weight loss for xiphosure (26%) than black coral (13%). This is related to the degradation of the polysaccharide structure of the molecule, in which aliphatic compounds (CH2, CH3, functional groups) were being separated from the chitin structural ring and additionally to the degradation of amides I and II (C¼O, NH) and phosphodiesters groups (COC, C O P, and P O P), (CO, P¼O, and PO2–). Another step was observed in xiphosure between 446 and 550 C which can be related to the complete denaturation of chitin [6,21–23,26]. 3.4. X-ray diffraction (XRD) Both the black coral and xiphosure exoskeleton were analyzed with XRD, and the results compared to standard commercial a-chitin (Fig. 5). Both species exhibited crystalline reflections characteristic of chitin, with peaks mainly at 9 and 19 (2u). However, under thermal treatment, the peaks were more intense in xiphosure than in coral; in particular the intensity of 19 (2u) peak increases, indicating that xiphosure has a more organized
Fig. 4. TGA thermograms and DTG curves of the exoskeleton of (a) black coral and (b) xiphosure. The highest loss weight (13–26%) and the relevant thermal transformations occur at 300 C on black coral exoskeleton and at 330 C on xiphosure exoskeleton.
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Fig. 5. X-ray diffractograms of the marine invertebrate exoskeleton with different thermal treatments (US: untreated sample, 110, 210, 270, and 310 C) respect to the standard a-chitin (inset). (a) Black coral and (b) xiphosure.
structure. XRD patterns became slightly sharper and more intense around 210 C in the coral and at 270 C in the xiphosure. Most of the peaks began to decrease at higher temperatures, and were nearly suppressed at 310 C, at which the polysaccharide crystalline structure is systematically destroyed (Fig. 5a and b). Crystallite size was analyzed for xiphosure exoskeleton with the Scherrer equation [27] obtaining a crystallite size of 3.27 nm at 25 C, very near to the reported previously for black coral extracted a-chitin of 3.34 nm [28]. Also the unit cell parameters were determined considering the orthorhombic P212121 symmetry of chitin using a Gaussian deconvolution of the peaks for planes (0 2 0), (11 0) and (2 11) [28,29]. When compared to standard commercial a-chitin (Table 1), crystallographic parameters showed the xiphosure skeleton to have an a-crystalline structure similar to previous reports for black coral chitin [6,28]. Exoskeleton crystallinity index (CI) was analyzed using the 19 (2u) peak, which corresponds to the hkl (11 0) plane. A method was used that previously developed to calculate the CI of cellulose, as follows [29,30], CI% = [Ic/(Ic + Ia)] 100; where Ic represents the crystalline region at 19 (2u ) corresponding to the hkl (11 0) plane; and Ia is the amorphous region, obtained from the XRD patterns.
Table 1 Crystallographic parameters of xiphosure exoskeleton in comparison to black coral chitina and the standard commercial a-chitin. Species Xiphosure Limulus polyphemus
Black coral chitina Antipathes caribbeana
standard a-chitin
a
2u ( ) (hkl)
d-Spacing (Å) Unit cell parameters (Å)
9.60 19.60 39.25
(020) 9.20 (110) 4.52 (211) 2.29
a = 4.67 b = 18.43 c = 17.13
9.4 12.5 19.6 26.3
(020) (021) (110) (013)
9.51 7.08 4.53 3.38
a = 4.58 b = 19.65 c = 10.56
9.28 (020) 9.53 12.66 (021) 6.99 19.3 (110) 4.59
a = 4.74 b = 19.06 c = 10.29
From Juárez-de la Rosa et al. [6].
CI values were 30% for L. polyphemus and 19% for A. caribbeana exoskeletons (Table 2). Morphologically, the observed XRD patterns suggest that higher temperature (210–270 C) may favor crystalline re-arrangement of the chitin of the exoskeleton (Fig. 5). Due to thermal treatment, this effect is common in sclerotized skeleton in which thermal treatments with specific temperature ranges are used to increase crystallinity [28]. The strong differences in the structure and crystallinity between the coral and xiphosure are consistent with our DSC measurements. Lower values in DH in xiphosure correspond to material with a higher crystallinity. In contrast coral exhibits higher values in DH and a lower crystallinity [21]. 3.5. Infrared analysis (FTIR) Both the xiphosure and black coral samples, exhibited FT-IR spectra distribution pattern similar to that of the standard commercial a-chitin (Fig. 6). The two species show bands at 3448 cm1 related to OH stretching mode of hydroxyl groups and 3290 cm1 associated to NH stretching vibration mode of the amide. The bands at 2886 and 2935 cm1, are correlated to aliphatic compounds, CH2 and CH3 asymmetric and symmetric stretching vibration modes. In the wavelength range from 1600 cm1 up to 1000 cm1, there are bands associated with amides I and II, the saccharide ring, phosphodiester groups and proteinaceous components [4,6,30,31]. These bands were more clearly defined when the temperature increases, intensity and sharpness were highest at 210 C for black coral and 270 C for
Table 2 Crystalline index as a function of thermal treatment in the exoskeleton of two marine invertebrates. Temperature (o C)
Untreated sample 110 210 270 310
Crystalline index (%) Black coral A. caribbeana
Xiphosure L. polyphemus
19.6 20 14.2 16.3 5.4
30.2 26.2 25.5 29.2 9.7
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Fig. 6. FTIR spectra of the marine invertebrate exoskeleton with different thermal treatments (US: untreated sample, 110, 210, 270, and 310 C) respect to the standard a-chitin (inset). (a) Black coral and (b) xiphosure.
xiphosure. The bands at 1646 cm1 and 1666 cm1 for amide I ( CN vibration and C¼O stretching modes), confirms the a-chitin structure [21]. The band at 1557 cm1 corresponding to amide II (NH stretch mode) begins to disappear above 210 C. The main changes were observed in the bands hydroxyl and amides groups after 210 C for black coral and 270 C for xiphosure, respectively. At 310 C, the presence of a new band at 2225 cm1, associated to CN vibrations mode, indicates that deacetylation is smaller in the xiphosure than in the black coral, for this temperature. Therefore, another processes began above 310 C, including degradation, depolymerization, and denaturation of the exoskeleton. The decreasing at high temperature of the FT-IR bands for the amides, are in accordance with the XRD and DSC measurements. In coral all the amide groups are destroyed above 210 C, in this case higher enthalpies and low crystallinity were observed. A strong contrast can be observed for xiphosure, the C N groups (2219 cm1 and 2225 cm1) can stand higher temperatures; in this case we have a material with high crystallinity and low enthalpy changes [21]. 4. Conclusions Thermomechanical, structural and thermal properties of two marine species a-chitin-based exoskeletons, black coral and xiphosure were studied. The mechanical response of both exoskeletons, obtained by DMTA, show a glass transition around 255 C, additionally black coral presented another transition around 175 C, associated to the acetamide group attached to the a-chitin chain. These results are consistent with the reported in the literature for a-chitin, although a shift in the temperature was observed, probably due to the presence of non-chitinous components in the skeleton matrix. DSC measurements revealed that the transition around 100 C, have enthalpies of nearly 30 times larger for black coral than for xiphosure, indicating strong differences between the macromolecular structure in relation to their water holding capacity and strength water–polymer interaction. X-ray diffraction shows that the crystallinity of xiphosure is larger than for coral, which is consistent with DSC and TGA measurements. Lower values in DH in xiphosure correspond to
material with a higher crystallinity. In contrast coral exhibits higher values in DH and a lower crystallinity. FTIR analysis shows clearly that both type skeletons are a-chitin structures, and allows following the thermal transformations of the matrix. At high temperature, the amide bands decrease and are destroyed eventually. However at the higher studied temperature, the C N groups remain, revealing that xiphosure possesses a tougher structure Acknowledgements Support for the XRD, DMA and FTIR analyses at the LANNBIO, Cinvestav Mérida, was provided by the projects FOMIX-Yucatán 2008-108160 and CONACYT LAB-2009-01 No. 123913. We are grateful to MSc. Daniel Aguilar-Treviño for the valuable technical support. References [1] R. Muzzarelli, C. Jeuniaux, G. Gooday, Chitin in Nature and Technology, Plenum Publishing Corporation, New York, USA, 1986. [2] R. Muzzarelli, Chitin: Formation and Diagenesis, 34, Springer, New York, USA, 2011. [3] S.P. Campana-Filho, D. De Britto, E. Curti, F. Abreu, M. Cardoso, M. Battisti, P. Sim, R. Lavall, Extraction, structures and properties of a- and b-chitin, Quim. Nova 30 (2007) 644–650. [4] M. Jang, B. Kong, Y. Jeong, C. Lee, J. Nah, Physicochemical characterization of a-chitin, b-chitin, and a-chitin separated from natural resources, J. Polym. Sci. A 42 (2004) 3423–3432. [5] J. Kumirska, M. Czerwicka, Z. Kaczýnski, A. Bychowska, K. Brzozowski, J. Thöming, P. Stepnowski, Application of spectroscopic methods for structural analysis of chitin and chitosan, Mar. Drugs 8 (2010) 1567–1636. [6] B. Juárez-de la Rosa, P. Quintana, P.-L. Ardisson, J. Yáñez-Limón, J. Alvarado-Gil, Effects of thermal treatments on the structure of two black coral species chitinous exoskeleton, J. Mater. Sci. 47 (2012) 990–998. [7] J.B. Gonzalez-Campos, E. Prokhorov, G. Luna-Barcenas, A. Mendoza-Galvan, I.C. Sanchez, S.M. Nuno-Donlucas, B. Garcia-Gaitan, Y. Kovalenko, Relaxations in chitin: evidence for a glass transition, J. Polym. Sci. B: Polym. Phys. 47 (2009) 932–943. [8] S. Kim, S. Kim, Y. Moon, Y. Lee, Thermal characteristics of chitin and hydroxypropyl chitin, Polymer 35 (1994) 3212–3216. [9] A. Glenn Richards, The Integument of Arthropods: The Chemical Components and Their Properties, the Anatomy and Development, the Permeability, North Central Publishing Co., St. Paul, USA, 1951. [10] W. Goldberg, Chemistry and structure of skeletal growth rings in the black coral Antipathes fiordensis (Cnidaria: Antipatharia), Hydrobiologia 216/217 (1991) 403–409.
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